RAB7HD/C5orf51 stabilizes newly synthesized RAB7A and facilitates its GTP loading

RAB7HD/C5orf51 stabilizes newly synthesized RAB7A and facilitates its GTP loading | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article RAB7HD/C5orf51 stabilizes newly synthesized RAB7A and facilitates its GTP loading Koji Yamano, Kei Okatsu, Haruhiro Sano, Reika Kikuchi, Mitsunori Fukuda, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8547387/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract RAB GTPases are essential molecular switches governing eukaryotic vesicular trafficking by cycling between active GTP-bound and inactive GDP-bound states. While conformational changes facilitate their function, exposure of the hydrophobic switch regions presents a risk, potentially leading to toxic aggregation and degradation, a process increasingly recognized as RAB quality control. In this study, we identified RAB7HD/C5orf51 as a novel RAB7A partner that plays a crucial role in RAB quality control. C5orf51 strongly binds to newly translated, guanine nucleotide-free (apo) RAB7A and acts as a specialized chaperone to maintain its solubility, thereby preventing aggregation. Furthermore, we determined the crystal structure of the C5orf51-apo RAB7A complex at 3.36 Å resolution, revealing a specific RAB7A intermediate. This structural and functional evidence provides new molecular insights into the initial regulation and quality control of RAB proteins, ensuring their proper folding and availability for vesicular trafficking. Biological sciences/Cell biology/Membrane trafficking/Small GTPases Biological sciences/Molecular biology/Protein folding/Chaperones Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Main A hallmark of eukaryotic cells is their compartmentalized endomembrane system. The maintenance and propagation of these endomembrane organelles require constant communication for the delivery of lipids, cargo proteins, and metabolites via vesicular trafficking. Among the various steps of this process, vesicle transport and tethering are primarily regulated by small RAB GTPases 1 2 3 . These proteins typically consist of 200–250 amino acids, with approximately 60 different members encoded by the human genome. Like other small GTPases, RAB proteins cycle between a GTP-bound active state and a GDP-bound inactive state. This cycling is catalyzed by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). GEFs on the target membrane are suggested to be major determinants of RAB localization 4 . Several other proteins are associated with RAB proteins. Following synthesis on cytosolic ribosomes, RAB proteins first bind to a RAB escort protein (REP) 5 . Their C-terminal cysteine residues are then irreversibly prenylated by geranylgeranyl transferase 6 , which enables RAB proteins to be inserted into membranes. On the target membrane, GTP-bound RAB proteins interact with effector proteins to exert their function in vesicular transport and/or membrane tethering. Finally, RAB proteins are extracted from the membranes with the help of a GDP dissociation inhibitor (GDI), which masks the prenyl moiety for the next round of the RAB cycle 7 . The G-domain common to small GTPases and RAB proteins form a globular fold with a 6-stranded b-sheet surrounded by 5 a-helices. The switch I and II regions of RAB proteins are known to undergo large conformational changes upon binding to GTP and GDP 8 . Recently, it was reported that the exposure of the hydrophobic switch I region in inactivated RAB8A is recognized by BAG6 and degraded via the ubiquitin-proteasome pathway 9 . This suggests that precise GTP-loading onto RAB proteins and/or preventing the exposure of the hydrophobic switch I region is crucial for RAB quality control. However, the mechanisms by which RAB quality control is maintained to prevent aggregate formation and/or unwanted degradation through the ubiquitin-proteasome pathway remain largely unknown. Among the 60 different RAB proteins in mammals, RAB7A is one of the most studied and is particularly important for lysosomal biogenesis 10,11 . RAB7A is localized mainly to late endosomes and lysosomes, and regulates vesicular trafficking from early endosomes to late endosomes and from late endosomes to lysosomes. The RAB7A GEF activity of the MON1-CCZ1 complex was demonstrated using purified yeast homologs (Mon1-Ccz1 complex) 12 . Several RAB7A effectors have been identified, including the HOPS complex 13 , RILP 14 , and FYCO1 15 , suggesting the multifaceted role of RAB7A. Furthermore, RAB7A has been reported to play a key role in the maturation of autophagosomes into autolysosomes by mediating fusion events with lysosomes 16 . In this study, we report the identification of C5orf51 as a novel binding partner of RAB7A. C5orf51 strongly binds to newly translated guanine nucleotide-free (apo) RAB7A, exhibiting chaperone-like activity that maintains its solubility. Furthermore, we determined the crystal structure of the C5orf51-apo RAB7A complex at a resolution of 3.36 Å. Thus, the specific chaperone for RAB7A provides a new insight into the regulation of RAB protein quality control. Results Identification of C5orf51 as a novel binding protein to guanine-nucleotide free form of RAB7A To identify factors involved in RAB protein quality control, we transiently expressed FLAG-tagged wild-type (WT) and nucleotide-free (apo) mutants of representative RAB proteins (RAB4A, RAB6A, RAB7A, RAB8A, RAB9A, RAB11A, and RAB18) in HeLa cells. The apo state was mimicked by a single amino acid substitution, Asparagine (N) to Isoleucine (I) 17 . Using HeLa cells without overexpression of RAB proteins as a control, we performed co-immunoprecipitation (co-IP) followed by mass spectrometry to identify interacting partners (Fig. 1A). Since factors that bind strongly to the structurally unstable apo form may are more likely to be involved in RAB protein quality control, the abundance ratios of co-IPed proteins between WT and apo form of each RAB protein were plotted (Fig. 1A). First, we observed that GDI1, GDI2, RABGGTA, CHM, and CHML bind to the RAB WT form but not the apo form (Fig. 1A), suggesting that lipid modification may be inhibited in the apo state. Among these RAB panels, RABIF/MSS4 was found to bind specifically to the apo form of RAB8A (Fig. 1A, RAB8A panel). This finding supports a report that RABIF acts as a chaperone involved in the folding of RAB8 family proteins 18 . On the RAB7A panel, the subunits of RAB7A-GEF complex, MON1B, CCZ1, and RMC1 were identified as proteins that bind specifically to apo RAB7A (Fig. 1A, RAB7A panel). This is consistent with a previous report 19 . Interestingly, C5orf51 was identified as a factor that interacts strongly with the apo RAB7A (Fig. 1A, RAB7A panel). Among the multiple RAB proteins tested, RABIF binds specifically to the apo RAB8A, while C5orf51 binds specifically and strongly to the apo RAB7A (Fig. 1B). A similar result was observed by immunoblotting (Fig. 1C). C5orf51 is a protein found across metazoan species, ranging from early-branching lineages such as Trichoplax sp. and N. vectensis to vertebrates such as D. rerio and H. sapiens . In contrast, it appears to have been lost in some ecdysozoa lineages including C. elegans and D. melanogaster (Fig. S1). Exceptionally, an ortholog of C5orf51 is present in the cryptophyte Guillardia theta , and it might have been acquired through horizontal gene transfer (Fig. S1). The human C5orf51 consists of 294 amino acid residues, and its molecular function including its cellular localization is unknown. C5orf51 binds to RAB7A in a MON1-CCZ1-independent manner. Next, we investigated the intracellular interaction between C5orf51 and RAB7A using the fluoppi (fluorescent-based technology detecting protein-protein interactions) system 20 21 . In this system, when a protein fused with a homo-tetrameric humanized azami-green (hAG) interacts with another protein fused with a homo-oligomeric assembly helper (HA-Ash) tag, they form large liquid-liquid phase separated (LLPS)-based foci in cells through multivalent interactions (Fig. 2A). When hAG-C5orf51 was co-expressed with a C-terminal Cys-truncated RAB7A (HA-Ash-RAB7A-DC WT), which was designed to maintain cytoplasmic solubility by preventing prenylation, both proteins were diffusely localized in the cytosol (Fig. 2B). A GTP-locked RAB7A mutant Q67L (HA-Ash-RAB7A-DC Q67L) also localized diffusely in the cytosol (Fig. 2B). In sharp contrast, both the GDP-bound RAB7A T22N and the apo-mimetic RAB7A N125I mutants formed fluoppi foci with hAG-C5orf51 (Figs. 2B and 2C), indicating a direct interaction. The interaction with C5orf51 was specific to RAB7A because RAB1A, which regulates vesicular transport from the ER to the Golgi apparatus 22 , did not interact with C5orf51 (Fig. 2B). Since 1) C5orf51 preferentially binds to apo or GDP-bound RAB7A (Fig. 2B), and 2) RMC1/C18orf8 that we previously found as an interacting protein with RAB7A T22N 23 was reported to be a component of the MON1-CCZ1 complex 19 , C5orf51 may be an additional subunit of MON1-CCZ1-RMC1 complex. We tested this possibility using fluoppi. As expected, hAG-MON1B and HA-Ash-CCZ1 formed fluoppi foci (Fig. 2D), indicating that they interact each other. However, neither HA-Ash-MON1B nor HA-Ash-CCZ1 formed foci with hAG-C5orf51 (Fig. 2D). Furthermore, hAG-RMC1, but not hAG-C5orf51, formed fluoppi foci with MON1B-CCZ1 (Figs. 2E and 2F). The similar results were obtained by co-IP. Both HA-tagged C5orf51 (3HA-C5orf51) and HA-tagged MON1-CCZ1 complex (3HA-MON1B and 3HA-CCZ1) were efficiently co-IPed with GDP-bound and apo forms of 3FLAG-RAB7A (Figs. 2G and 2H). On the other hands, MON1-CCZ1 complex (3HA-MON1B and 3HA-CCZ1) were co-IPed by 3FLAG-RMC1, but not by 3FLAG-C5orf51 (Fig. 2I). These results indicate that, while RMC1 is a component of MON1-CCZ1 complex, C5orf51 interacts with RAB7A in a MON1-CCZ1-independent manner. Specificity of C5orf51 interaction with mammalian RAB GTPases. To comprehensively identify the RAB-binding specificity of C5orf51, we next performed a yeast-two hybrid (Y2H) assay using 62 different GTP-locked or apo RAB mutants 24 25 . Based on the growth rate of the yeast colonies, we found that C5orf51 almost exclusively interacted with the apo form of RAB7A (Fig. 3A). While a marginal interaction with the apo form of RAB32 was observed in the Y2H screen, this interaction could not be confirmed in a subsequent fluoppi assay (Figs. 3B-3D). Furthermore, we could not detect an interaction via fluoppi with other RAB proteins phylogenetically close to RAB7A including RAB9A, RAB9B, RAB23 and RAB38 (Figs. 3B-3D). To further investigate the binding partner of C5orf51 in a more physiological context, we performed an IP-MS assay. 3FLAG-C5orf51 stably expressed in HeLa cells were immunoprecipitated with anti-FLAG antibody, and the interacting proteins were analyzed by mass spectrometry (Fig. 3E). The resulting volcano plot revealed that RAB7A was one of the key interacting proteins with C5orf51. On the other hand, other factors known to be involved in the RAB cycle, such as GEFs, GAPs, and effectors, were not identified in our analysis (Fig. 3E). These results collectively suggest that C5orf51 specifically interacts with RAB7A. C5orf51 directly interacts with the apo form of RAB7A. The above results strongly suggest that C5orf51 directly binds to RAB7A. To test this possibility, we first narrowed down the interacting region of RAB7A with C5orf51. Since The C-terminal Cys-deleted RAB7A T22N (RAB7A-DC) formed fluoppi foci with hAG-C5orf51, prenylation is not required for the interaction (Figs. 2B and S2). Furthermore, truncated RAB7A 1-176 (residues 1-176) still had an ability to interact with C5orf51, indicating that the hypervariable region is dispensable (Fig. S2). Next, to investigate the direct interaction, we prepared recombinant C5orf51-His 6 and GST-RAB7A 1-176 (WT, Q67L and T22N) while apo mimetic RAB7A 1-176 N125I could not be prepared because of severe aggregate-prone property. When GST and GST-RAB7A 1-176 were pulled down by glutathione Sepharose, C5orf51 was eluted only with GST-RAB7A 1-176 (Fig. 3F), indicating that C5orf51 directly binds to RAB7A. The elution efficiency of C5orf51 with RAB7A T22N were higher than those with RAB7A WT or Q67L (Fig. 3F). Furthermore, different eluted efficiencies of C5orf51 due to a different RAB7A nucleotide-binding state were nullified when guanine nucleotides and Mg 2+ ion were dissociated from RAB7A by EDTA treatment (Fig. 3G). When the apo, GDP-bound, and GppNHp (non-hydrolyzable GTP analog)-bound states were prepared using RAB7A 1-176 WT, the apo form strongly bounds to C5orf51 (Fig. 3H). These results indicate that C5orf51 directly interacts with RAB7A preferentially at the guanine-nucleotide free state. Loss of C5orf51 in cells reduces RAB7A protein amount. To investigate cellular function of C5orf51, we first examined its intracellular localization. GFP-tagged RAB7A (GFP-RAB7A WT) was found primarily on late endosomes and lysosomes as confirmed by co-staining anti-LAMP2 antibody (Fig. 4A). In contrast, C5orf51-mCherry was distributed throughout the cells (Fig. 4A). When GFP-RAB7A T22N, which interacts with C5orf51, was overexpressed, the nuclear signal of C5orf51-mCherry was significantly reduced, while its cytosolic signal remained unchanged (Fig. 4A). This suggests that the interaction between C5orf51 and RAB7A T22N occurs in the cytosol, drawing C5orf51 out of the nucleus. To further elucidate the role of C5orf51, we generated C5orf51 knockout (KO) HeLa cells using CRISPR/Cas9 gene editing. While the deletion of C5orf51 did not alter lysosomal morphology or pH (Fig. S3), we discovered that the loss of C5orf51 consistently reduced the amount of RAB7A protein (Figs. 4B and 4C). Specifically, the amount of RAB7A in two independent C5orf51 KO clones (#2-43 and #3-2) was reduced to approximately 60-70% of the levels in WT HeLa cells (Figs. 4B and 4C). Importantly, the reduced RAB7A protein levels in both clones were rescued by the exogenous expression of 3FLAG-C5orf51 (Figs. 4B and 4C). As a control, the protein levels of CCZ1, a subunit of the RAB7A GEF complex, and TOMM20, a mitochondrial protein, were not affected by C5orf51 deletion (Figs. 4B and 4C). Furthermore, we obtained similar results in C5orf51-/- HCT116 cells (Figs. 4D and 4E), confirming that this phenomenon is not cell-type specific. Given that RAB7A protein levels are reduced in C5orf51 KO cells, we anticipated that RAB7A-mediated lysosomal and autophagic functions would be impaired. However, C5orf51 deletion alone did not affect lysosomal morphology or acidification (Fig. S3), and it did not inhibit amino acid starvation-induced autophagy as measured by a Halo-tag processing assay (Figs. 4F and 4G). Previous studies have demonstrated that RAB7A, RAB9A and RAB9B have functional redundancy, and severe defects in lysosomal function are observed only when all three are knocked out 26 . Therefore, we established a RAB9A_RAB9B_C5orf51 triple KO (9A/B/C5 TKO) cell line, and then measured amino acid starvation-induced autophagy. The TKO cells showed a significant decrease in the production of the processed Halo fragment compared to WT or RAB9A/B DKO cells (Figs. 4H and 4I). Importantly, the reduced Halo fragments in the TKO cells were recovered upon exogenous expression of 3FLAG-C5orf51 (Figs. 4H and 4I). Therefore, these results indicate that loss of C5orf51 reduces RAB7A and affects an autophagy-lysosome function under a particular condition. C5orf51 enhances thermal stability of the nucleotide-free RAB7A. Previous studies have shown that small RAB GTPases without a bound guanine nucleotide are more susceptible to urea denaturation followed by proteolytic degradation 27 28 29 . To confirm the nucleotide-dependent stability of RAB7A, we first performed a thermal shift assay. Recombinant GST-RAB7A 1-176 , which was overexpressed and purified form E.coli BL21(DE3) cells, was incubated with a thermofluor dye that binds to exposed hydrophobic regions of the protein. The melting temperature (Tm) was determined by monitoring fluorescence as the temperature was gradually increased. The Tm values for GST and GST-RAB7A 1-176 WT were 52.0°C and 49.5°C, respectively (Fig. S4A). When EDTA was added to chelate Mg 2+ ion and dissociate bound guanine nucleotides, the Tm of RAB7A 1-176 WT was lowered to 45.5°C, while the Tm of GST alone remained unchanged (Fig. S4A). Furthermore, the GST-RAB7A 1-176 T22N mutant, which has a reduced affinity for guanine nucleotides, exhibited a lower Tm of 47.5°C that was not affected by EDTA treatment (Fig. S4A). These results indicate that both the purified RAB7A T22N and the nucleotide-free apo form of RAB7A exhibit low resistance to thermal denaturation. Next, we investigated whether C5orf51 could suppress heat-induced aggregation of the apo form of RAB7A or the T22N mutant. For this purpose, recombinant GST or GST-RAB7A 1-176 T22N was incubated at various temperatures (40-64°C), and protein aggregates were then pelleted by centrifugation. We found that GST began to appear in the pellet fraction at 54.5°C, while GST-RAB7A 1-176 T22N started to aggregate at a lower temperature of 49.1°C (Figs. S4B Frac. #5 and S4C Frac. #4). The addition of recombinant C5orf51 reduced the aggregation of GST-RAB7A 1-176 T22N, resulting in more soluble RAB7A remaining in the supernatant fraction, while the phenomenon was not observed with GST (Figs. S4B and S4C). Further analysis at temperatures ranging from 43°C to 56°C revealed that C5orf51 exhibited chaperone activity toward the apo form of RAB7A 1-176 WT. This effect was not observed for RAB7A 1-176 bound to GppNHp, a non-hydrolyzable GTP analog (Figs. S4D-S4F). These findings suggest that C5orf51 interacts specifically with nucleotide-free RAB7A to prevent it from aggregating. C5orf51 acts as a chaperone to facilitate correct folding of nascent RAB7A. Given that nucleotide-free (apo) RAB proteins may exist transiently during translation before GTP loading, we investigated the requirement of C5orf51 for RAB7A’s proper folding using the following two distinct approaches. First, we utilized an E.coli expression system, as procaryotes lack RAB-related factors including REPs. Although the RAB7A 1-176 is known to form a compact structure without intrinsically disordered region 30 , over half of the expressed HA-tagged RAB7A 1-176 was found in inclusion bodies (pellet fractions) upon induction at 28°C (Fig. 5A). This aggregation occurred regardless of the guanine binding state, as approximately half of the WT, Q67L, and T22N were recovered in the pellet fractions (Figs. 5A and 5B). As expected, the extent of aggregation was dependent on the culture temperature (Figs. 5A, 5B, S5A and S5B). In sharp contrast, the co-expression of C5orf51 significantly suppressed RAB7A aggregation, with nearly all of the WT and Q67L RAB7A 1-176 recovered in the soluble supernatant fraction at all temperatures tested (Figs. 5A, 5B, S5A and S5B). A similar chaperone-like activity was observed for full-length RAB7A (Figs. S5C and S5D). Importantly, once RAB7A 1-176 WT forms a stable structure, it no longer interacts with C5orf51; when C5orf51-His 6 was pulled down using a nickel affinity resin, RAB7A 1-176 T22N was found in the bound fraction, whereas RAB7A 1-176 WT was not (Fig. 5C). We also demonstrated that C5orf51 chaperone activity is specific to RAB7A, as the co-expression of C5orf51 did not prevent the aggregate formation of other RAB proteins (Figs. 5D and 5E). Furthermore, not only human C5orf51, but also its ortholog from Danio rerio (Zf C5orf51) had the same ability to prevent RAB7A from aggregate formation (Fig. S5E), indicating that this specific chaperone activity is evolutionarily conserved. For another approach to precisely monitor C5orf51 chaperone activity, we employed the PUREfrex, an in vitro coupled transcription/translation system. PUREfrex is a "bottom-up" reconstitution system that contains individually purified translational factors and ribosomes but lacks chaperones such as Hsp70 and GroEL. This allowed us to specifically monitor C5orf51-dependent chaperone activity during the translation of RAB7A. When GST-RAB7A 1-176 was translated in the presence of BSA as a control, more than half of the RAB7A WT, T22N, and Q67L proteins formed insoluble aggregates and were recovered in the pellet fraction after centrifugation (Figs. 5F and 5G). However, in the presence of recombinant C5orf51, these RAB7A proteins were translated almost completely as soluble proteins (Figs. 5F and 5G). Even the highly aggregate-prone nucleotide-free N125I mutant was rendered soluble when translated with C5orf51 (Figs. 5H and 5I). These results strongly suggest that C5orf51 acts as a specific chaperone for RAB7A and promotes its proper folding immediately after translation. Crystal structure of C5orf51 in complex with RAB7A To gain insight into the structural basis for the recognition of RAB7A by C5orf51, we set out to determine the C5orf51-RAB7A complex structure by crystallography. The complex was formed by mixing C5orf51 with apo RAB7A 1-176 (hereafter referred to as RAB7A) that had been pretreated with EDTA. The resulting C5orf51-RAB7A complex was purified and crystallized upon concentration (Fig. S6). We determined the crystal structure of the C5orf51–RAB7A complex at a resolution of 3.36 Å (Figs. 6A­-6C and Table S1). The analysis of structural homologs of C5orf51 using the DALI server 31 revealed that C5orf51 shares similarity with tetratricopeptide repeat (TPR) proteins including LNG, CNS1, and TONSOKU (Figs. S7A and S7B). The C5orf51 consists of ten a-helices and two insertion loops (residues 179–203 and 225–252) (Fig. 6B). The first insertion contained a-helix (a7) while the second insertion was disordered (Fig. S7C). In other TPR proteins such as LNG and TONSOKU, the concave surfaces are crucial for recognizing interacting partners (Figs. S7D and S7E). Similarly, the concave surface of C5orf51, which is evolutionarily highly conserved (Fig. S8A), directly interacts with RAB7A (Fig. S8B). The switch I region of RAB7A adopts a chaperone-specific conformation. RAB7A in the complex consists of a central six-stranded b-sheet (b1- b6) flanked by five a-helices (a1- a5) (Fig. 6C). Notably, the canonical a1 helix of RAB7A is unfolded in the present structure. Instead, the switch I region forms a new a-helix (referred to as aSI), which directly interacts with the concave surface of C5orf51 (Figs. 6A and 6C­­). This a1-switch I conformation in the C5orf51-RAB7A complex is distinct from other previously reported states of RAB7A (Figs. 6D-6H). The local unfolding of the a1 helix suggests an intermediate state, which is structurally different from the nucleotide-bound (GTP or GDP) forms of RAB7A (Figs. 6G and 6H). The yeast RAB7A homolog Ypt7 structure with MON1-CCZ1 also shows a distinct a1-switch I conformation due to the interaction with the GEF (Fig. 6F), suggesting the different structural dynamics of this region across the various functional states of RAB7A. We then investigated whether similar unfolded α1 and formed aSI conformation have been observed in other RAB proteins. RABIF/MSS4 was previously shown to recognize RAB8A in such a conformation, where the a1 helix is unfolded and the switch I forms a new aSI helix (Fig. S9A). While the conformation of RAB8A bound to RABIF/MSS4 closely resembles that of RAB7A bound to C5orf51 (Fig. S9B), the structures themselves (C5orf51 and RABIF/MSS4) are substantially different (Figs. S9C and S9D). Despite the similar switch I conformations, RAB7A and RAB8A are recognized at the different binding sites on their primary sequence (Fig. S9E). As a result, RAB7A has a more bent conformation with C5orf51 compared to RAB8A bound to RABIF/MSS4. The interface between C5orf51 and RAB7A. The RAB7A-binding interface of C5orf51 is composed of three distinct hydrophobic pockets located on its concave surface (Figs. 6I-6M and S10A). The first pocket extensively accommodates and captures the aSI region of RAB7A (Figs. 6J, 6K and S10). This interaction is mediated by bulky hydrophobic residues of RAB7A (Y28, F33, and Y37). M25 and V29 of RAB7A are also oriented toward C5orf51. C5orf51 provides a conserved hydrophobic concavity formed by Y76, C130, Y140, M141, L209, M212, Y213, and W277, which protects the hydrophobic side of RAB7A from solvent exposure. The second hydrophobic pocket of C5orf51, formed by W277, L197, L208, and L272, covers invariant residues (F45 and W62) in the switch I and inter-switch regions of RAB7A (Figs. 6L and S10). The third pocket of C5orf51 is comprised of I181, V187, F189, L197, and L198, which are located within the first insertion loop (Figs. 6M and S10). This pocket is buried by L8 and T47 from the b1 strand of RAB7A (Figs. 6M and S10). The hydrophilic interaction is also observed between K38 of RAB7A and D205 of C5orf51, with a proximity of 3.0 Å although the electron density for K38 is relatively weak (Fig. 6L). GTP loading affects the interaction of C5orf51 with RAB7A. To complement the biochemical and crystallographic analyses, we next conducted molecular dynamics (MD) simulations of both the C5orf51-apo RAB7A complex and the C5orf51-GTP-bound RAB7A complex. We prepared the structure of the GTP-bound form based on the crystal structure of the apo form (see ‘Methods’) and noted that the GTP-binding site is the opposite side from the interface between C5orf51 and RAB7A and that the C5orf51-RAB7A complex can accommodate GTP though the switch I region adopts its distinctive conformation which is structurally different from the nucleotide-bound (GTP or GDP) forms of RAB7A monomer. The results of the simulations showed that, in GTP-bound RAB7A, the C-terminal region (a4- a5 and b5- b6) containing guanine base recognition (G4-G5) motifs bent towards the N-terminal region (a1- a2) around the phosphate-binding (G1-G3) motifs, compared to the apo form (Fig. 6N). Moreover, torsion of the N-terminal region opened a pathway to the binding site, enabling access for GTP. Over the 1 ms simulation, the guanosine moiety of GTP was moving in and out of the site located between the G1 (P-loop) and G4–G5 regions, while the triphosphate groups of GTP was anchored to the P-loop (Fig. S11A). These results suggest that the C5orf51-RAB7A complex can load GTP onto apo RAB7A while maintaining chaperone-specific conformations. Regarding interactions within the complex, the number of hydrogen bonds between C5orf51 and RAB7A, as well as the buried surface area of the complex, tended to decrease in the GTP-bound form compared to the apo form (Figs. 6O and 6P), indicating a reduction in the interaction between C5orf51 and RAB7A. Especially, the number of hydrogen bonds to C5orf51 in the unfolded a1 region of RAB7A (M25, Y28, and V29), which is critical for C5orf51 recognition as described below, decreased in the GTP-bound form (Table S2). This is mainly because the changes in the P-loop conformation during GTP binding lead to more extensive unfolding of the helix around the a1 region (Fig. S11B). These results collectively suggest that the C5orf51-RAB7A complex can load GTP into the binding site of apo RAB7A and that C5orf51 and RAB7A dissociate coupled with loading GTP. The recognition of RAB7A by the concave surface of C5orf51 is required for chaperone activity. To assess the recognition mechanism revealed by the structural analysis of C5orf51-RAB7A, we introduced structure-based mutations into residues of C5orf51 that face RAB7A and tested their interactions by fluoppi assay (Fig. 7A). Alanine substitutions of Y140, L209, Y213, and W277 in C5orf51 abolished fluoppi foci formation with RAB7A, indicating a loss of interaction. The small side-chain mutation C130A in C5orf51 had no effect on the interaction, whereas the replacement with a bulky side chain (C130W) impaired the interaction. The substitution of Y76 and M212 of C5orf51 with a small hydrophilic serine residue reduced the interaction with RAB7A. The L272 of C5orf51 was substituted with an asparagine residue based on its proximity to RAB7A and its hydrophobic character. However, the L272N mutation in C5orf51 did not impair RAB7A recognition. Similarly, the D205A mutation, which was designed to disrupt the electrostatic interaction between D205 of C5orf51 and K38 of RAB7A, did not affect the interaction. Alanine substitution of the five hydrophobic residues (I181, V187, F189, L197, and L198) in the insertion1 of C5orf51 also had no effect on the interaction. Moreover, deletion of the entire hydrophobic cluster (residues 181–199) did not impair the interaction. We next examined the effect of C5orf51 mutants on RAB7A solubility. The C5orf51 mutants Y76S, C130W, L209A, M212S, Y213A, and W277A were prepared as a recombinant protein and used for in vitro PUREfrex assays (Fig. 7B). The L209A and W277A mutants of C5orf51 almost completely lost their chaperone activity toward RAB7A (Figs. 7B and 7C). The Y76S, C130W, M212S, and Y213A mutants of C5orf51 retained partial activity, but they showed lower activity than wild-type C5orf51 (Figs. 7B and 7C). These results indicate that residues composing the concave surface of C5orf51 are important for RAB7A recognition (Fig. 7D). The a SI region of RAB7A is essential for recognition and solubilization by C5orf51. To identify the residues of RAB7A critical for the interaction with C5orf51, we examined both the in vitro solubilization efficiency and intracellular interaction using RAB7A mutants. Substitutions of residues within the aSI region (Y28A, V29A, F33A, and Y37A), which bind to the hydrophobic pocket 1 of C5orf51, significantly abrogated C5orf51-dependent solubilization (Figs. 7E and 7F). Moreover, the Y28A and Y37A mutants showed no detectable interaction with C5orf51 in the fluoppi assay, and the V29A and F33A mutants exhibited reduced foci formation (Figs. 7G and 7H). On the other hands, alanine mutations at L8, F45, and W62 of RAB7A, which contact the hydrophobic pockets 2 and 3 of C5orf51, showed in vitro solubility comparable to that of wild-type RAB7A (Figs. 7E and 7F) and formed fluoppi foci with C5orf51 (Figs. 7G and 7H). Taken together, these results indicate that the aSI region of RAB7A is a primary recognition site for C5orf51 (Figs. 7I and S10). Among the four RAB7A residues (Y28A, V29A, F33A, and Y37A) critical for C5orf51 recognition, V29 and Y37 are less conserved among RAB family proteins (Fig. 7J). This sequence comparison supports the idea that non-conserved residues in RAB7A contribute to the substrate specificity of C5orf51. Given that mutations in the structural core of RAB7A did not affect its interaction with and solubilization by C5orf51, we hypothesized that the N-terminal region of RAB7A might be sufficient for recognition by C5orf51. Indeed, using fluoppi assays, we found that the N-terminal 38 residues of RAB7A is sufficient for the interaction with C5orf51 (Figs. 7G and 7H). This finding supports the idea that C5orf51 primarily recognizes the N-terminal region of RAB7A. To further test the sufficiency of the RAB7A aSI region, we constructed chimeric proteins in which the a1-switch I segments (residues 25-38 in each case) of RAB8A and RAB9A were replaced with the corresponding region of RAB7A (Fig. 7K). While C5orf51 interacted with neither RAB8A nor RAB9A, these chimeric proteins were recognized by C5orf51 in cells as shown by fluoppi assay (Figs. 7L and 7M). These results demonstrate that C5orf51 discriminates RAB7A from the other RABs by recognizing the a1-switch I sequence. Discussion While the structural stability of Rab GTPases was previously known to be influenced by their guanine-nucleotide binding state 27 , our understanding of the cellular quality control mechanisms that support Rab protein stability has been limited. In this study, we identified C5orf51 as a factor that binds strongly to the nucleotide-free state of RAB7A, which is critical for lysosome and autophagy regulation. Because C5orf51 also binds to the GDP-bound mutant RAB7A T22N, it may function as a guanine nucleotide exchange factor (Fig. 2 B). However, C5orf51 is localized diffusely in the cytoplasm (Fig. 4 A), unlike the endomembrane-associated RAB7A GEF, the MON1–CCZ1–RMC1 complex 32 . C5orf51 was previously suggested to be a new subunit of the MON1-CCZ1 complex 33 and was designated RIMOC1 (RAB7A-interacting MON1-CCZ1 complex subunit 1). Our fluoppi assay and co-immunoprecipitation experiments, however, did not detect an interaction between C5orf51 and the MON1-CCZ1 complex (Figs. 2 D- 2 I). Furthermore, structural superposition of the RAB7-MON1-CCZ1 complex and our RAB7A-C5orf51 complex revealed a substantial overlap between the binding surfaces of MON1-CCZ1 and C5orf51 on RAB7A (Fig. S12). These observations strongly suggest that C5orf51 and the MON1-CCZ1 complex do not function cooperatively but rather compete for binding to RAB7A. Therefore, the name RIMOC1 is not appropriate as it does not reflect the actual function of C5orf51. In this study, we discovered that C5orf51 acts as a chaperone-like protein, binding to the structurally unstable, nucleotide-free form of RAB7A to increase its solubility. The term “holdase” is used for a molecular chaperone that binds to an unfolded or partially folded substrate to prevent aggregation without using ATP hydrolysis energy. Therefore, we propose to name C5orf51 as RAB7HD, for RAB7A -specific H ol D ase. RAB7HD/C5orf51 increased the solubility of RAB7A (Fig. 5 ). Interestingly, even the wild-type and GTP-locked forms of RAB7A, which do not stably interact with C5orf51, also require C5orf51 to facilitate their folding to become stable structure. Our results strongly suggest that RAB7HD/C5orf51-RAB7A complex forms immediately after RAB7A translation, and they dissociate when GTP is loaded onto apo-RAB7A. C5orf51 recognizes the N-terminal region of RAB7A (Figs. 7 G and 7 H), which is consistent with the idea that C5orf51 binds to RAB7A during the early stages of translation, interacting with the nascent polypeptide chain to prevent aggregation. Interestingly, the RAB7A-RAB7HD/C5orf51 complex shows structural similarities to the RAB8A-RABIF/MSS4 complex (Fig. S9). While RABIF/MSS4 was initially reported as a GEF for RAB8A 34 , a recent study showed that it also functions as a chaperone that contributes to RAB8A stabilization in cells 18 . Although there is no structural similarity between the individual RAB7HD/C5orf51 and RABIF/MSS4, it is intriguing that they both induce a functional and structural similarity upon binding to their respective RAB proteins: the unfolding of the RAB a1 helix and the formation of a new a-helix in the Switch I region. The bulky hydrophobic residues F45 and W62 of RAB7A are located at the interaction interfaces not only in a complex with RAB7HD/C5orf51 but also in the complexes with the GEF MON1-CCZ1 and effector proteins like RILP and PDZD8 30 35 . This suggests that RAB7HD/C5orf51 may transiently cover the interaction surface with other binding partners, protecting the hydrophobic surface of RAB7A from non-specific interactions or premature binding. In this way, RAB7HD/C5orf51 functions consistent with the classical definition of a molecular chaperone, assisting the proper folding of RAB7A by preventing misfolding and aggregation without becoming a part of the final structure or influencing its final conformation. Materials and methods Cell lines, antibodies, plasmid DNAs and reagents used in this study are listed in Table S3, S4, S5 and S6, respectively. Plasmid Construction Human C5orf51 , RMC1 , RAB1A , RAB4A , RAB6A , RAB8A , RAB11A and RAB18 coding sequences were amplified by PCR from HeLa cDNA library. Mouse RAB9A , RAB9B , RAB23 , RAB32 and RAB38 coding sequences were purchased from RIKEN BRC DNA bank. For preparation of recombinant proteins, human C5orf51 , zebrafish C5orf51 and human RAB7A genes, which are codon-optimized for Escherichia coli K12, were purchased from Eurofins. For biochemical assay, codon-optimized C5orf51 gene was inserted into BamHI/NotI sites of a pET21a vector. Codon-optimized human RAB7A 1–176 (WT, Q67L, T22N, and N125I) genes were inserted into BamHI/EcoRI sites of a pGEX6P1 vector. For structure determination, RAB7A 1–176 and C5orf51 genes were subcloned into pET21a and pET16b vectors to produce the C-terminal His 6 -tagged RAB7A 1–176 and N-terminal His 10 -tagged C5orf51, respectively. For yeast two-hybrid assay, C5orf51 gene was inserted into pAct2 plasmid (TaKaRa Bio) and pGBD-C1-Rab1-43(GTP-locked or Apo)-DCys (RIKEN) were prepared as described previously 24 25 . Mutations were introduced by PCR-based DNA mutagenesis. DNA sequences inserted into vectors were confirmed by Sanger sequencing. Cell culture and transfection HeLa and HEK293T cells were cultured in DMEM supplemented with 10% (v/v) FBS, 1 mM sodium pyruvate, nonessential amino acids, and Penicillin-Streptomycin-Glutamine. HCT116 cells were cultured in McCoy’s 5A medium supplemented with 10% (v/v) FBS, nonessential amino acids, and 2 mM GlutaMax. Cell lines used in this study were authenticated and tested for mycoplasma contamination. Stable cell lines were made by recombinant retrovirus infection 21 . FuGENE6 and FuGENE HD reagents were used for plasmid transfection according to the manufacturer’s instructions. Anti-C5orf51 Antibody Anti-C5orf51 antiserum was acquired by immunizing rabbits with the purified C5orf51-His 6 and the anti-C5orf51 antibody was purified using CNBr-activated Sepharose 4B coupled to recombinant C5orf51 (Eurofins Genomics). CRISPR/Cas9-edited gene knockout. C5orf51-/- HCT116 cell lines were established using a CRISPR/Cas9-based genome editing with an antibiotic selection strategy described previously 36 . Three different guide-RNAs (gRNAs) targeted to the exon 1 in C5orf51 gene (5’- ACT AGA GAC TGC GGC CGC CA -3’, 5’- GGC GGC CGC AGT CTC TAG TG -3’, and 5’- CTT CGC TAA GTT GCT GTA TG -3’) were designed using an online CRISPR design tool: CRISPOR ( http://crispor.tefor.net/ ). Three pairs of DNA oligonucleotides: C5orf51-ex1-PX459-F1/R1 (5’- cac cgA CTA GAG ACT GCG GCC GCC A -3’ and 5’-aaa cTG GCG GCC GCA GTC TCT AGT c -3’), C5orf51-ex1-PX459-F2/R2 (5’- cac cgG GCG GCC GCA GTC TCT AGT G -3’ and 5’-aaa cCA CTA GAG ACT GCG GCC GCC c -3’), and C5orf51-ex1-PX459-F3/R3 (5’- cac cgC TTC GCT AAG TTG CTG TAT G -3’ and 5’- aaa cCA TAC AGC AAC TTA GCG AAG c -3’) were annealed and inserted into BpiI sites of PX459 vector. To construct donor plasmids, 500 bp sequences, which include 247 bp of the 5’ and 3’ of the gRNA targeting regions as homologous arms and a BamHI site in the middle instead of the gRNA sequence were synthesized in a pEX-A2J2 vector (Eurofins Genomics). The neomycin-resistant gene (NeoR) and the hygromycin-resistant gene (HygroR), including the appropriate promoter and terminator 37 were inserted into the BamHI site of the pEX-A2J2 plasmids. The resultant PX459_C5orf51-ex1 plasmids and pEX-A2J2 donor plasmids containing NeoR or HygroR were transfected into HCT116 cells using FuGENE HD. After 48 hours transfection, cells were grown in McCoy’s 5A media containing 700 µg/ml G418 and 100 µg/ml hygromycin B. Single colonies were isolated into a 24-well plate. Genomic DNA was extracted using a Microprep Kit Quick-gDNA and neomycin-resistant and hygromycin-resistant gene insertions into the target region were verified by PCR. C5orf51 KO, RAB9A_RAB9B double knockout (DKO), C5orf51_RAB9A_RAB9B triple knockout (TKO) HeLa cell lines were established as follows. The gRNA target sequences (5’- GAT TGA CAT AAG CGA ACG GC -3’ and 5’- GAG TTC TTG CTA CCG AGG AT -3’ ) for RAB9A exon 3, (5’- CTG CCG ATC ATC CAC GCT GA -3’ and 5’- CAA ATT TGT TGG TTA CGT AA -3’) for RAB9B exon 3 were designed using CRISPOR. These DNA oligonucleotides were inserted into BpiI sites of PX459 vector. The same PX459 plasmids described above were used for knocking out C5orf51 genes in HeLa cells. The PX459 plasmids were transfected into HeLa cells, and puromycin-resistant cells were seeded onto 96-well plates. The knockout of C5orf51 was analyzed by immunoblotting and the knockout of RAB9A and RAB9B genes were analyzed by sanger sequencing of the genome extracted from single clones. Immunoblotting Cells grown in 6-well plates were washed twice with Phosphate Buffered Saline (PBS) and solubilized with 2% CHAPS buffer (25 mM HEPES-KOH pH7.5, 300 mM NaCl, 2% [w/v] CHAPS, a protease inhibitor cocktail) on ice for 10 min. After centrifugation at 12,000 ×g for 2 min at 4°C, the supernatants were collected, and protein concentrations were determined on a DS-11 + spectrophotometer (DeNovix). SDS-PAGE sample buffer with DTT was added to the supernatants, which were then incubated at 42°C for 5 min. To detect hAG constructs, the samples were boiled at 95°C for 5 min. Total cell lysates were loaded on NuPAGE 4–12% Bis-Tris gels and electrophoresed using MES or MOPS running buffer. Proteins were transferred to PVDF membranes that were blocked with 2% (w/v) skim-milk/TBS-T and then incubated with primary and HRP-conjugated secondary antibodies. Proteins were detected using a Western Lighting Plus-ECL Kit on an ImageQuant LAS4000 (GE Healthcare) or a FUSION SOLO S system (VILBER). ImageJ was used to quantify protein bands. Immunostaining and immunofluorescence microscopy Cells grown on 35-mm glass-bottom dishes were fixed with 4% paraformaldehyde (PFA) in PBS for 20 min at room temperature. The cells were then permeabilized with 0.15% (v/v) TritonX-100 for 15 min at room temperature, and incubated with 0.1% (w/v) gelatin in PBS for 30 min. The cells were incubated with primary antibodies diluted in 0.1% gelatin for 2 hours at room temperature. After washing with PBS-T (PBS with Tween 20), the cells were incubated with Alexa Fluor-conjugated secondary antibodies diluted in 0.1% gelatin for 1 hour. Primary antibodies and Alexa Fluor-conjugated secondary antibodies used in this study are listed in Table S3. 4',6-diamidino-2-phenylindole (DAPI) was used for nuclear staining. The images were acquired using an inverted confocal microscope LSM780 (Carl Zeiss) with a Plan-Apochromat 63×/1.4 oil lens, an FV3000 (EVIDENT) with a PlanApo N 60x/1.4 oil objective lens, or EVOS M5000 imaging system (Thermo Fisher Scientific). For image analysis, ZEN microscope software and Photoshop (Adobe) were used. Co-Immunoprecipitation HeLa cells expressed with FLAG-tagged proteins were grown in a 6-well plate. The cells were washed with PBS twice and solubilized on ice for 15 min in IP buffer (50 mM Tris-HCl pH7.5, 150 mM NaCl, 0.5% (w/v) sodium deoxycholate, 1% (v/v) Triton X-100, a protease inhibitor cocktail). Insolubilized debris was removed by centrifugation at 16,000 ×g, 4°C for 10 min. The supernatants were incubated with anti-DDDDK-tag mAb magnetic beads or anti-FLAG M2 magnetic beads for 1 hour at 4°C under gentle rotation. 5–30% of the supernatants was collected as an input. Beads were washed with IP buffer three times and bound proteins were eluted with SDS-PAGE sample buffer. Mass spectrometry (MS) analysis To identify nucleotide-dependent RAB interacting proteins (Fig. 1 A), samples for MS analysis were prepared as follows. HeLa cells transiently expressing 3FLAG-TEV-RAB proteins grown in a well of 6-well plate were washed with PBS and solubilized on ice for 10 min in TX-100 buffer (50 mM Tris–HCl pH7.5, 150 mM NaCl, 1% (v/v) Triton X-100, a protease inhibitor cocktail). To identify C5orf51 binding proteins (Fig. 3 E), HeLa cells (control) and those stably expressing 3FLAG-TEV-C5orf51 were grown in a 10 cm dish. The cells were washed with PBS with three times and solubilized on ice for 10 min in TX-100 buffer. After centrifugation at 14,000 ×g, 4°C for 10 min, the supernatants were incubated with equilibrated anti-FLAG M2 magnetic beads for 1.5 hours at 4°C under gentle rotation. The beads were collected using a magnetic stand, washed three times with TX-100 buffer, and another three times with 50 mM ammonium bicarbonate. Proteins bound to the beads were digested with 200 ng of trypsin/Lys-C mix for 16 hours at 37°C. The digests were reduced, alkylated, acidified with trifluoroacetic acid, and desalted using GL-Tip SDB. The eluates were evaporated in a SpeedVac concentrator and dissolved in 0.1% TFA and 3% acetonitrile (ACN). LC-MS/MS analysis of the resultant peptides was performed using an EASY-nLC 1200 UHPLC connected to an Orbitrap Fusion mass spectrometer equipped with a nanoelectrospray ion source (Thermo Fisher Scientific). The peptides were separated on a 75 µm inner diameter × 150 mm C18 reversed-phase column using a linear 4–32% ACN gradient for 0-100 min followed by an increase to 80% ACN for 10 min and a final hold at 80% ACN for 10 min. The mass spectrometer was operated in data-dependent acquisition mode with a maximum duty cycle of 3 s. MS1 spectra were measured at a resolution of 120,000, an automatic gain control (AGC) target of 4 × 10 5 , and a mass range from 375 to 1,500 m / z . HCD MS/MS spectra were acquired in the linear ion trap with an AGC target of 1 × 10 4 , an isolation window of 1.6 m / z , a maximum injection time of 35 ms, and a normalized collision energy of 30. Dynamic exclusion was set to 20 s. Raw data were directly analyzed against the SwissProt database restricted to H. sapiens using Proteome Discoverer version 2.5 (Thermo Fisher Scientific) with Sequest HT search engine for identification and label-free precursor ion quantification. The search parameters were as follows: (i) trypsin as an enzyme with up to two missed cleavages; (ii) precursor mass tolerance of 10 ppm; (iii) fragment mass tolerance of 0.6 Da; (iv) carbamidomethylation of cysteine as a fixed modification; and (v) acetylation of the protein N-terminus and oxidation of methionine as variable modifications. Peptides and proteins were filtered at a false discovery rate (FDR) of 1% using the percolator node and the protein FDR validator node, respectively. Normalization was performed such that the total sum of abundance values for each sample over all peptides was the same. Yeast two hybrid The yeast strain (PJ69-4A), medium, culture conditions, and transformation protocol used were described previously 38 . Yeast cells containing pGBD-C1-Rab(GTP-locked or Apo)-DCys and pAct2-C5orf51 were streaked on the synthetic complete medium lacking adenine, histidine, leucine and tryptophan were incubated at 30°C for 4–6 days. Preparation of recombinant proteins for biochemical assay Recombinant C5orf51 was prepared as follows. pET21a plasmid harboring C5orf51 was introduced into BL21-CodonPlus(DE3)-RIL. The transformants were cultured in LB media at 37°C until the logarithmic growth phase. Protein expression was induced with 100–200 µM Isopropyl b-D-1-thiogalactopyranoside (IPTG) overnight at 18°C. The cells were collected by centrifugation, lysed with TBS buffer (50 mM Tris-HCl pH7.5, 120 mM NaCl, 50 µg/ml lysozyme, 1 µg/ml DNaseI, 1 mM DTT, 1 mM MgCl 2 , a protease inhibitor cocktail) and sonicated. After centrifugation at 10,000 ×g, 4°C for 10 min, the supernatants were incubated with Ni-NTA agarose for 30 min at 4°C under gentle rotation. The agarose was washed three times with TBS + TCEP (TBS with 1mM tris(2-carboxyethl)phosphine) buffer, and bound proteins were eluted with TBS + TCEP buffer containing 40, 80, and 200 mM imidazole in a stepwise fashion. Recombinant His 6 -tagged C5orf51 (C5orf51-His) was then concentrated using an Amicon-Ultra-15, and imidazole was removed using PD midiTrap G-25. Recombinant GST-tagged RAB7A was prepared as follows. pGEX6P1 plasmids were introduced into Chaperone competent cells pGro7/BL21. The transformants were cultured in LB media at 37°C until the logarithmic growth phase. Expressions of GroES-GroEL and GST-RAB7A were induced with 2 mg/ml L-(+)-arabinose and 100 µM IPTG overnight at 18°C. The cells were collected by centrifugation, lysed with TBS buffer (50 mM Tris-HCl pH7.5, 120 mM NaCl, 50 µg/ml lysozyme, 1 µg/ml DNaseI, 1 mM DTT, 1 mM MgCl 2 , a protease inhibitor cocktail) and sonicated. After centrifugation at 10,000 ×g, 4°C for 10 min, the supernatants were incubated with glutathione Sepharose 4B for 30 min at 4°C under gentle rotation. The Sepharose was washed three times with TBS + TCEP buffer, and bound proteins were eluted with TBS + TCEP buffer containing 20 mM L-glutathione reduced (GSH). Recombinant GST-RAB7A was then concentrated using an Amicon-Ultra and GSH was removed using PD midiTrap G-25. Preparation of recombinant proteins for structure determination Escherichia coli strain Rosetta (DE3) cells were transformed with pET21a and pET16b expression vector containing the gene encoding RAB7A 1–176 -His 6 and His 10 -C5orf51, and cultured in LB medium containing 100 mg/L ampicillin at 37°C until optical density at 600 nm (OD 600 ) reached ~ 0.8. The expression of RAB7A 1–176 -His 6 and His 10 -C5orf51 was induced by adding IPTG at the final concentration of 0.1 mM The culture was further continued for 18 h at 15°C. The cells were collected by centrifugation at 7,000 ×g for 10 min and then disrupted by sonication in 50 mM Tris-HCl buffer (pH 8.0) containing 150 mM NaCl, 0.5%(v/v) Triton X-100 and 20 mM imidazole (buffer A). The lysates were centrifuged at 17,000 rpm for 60 min, and the supernatants were loaded onto a Ni-NTA agarose column. After the column was washed with buffer A and buffer A without Triton X-100, the proteins were eluted with 50 mM Tris-HCl buffer (pH 8.0) containing 150 mM NaCl, 1 mM DTT and 200 mM imidazole. RAB7A 1–176 -His 6 incubates with 5 mM EDTA for 30 min on ice, and then mix with His 10 -C5orf51 in an equimolar ratio. The mixture of RAB7A 1–176 -His 6 and His 10 -C5orf51 were further purified by size exclusion chromatography using Hiload 16/600 Superdex200 prep grade (GE healthcare) with 10 mM Tris-HCl (pH 7.2) buffer containing 50 mM NaCl and 5 mM b-mercaptoethanol (buffer B). The fractions abundant in RAB7A 1–176 -His 6 and His 6 -C5orf51 proteins were collected and concentrated to 2 g/L using an Amicon Ultra-15 (Millipore). For data collection, the crystals of the RAB7A 1–176 -His 6 and His 10 -C5orf51 complex were soaked in the buffer B containing 30% glycerol and then flash frozen in liquid N 2 . Structure determination and refinement. Diffraction data sets of the RAB7A 1–176 and C5orf51 were collected at beamline BL45XU in SPring-8 (Hyogo, Japan) and processed with XDS 39 and the CCP4i2 program suite 40 . The complex structure was determined by molecular replacement using MolRep 41 . The structures of C5orf51 and RAB7A 1–176 predicted by AlphaFold2 were used as the search models, respectively. Atomic models were corrected using Coot 42 with careful inspection. Structure refinement was carried out using the program Phenix 43 with iterative correction and refinement of the atomic models. Data collection and refinement statistics are shown in Table S1. All molecular graphics were prepared with PyMOL (DeLano Scientific; http://www.pymol.org ). Preparation of apo or nucleotide-bound RAB7A in vitro GST-RAB7A WT was incubated with glutathione Sepharose in TBS-T-E buffer (50 mM Tris-HCl pH7.5, 120 mM NaCl, 1 mM TCEP, 5 mM EDTA) for the apo form, in TBS-T-E containing 40 µM GppNHp for the GTP-bound form, or in TBS-T-E containing 40 µM GDP for the GDP-bound form, for 2 hours at room temperature. After adding 10 mM MgCl 2 , the resin was washed 3 times with GEF-TX buffer (50 mM Tris-HCl pH7.5, 120 mM NaCl, 1 mM TECP, 0.5 mM MgCl 2 , 0.1%[v/v] TX-100) for the apo form, with GEF-TX buffer containing 100 µM GppNHp for the GTP-bound form, and with GEF-TX buffer containing 100 µM GDP for the GDP-bound form. Thermal shift assay Determination of thermal stability of recombinant RAB7A was undertaken using Protein Thermal Shift Dye (Thermo Fisher Scientific). GST or GST-RAB7A 1–176 (WT or T22N) (30 µM) were added to the reaction buffer containing 0.01% thermal shift dye with or without 5 mM EDTA. Fluorescence was measured using the ROX channel of a BioRad CFX96 Real Time PCR machine, with a 0.5°C/15 s per step (20–95°C) melting curve. Peaks were determined using BioRad CFX Manager software. Fluorescence intensity was measured using the ROX reporter of a StepOnePlus™ Real-Time PCR system, with a ramp rate of 0.05°C/s per step (25–99°C). Melting temperature were determined using Protein Thermal Shift™ Software (Thermo Fisher Scientific). Heat-stress chaperone assay Holdase activity of C5orf51 for RAB7A against heat-stress was performed as follows. Recombinant GST (11 µM) or GST-RAB7A 1–176 (7 µM) with or without recombinant C5orf51-His 6 (11 µM) in TBS buffer were divided equally into 8 samples and incubated at 40–64°C or 43–56°C gradient for 5min using a BioRad T100 thermal cycler. The samples were subsequently separated into supernatants and pellets by centrifugation at 20,000 ×g for 10min. PUREfrex chaperone assay PUREfrex®1.0 was used for in vitro transcription/translation reactions to test chaperone-like activity. GST-RAB7A 1–176 -coding DNA fragment was amplified via 3-step PCR reactions. For the 1st PCR, pGEX6P1_Opt_RAB7A(1-176) plasmid was used for a template, and FOR-GST-F (5’- AAG GAG ATA TAC CAA TGT CCC CTA TAC TAG GTT A -3’) and pGEX-PURE-R (5’- CGC TCG AGT CGA CCC GGT TA -3’) were used for primers. INTR-PURE-F (5’- GGT TTC CCT CTA GAA ATA ATT TTG TTT AAC TTT AAG AAG GAG ATA TAC CA -3’) and pGEX-PURE-R was used for the 2nd PCR, and T7pro-PURE-F (5’- GAA ATT AAT ACG ACT CAC TAT AGG GAG ACC ACA ACG GTT TCC CTC TAG AAA − 3’) and pGEX-PURE-R was used for the 3rd PCR to fuse T7 promoter and ribosome binding site sequences to the GST-RAB7A 1–176 . In vitro transcription/translation reaction carried out according to the manufacture’s instruction in the presence of 3 ng/µL DNA fragments and 0.5 mg/mL BSA or recombinant C5orf51-His 6 . After 2 hours of 37°C incubation, supernatants and pellets were separated by centrifugation at 20,000 ×g, 10min, 4°C. Co-expression of RAB GTPases and C5orf51 in bacterial cells pETDuet-1 plasmids were introduced into BL21-CodonPlus(DE3)-RIL Competent Cells and ampicillin/chloramphenicol resistant cells were isolated. The 2 mL saturation culture was first prepared by growing the cells in LB media containing ampicillin/chloramphenicol (LB/amp + Cm) overnight. 100 µL saturation culture was then diluted in 2 mL of fresh LB/amp + Cm media and grown at 37°C for 2 hours. IPTG (final 200 µM) was added to the culture and the cells were grown for 5 hours at 28°C and 3 hours at 32°C or 37°C. 400 µL of culture was transferred to 1.5 mL tube and the cells collected by centrifugation at 12,000 ×g for 2 min were resuspended in 200 µL TBS containing 1 µg/mL DNaseI, 50 µg/mL lysozyme, 1 mM DTT, 1mM MgCl 2 and protease inhibitor cocktail, frozen with liquid N 2 and kept in -20°C. The frozen cells were thawed on ice and subjected to sonication (ultrasonic disrupter UR-21P; TOMY). The supernatants and pellets were separated by centrifugation at 14,000 ×g for 5 min, and solubilized with SDS-PAGE sample buffer. Halo-tag processing assay Cells stably expressing Halo-GFP were pre-treated with 100 nM TMR-conjugated Halo ligand for 20 min. After washing twice with PBS, the cells were incubated in amino-acid free DMEM for 9 hours. Measurement of Lysosomal activity Cells grown on 35-mm glass-bottom dishes were treated with 75 nM LysoTracker Green DND-26 for 1 hour. DMEM medium was replaced with fresh medium, and the cells were observed using an inverted confocal microscope (LSM780; Carl Zeiss) equipped with a Plan-Apochromat 63×/1.4 oil lens. For signal quantification, images were analyzed using ImageJ software. A threshold value of 20/255 was applied to the images. The total fluorescence-positive area (Area value) was measured and then normalized by the number of cells to obtain the area value per cell. 10 microscopic images were quantified for each experimental condition, with each image containing 10–30 cells. Evolutionary profile We generated the evolutionary profile based on presence or absence of ortholog of C5orf51. Orthologs of C5orf51 were obtained from Ortholog Group, OG6_113094 in OrthoMCL DB version 6.5 44 . We searched homologs of C5orf51 using jackhammer in HMMER 3.3.2 45 against reference proteomes of UniProt 46 if organism is not included in OrthoMCL DB. We then performed orthology inference using reciprocal best hit methodology 47 . Molecular dynamics simulation Molecular dynamics (MD) simulations were carried out for the apo and GTP-bound forms of the RAB7A-C5orf51 complex structure, whose undetermined regions were completed using MODELLER 48 . The systems consisted of one molecule of RAB7A and one molecule of C5orf51, and more than 33,000 TIP3P water molecules 49 . Approximately 100 Na⁺ and 100 Cl⁻ were added to neutralize the system at a physiological concentration of 0.15 mol/L. In the GTP-bound form, they additionally contained one GTP whose position was calculated using DiffDock 50 and one Mg 2+ , superimposed onto the RAB7A conformation with a crystallographic structure of RAB7A in the GTP bound state (PDB: 1T91) 30 as a reference. All these molecules were placed in an isotropic box with the size of 108 Å ×108 Å ×108 Å, to ensure a minimum distance of more than 10 Å from non-solvent molecules. As the force field of the system, AMBER ff14SB 51 for protein atoms, Carlson 2003 model 52 in AMBER parameter database for GTP, Sorensen 2012 model 53 in AMBER parameter database for Mg 2+ , and Joung-Cheatham model for Na + /Cl − 54 were used. The systems (Table S7) were subjected to energy minimization with the positional restraints on non-hydrogen atoms of protein molecules, GTP, and Mg 2+ , gradually relaxed to the unrestrained condition. Subsequently, temperature annealing protocol was applied to the system from 0 K to 300 K for 10,000 steps with a time step of 2 fs under NVT ensemble with the velocity-rescale thermostat 55 as temperature coupling with a coupling constant (t t ) of 2 ps. After that, NVT equilibration was performed for total 20,000 steps with a time step of 2 fs under the same condition, gradually relaxing restraints on heavy atoms. After reaching NVT equilibrium, NPT equilibration was conducted for 500,000 steps with a time step of 2 fs without any restraints. The subsequent production runs were carried out for 1 µs simulation with a time step of 2 fs. Simulations, energy minimization and equilibration of the system using the GROMACS software 56 . The electrostatic interactions were computed by the particle mesh Ewald method under the periodic boundary conditions. In addition, analysIes, including monitoring the number of hydration bonds and the Buried Surface Area ( BSA ) between RAB7A and C5orf51 over the simulation time, were performed using GROMACS tools. The BSA was defined as follows: $$\:BSA=SASA\left(\text{R}\text{A}\text{B}7\text{A}\right)+SASA\left(\text{C}5\text{o}\text{r}\text{f}51\right)-SASA(\text{R}\text{A}\text{B}7\text{A}+\text{C}5\text{o}\text{r}\text{f}51),$$ where SASA represents solvent accessible surface area. Data availability The coordinates and structure factors of C5orf51 in complex with RAB7A have been deposited in the Protein Data Bank under the accession code 9X2L. Other data are available from the corresponding author upon reasonable request. Statistical analysis Statistical analysis was performed using data obtained from three or more biologically independent experimental replicates. Student’s t -test was used for comparisons between two groups and Dunnett’s test was used for multiple comparisons using GraphPad Prism (n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001). Declarations Declaration of interests We declare no competing financial interests. Acknowledgments We thank the beamline staff of BL45XU in SPring-8 (Hyogo, Japan) for technical help during data collection. We are also grateful to Dr. Kohei Nishino (Tokushima University) for his assistance with mass spectrometry analysis and to Dr. Tomohide Saio for valuable discussion. This work was supported by JSPS KAKENHI (21K15084, 24H01894 to K.O., 18H05501 to S.F., 22K15045 to W.K., 23K23841, 23H04923 to K.Y., 25K02267 to M. F., 21H03551, 25K03224 to K.I., 19H05712 to N.M.), Medical Research Center Initiative for High Depth Omics to H.K., Research Support Project for Life Science and Drug Discovery (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under Grant Number JP25ama121028 (support number 5738) to K.T., and JP25ama121029j0004 to K.I., AMED CREST Grant JP24gm1410004 and Nanken-Kyoten Foundation in Science Tokyo, and Medical Research Center Initiative for High Depth Omics to N.M., and AMED PRIME Grant JP25gm6910030h0001 to K.Y. References Homma, Y., Hiragi, S., and Fukuda, M. (2021). Rab family of small GTPases: an updated view on their regulation and functions. FEBS J 288 , 36–55. 10.1111/febs.15453. Zhen, Y., and Stenmark, H. (2015). Cellular functions of Rab GTPases at a glance. J Cell Sci 128 , 3171–3176. 10.1242/jcs.166074. Hutagalung, A.H., and Novick, P.J. (2011). Role of Rab GTPases in membrane traffic and cell physiology. 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GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX 1-2 , 19–25. https://doi.org/10.1016/j.softx.2015.06.001. Additional Declarations There is NO Competing Interest. Supplementary Files TableS7.docx Table S7 TableS3Cell251002.xlsx Table S3 TableS2.docx Table S2 TableS5Plasmid251002.xlsx Table S5 TableS4Antibody251002.xlsx Table S4 D1300064347valreportannotateP1.pdf X-ray structure validation report TableS6Reagents251002.xlsx Table S6 C5NSMBsuppleFiglegends260108.docx supple Fig legends SuppleFis251017low.pdf supple Figures TableS1Datacollectionandrefinementstatistics251006.docx Table S1 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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06:05:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8547387/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8547387/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":100950785,"identity":"e2f8e5a8-e898-452d-9467-72a20c664462","added_by":"auto","created_at":"2026-01-23 07:09:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":600968,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProteomic analyses of nucleotide-dependent RAB interacting proteins\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Proteomic analysis of RAB nucleotide-binding-dependent interacting proteins. The log\u003csub\u003e2\u003c/sub\u003e (abundance ratio of RAB vs control) for GTP-bound and apo RAB are shown on the x- and y-axis, respectively.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eB\u003c/strong\u003e) Abundance of C5orf51 and RABIF/MSS4 are shown against RAB GTP-bound and apo forms.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eC\u003c/strong\u003e) Proteins co-immunoprecipitated by the indicated FLAG-RAB were analyzed by immunoblotting (IB). Data shown are representative of two experiments.\u003c/p\u003e","description":"","filename":"MainFigs251029low1.png","url":"https://assets-eu.researchsquare.com/files/rs-8547387/v1/44d3c2114b4f2b7a79d81e6e.png"},{"id":100950365,"identity":"e7f7236a-9e74-49b7-961f-bed40777645d","added_by":"auto","created_at":"2026-01-23 07:07:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1154496,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eC5orf51 interacts with apo RAB7A in a MON1-CCZ1-independent manner.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Schematic representation of fluoppi assay.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eB\u003c/strong\u003e) The indicated hAG and HA-Ash constructs were expressed in HeLa cells and the cells were immunostained. Bars, 10 mm. Data shown are representative of two experiments.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eC\u003c/strong\u003e) The percentage of cells having RAB7A fluoppi foci in (B) were quantified. Error bars represent mean ± s.d. of three independent experiments.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eD, E\u003c/strong\u003e) fluoppi assay of the indicated constructs. Bars, 10 mm. Data shown are representative of two experiments.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eF\u003c/strong\u003e) The percentage of cells having fluoppi foci with MON1B-CCZ1 in (E) were quantified. Error bars represent mean ± s.d. of three independent experiments.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eG, H, I\u003c/strong\u003e) The indicated constructs were transiently expressed in HeLa cells and co-immunoprecipitation was performed with anti-FLAG antibody and analyzed by immunoblotting (IB). Asterisks indicate IgG. Data shown are representative of two experiments.\u003c/p\u003e","description":"","filename":"MainFigs251029low2.png","url":"https://assets-eu.researchsquare.com/files/rs-8547387/v1/45e3b62eb51a135fda169c90.png"},{"id":100881331,"identity":"c6e0278e-300e-42bc-b321-5ea20ee75cb2","added_by":"auto","created_at":"2026-01-22 11:13:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":494277,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpecificity of C5orf51 interaction with RAB7A.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) RAB-binding specificity of C5orf51 by yeast two-hybrid assay. The interaction was detected by the growth of yeast cells. Positions of a GTP-locked and apo form of each RAB are indicated in the left panel. Positive patches are boxed. Data shown are representative of two experiments.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eB\u003c/strong\u003e) Fluoppi assay of hAG-C5orf51 with the indicated HA-Ash-RAB constructs.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eC\u003c/strong\u003e) Transient expression of HA-Ash-RAB constructs in (B) were confirmed by immunoblotting (IB).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eD\u003c/strong\u003e) The percentage of cells having fluoppi foci in (B) were quantified. Error bars represent mean ± s.d. of three independent experiments.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eE\u003c/strong\u003e) The volcano plot shows the abundance ratios (FLAG-C5orf51 vs control) plotted against the p-value in a -long\u003csub\u003e10\u003c/sub\u003e scale for three independent experiments.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eF\u003c/strong\u003e) Pull-down assays using recombinant GST or GST-RAB7A\u003csup\u003e1-176\u003c/sup\u003e (WT, Q67L or T22N) were performed to assess the interaction with C5orf51. The input and bound proteins were analyzed by SDS-PAGE followed by CBB staining. Data shown are representative of two experiments.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eG\u003c/strong\u003e) Recombinant GST or GST-RAB7A\u003csup\u003e1-176\u003c/sup\u003e bound to glutathione Sepharose were treated with EDTA before the pull-down assays. The 10% input of C5orf51 and eluted fractions were analyzed by SDS-PAGE followed by CBB staining. Data shown are representative of two experiments.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eH\u003c/strong\u003e) Pull-down assays using GppNHp-bound or GDP-bound or apo form of GST-RAB7A\u003csup\u003e1-176\u003c/sup\u003e WT were performed to assess the interaction with C5orf51. The 20% input of C5orf51 and eluted fractions were analyzed by SDS-PAGE followed by CBB staining. Data shown are representative of two experiments.\u003c/p\u003e","description":"","filename":"MainFigs251029low3.png","url":"https://assets-eu.researchsquare.com/files/rs-8547387/v1/1d444a6cd8bc82d52d8d0e77.png"},{"id":100881319,"identity":"d8f0928f-68da-4374-99a7-429f9db04dad","added_by":"auto","created_at":"2026-01-22 11:13:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":521368,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLoss of C5orf51 reduces RAB7A protein amount.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) GFP-RAB7A with or without C5orf51-mCherry were expressed in HeLa cells, and then immunostained. Magnified images were also shown. Bars, 10 mm. Data shown are representative of two experiments.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eB\u003c/strong\u003e) Total cell lysates from WT and \u003cem\u003eC5orf51\u003c/em\u003eKO HeLa cells with or without stable expression of 3FLAG-C5orf51 were analyzed by immunoblotting (IB).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eC\u003c/strong\u003e) Protein levels in (B) were quantified. Each protein of WT cells was set to 100. Error bars represent mean ± s.d. of three independent experiments.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eD\u003c/strong\u003e) Total cell lysates from WT and \u003cem\u003eC5orf51-/- \u003c/em\u003eHCT116 cells with or without stable expression of 3FLAG-C5orf51 were analyzed by immunoblotting.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eE\u003c/strong\u003e) Protein levels in (D) were quantified. Each protein of WT cells was set to 100. Error bars represent mean ± s.d. of three independent experiments.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eF\u003c/strong\u003e) Schematic representation of HaloTag processing assay.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eG, H\u003c/strong\u003e) The indicated HeLa cells stably expressing Halo-GFP were treated with a Halo ligand and then incubated in normal and starvation media for 9 hrs. Total cell lysates were analyzed by immunoblotting. Data shown are representative of two experiments.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eI\u003c/strong\u003e) Starvation autophagy rates in (H) were quantified as the ratio of processed Halo to total Halo-GFP. Halo processing in WT cells upon starvation was set to 100. Error bars represent mean ± s.d. of three independent experiments.\u003c/p\u003e","description":"","filename":"MainFigs251029low4.png","url":"https://assets-eu.researchsquare.com/files/rs-8547387/v1/a867f551019e90ab6ddcb355.png"},{"id":100881307,"identity":"0575df29-7811-45d3-8480-09f3b38eedd7","added_by":"auto","created_at":"2026-01-22 11:13:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":504286,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eC5orf51 acts as a chaperone for newly synthesized RAB7A.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) The indicated RAB7A\u003csup\u003e1-176\u003c/sup\u003e with an HA-tag were expressed in \u003cem\u003eE.coli\u003c/em\u003e BL21(DE3) cells with or without C5orf51 at 28°C for 5 hrs. The cells were sonicated, and supernatant (S) and pellet (P) fractions were separated by centrifugation and analyzed by SDS-PAGE followed by CBB staining, and RAB7A-HA and C5orf51 were analyzed by immunoblotting (IB).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eB\u003c/strong\u003e) Proteins recovered in supernatant (sup) in (A) were quantified. Total (S+P) amounts were set to 100. Error bars represent mean ± s.d. of three independent experiments.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eC\u003c/strong\u003e) Upper: RAB7A\u003csup\u003e1-176\u003c/sup\u003e-HA (WT or T22N) and C5orf51-His were co-expressed in \u003cem\u003eE.coli\u003c/em\u003e cells. Following cell sonication, supernatant (S) fractions were subjected to Ni-affinity pull down assays. The supernatant (S), flow-through (FT), and bound (B) fractions were analyzed by CBB staining and immunoblotting. Data shown are representative of two experiments. Lower: A schematic representation of the nucleotide-dependent interaction between RAB7A and C5orf51.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eD\u003c/strong\u003e) The indicated RAB proteins were expressed at 37°C as in (A).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eE\u003c/strong\u003e) Proteins recovered in the supernatant (sup) in (D) were quantified. Total (S+P) amounts were set to 100. Error bars represent mean ± s.d. of three independent experiments.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eF\u003c/strong\u003e) GST-RAB7A\u003csup\u003e1-176\u003c/sup\u003e (WT, T22N, and Q67L) were synthesized with BSA (control) or recombinant C5orf51 using PUREfrex. The supernatant (S) and pellet (P) fractions separated by centrifugation were analyzed by immunoblotting.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eG\u003c/strong\u003e) GST-RAB7A recovered in the supernatant (sup) in (F) were quantified. Total (S+P) amounts were set to 100. Error bars represent mean ± s.d. of three independent experiments.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eH\u003c/strong\u003e) GST-RAB7A\u003csup\u003e1-176\u003c/sup\u003e (WT and N125I) were synthesized as in (F).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eI\u003c/strong\u003e) GST-RAB7A recovered in the supernatant (sup) in (H) were quantified. Error bars represent mean ± s.d. of three independent experiments.\u003c/p\u003e","description":"","filename":"MainFigs251029low5.png","url":"https://assets-eu.researchsquare.com/files/rs-8547387/v1/462f4a41b6ed39c4993dc4ee.png"},{"id":100881345,"identity":"c3042cca-4f38-4e74-9b5a-4cc0059edda8","added_by":"auto","created_at":"2026-01-22 11:13:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1524507,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCrystal structure of C5orf51 in complex with apo RAB7A.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Overall structure of C5orf51-RAB7A complex. C5orf51 and RAB7A are colored cyan and green, respectively. The a1 and switch I region of RAB7A are highlighted in red and orange, respectively. Disordered regions are shown as dotted lines.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eB\u003c/strong\u003e) Top view of C5orf51. Nine a-helices and two insertion regions are indicated. (\u003cstrong\u003eC\u003c/strong\u003e) Two orientations of the C5orf51-RAB7A complex rotated by 90°. C5orf51 and RAB7A are shown as a surface representation and a cartoon, respectively.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eD-H)\u003c/strong\u003e The coloring scheme of subdomain of RAB7A is as follows: phosphate-binding loop (P-loop), purple; a1 helix, red; Switch I, orange; Switch II, green; Glycine brace, yellow; Guanine-binding loop, cyan. Amino acid sequence of human RAB7A (\u003cstrong\u003eD\u003c/strong\u003e). The structure of apo RAB7A in complex with C5orf51 (\u003cstrong\u003eE\u003c/strong\u003e, this study), apo yeast RAB7A homolog (Ypt7) in complex with Mon1/Ccz1 (\u003cstrong\u003eF\u003c/strong\u003e, PDB: 5LDD), GTP-bound RAB7A (\u003cstrong\u003eG\u003c/strong\u003e, PDB: 1T91) and GDP-bound RAB7A (\u003cstrong\u003eH\u003c/strong\u003e, PDB: 1VG1).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eI\u003c/strong\u003e) Overall view of the interaction between C5orf51 and RAB7A. The residues involved in C5orf51-RAB7A interaction are shown as sticks.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eJ-M\u003c/strong\u003e) The close-up views of the regions indicated by the boxes in (I), highlighting the molecular interface between C5orf51 and RAB7A.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eN\u003c/strong\u003e) Schematic illustrations of the C5orf51-RAB7A complex in its apo (pale green/green) and GTP-bound (pale orange/gray) forms obtained from snapshots of the MD trajectories.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eO\u003c/strong\u003e) Frequency distributions of the number of hydration bonds between C5orf51 and apo RAB7A (black) and between C5orf51 and GTP-bound RAB7A (red) throughout the MD trajectories.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eP\u003c/strong\u003e) Frequency distributions of the buried surface area of the C5orf51-RAB7A complex in its apo (black) and GTP-bound (red) forms throughout the MD trajectories.\u003c/p\u003e","description":"","filename":"MainFigs251029low6.png","url":"https://assets-eu.researchsquare.com/files/rs-8547387/v1/0a6373b65a997b54fbfcd9e6.png"},{"id":100881275,"identity":"8663f7fc-95b3-43c6-bd1b-e6472eddf0ce","added_by":"auto","created_at":"2026-01-22 11:13:01","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":819295,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMutational analyses to assess C5orf51-RAB7A interaction.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) The percentage of cells having fluoppi foci composed of HA-Ash-RAB7A-DC (T22N) and hAG-C5orf51 with the indicated mutations were quantified. Error bars represent mean ± s.d. of three independent experiments.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eB\u003c/strong\u003e) GST-RAB7A\u003csup\u003e1-176\u003c/sup\u003e were synthesized in the presence of the indicated recombinant C5orf51 using PUREfrex. The supernatant (S) and pellet (P) fractions separated by centrifugation were analyzed by immunoblotting (IB).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eC\u003c/strong\u003e) GST-RAB7A recovered in the supernatant (sup) in (B) were quantified. Total (S+P) amounts were set to 100. Error bars represent mean ± s.d. of three independent experiments.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eD\u003c/strong\u003e) Residues of C5orf51 located at the interface with RAB7A are shown as sticks. Asterisks indicate residues whose mutation affected function.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eE\u003c/strong\u003e) The indicated GST-RAB7A\u003csup\u003e1-176\u003c/sup\u003e were synthesized with BSA or recombinant C5orf51 using PUREfrex as in (B).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eF\u003c/strong\u003e) GST-RAB7A recovered in the supernatant (sup) in (E) were quantified. Total (S+P) amounts were set to 100. Error bars represent mean ± s.d. of three independent experiments.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eG\u003c/strong\u003e) The percentage of cells having fluoppi foci composed of hAG-C5orf51 and HA-Ash-RAB7A-DC (T22N) with the indicated mutations were quantified. Error bars represent mean ± s.d. of three independent experiments.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eH\u003c/strong\u003e) Expression of HA-Ash-RAB7A-DC (T22N) used in (G) were confirmed by immunoblotting.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eI\u003c/strong\u003e) C5orf51 is shown as a surface representation. Residues of RAB7A located at the interface with C5orf51 are shown as sticks. Asterisks indicate residues whose mutation affected function.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eJ\u003c/strong\u003e) Sequence alignment of RAB7A with representative RAB family GTPases using clustal omega. Conserved residues are indicated using standard Clustal symbols (* and :). Black asterisks indicate identical residues. Colons indicate conserved substitutions. Blue and red asterisks above the alignment indicate residues in RAB7A whose mutations maintained and impaired the chaperone activity, respectively.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eK\u003c/strong\u003e) Schematic representation of RAB chimeric constructs.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eL\u003c/strong\u003e) The indicated HA-Ash-RAB constructs were expressed with hAG-C5orf51 in HeLa cells and the cells were immunostained. Bars, 10 mm.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eM\u003c/strong\u003e) The percentage of cells having fluoppi foci in (L) were quantified. Error bars represent mean ± s.d. of three independent experiments.\u003c/p\u003e","description":"","filename":"MainFigs251029low7.png","url":"https://assets-eu.researchsquare.com/files/rs-8547387/v1/021e754d5cbe65ad38c97bfc.png"},{"id":102294795,"identity":"f7d6abcd-800e-414e-9749-584027c96fca","added_by":"auto","created_at":"2026-02-10 09:58:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7197543,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8547387/v1/1bc9d35e-ca39-4fc8-be50-6af64eafbd83.pdf"},{"id":100881343,"identity":"e0afcad2-205d-4e2b-8ee4-3b8003d3dc02","added_by":"auto","created_at":"2026-01-22 11:13:11","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":23355,"visible":true,"origin":"","legend":"Table S7","description":"","filename":"TableS7.docx","url":"https://assets-eu.researchsquare.com/files/rs-8547387/v1/bb5d7cd7a5356e0190d22e88.docx"},{"id":100881270,"identity":"4cd1d00b-83a7-41eb-8b35-3161b9ec78aa","added_by":"auto","created_at":"2026-01-22 11:13:01","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":10628,"visible":true,"origin":"","legend":"Table S3","description":"","filename":"TableS3Cell251002.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8547387/v1/848a7239725dd6f367a031cd.xlsx"},{"id":100881317,"identity":"83f27bfb-b174-4940-9031-16eafb693a92","added_by":"auto","created_at":"2026-01-22 11:13:05","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":23851,"visible":true,"origin":"","legend":"Table 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Figures","description":"","filename":"SuppleFis251017low.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8547387/v1/49c3f9ee7e5859d1fac947e9.pdf"},{"id":100881303,"identity":"80d7212b-fc0e-4684-93fe-390eff151365","added_by":"auto","created_at":"2026-01-22 11:13:03","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":18516,"visible":true,"origin":"","legend":"Table S1","description":"","filename":"TableS1Datacollectionandrefinementstatistics251006.docx","url":"https://assets-eu.researchsquare.com/files/rs-8547387/v1/5784c96090d47050acb9f689.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"RAB7HD/C5orf51 stabilizes newly synthesized RAB7A and facilitates its GTP loading","fulltext":[{"header":"Main","content":"\u003cp\u003eA hallmark of eukaryotic cells is their compartmentalized endomembrane system. The maintenance and propagation of these endomembrane organelles require constant communication for the delivery of lipids, cargo proteins, and metabolites via vesicular trafficking. Among the various steps of this process, vesicle transport and tethering are primarily regulated by small RAB GTPases \u003csup\u003e1 2 3\u003c/sup\u003e. These proteins typically consist of 200\u0026ndash;250 amino acids, with approximately 60 different members encoded by the human genome. Like other small GTPases, RAB proteins cycle between a GTP-bound active state and a GDP-bound inactive state. This cycling is catalyzed by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). GEFs on the target membrane are suggested to be major determinants of RAB localization \u003csup\u003e4\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSeveral other proteins are associated with RAB proteins. Following synthesis on cytosolic ribosomes, RAB proteins first bind to a RAB escort protein (REP) \u003csup\u003e5\u003c/sup\u003e. Their C-terminal cysteine residues are then irreversibly prenylated by geranylgeranyl transferase \u003csup\u003e6\u003c/sup\u003e, which enables RAB proteins to be inserted into membranes. On the target membrane, GTP-bound RAB proteins interact with effector proteins to exert their function in vesicular transport and/or membrane tethering. Finally, RAB proteins are extracted from the membranes with the help of a GDP dissociation inhibitor (GDI), which masks the prenyl moiety for the next round of the RAB cycle \u003csup\u003e7\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe G-domain common to small GTPases and RAB proteins form a globular fold with a 6-stranded b-sheet surrounded by 5 a-helices. The switch I and II regions of RAB proteins are known to undergo large conformational changes upon binding to GTP and GDP \u003csup\u003e8\u003c/sup\u003e. Recently, it was reported that the exposure of the hydrophobic switch I region in inactivated RAB8A is recognized by BAG6 and degraded via the ubiquitin-proteasome pathway \u003csup\u003e9\u003c/sup\u003e. This suggests that precise GTP-loading onto RAB proteins and/or preventing the exposure of the hydrophobic switch I region is crucial for RAB quality control. However, the mechanisms by which RAB quality control is maintained to prevent aggregate formation and/or unwanted degradation through the ubiquitin-proteasome pathway remain largely unknown.\u003c/p\u003e \u003cp\u003eAmong the 60 different RAB proteins in mammals, RAB7A is one of the most studied and is particularly important for lysosomal biogenesis \u003csup\u003e10,11\u003c/sup\u003e. RAB7A is localized mainly to late endosomes and lysosomes, and regulates vesicular trafficking from early endosomes to late endosomes and from late endosomes to lysosomes. The RAB7A GEF activity of the MON1-CCZ1 complex was demonstrated using purified yeast homologs (Mon1-Ccz1 complex) \u003csup\u003e12\u003c/sup\u003e. Several RAB7A effectors have been identified, including the HOPS complex \u003csup\u003e13\u003c/sup\u003e, RILP \u003csup\u003e14\u003c/sup\u003e, and FYCO1 \u003csup\u003e15\u003c/sup\u003e, suggesting the multifaceted role of RAB7A. Furthermore, RAB7A has been reported to play a key role in the maturation of autophagosomes into autolysosomes by mediating fusion events with lysosomes \u003csup\u003e16\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we report the identification of C5orf51 as a novel binding partner of RAB7A. C5orf51 strongly binds to newly translated guanine nucleotide-free (apo) RAB7A, exhibiting chaperone-like activity that maintains its solubility. Furthermore, we determined the crystal structure of the C5orf51-apo RAB7A complex at a resolution of 3.36 \u0026Aring;. Thus, the specific chaperone for RAB7A provides a new insight into the regulation of RAB protein quality control.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eIdentification of C5orf51 as a novel binding protein to guanine-nucleotide free form of RAB7A\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo identify factors involved in RAB protein quality control, we transiently expressed FLAG-tagged wild-type (WT) and nucleotide-free (apo) mutants of representative RAB proteins (RAB4A, RAB6A, RAB7A, RAB8A, RAB9A, RAB11A, and RAB18) in HeLa cells. The apo state was mimicked by a single amino acid substitution, Asparagine (N) to Isoleucine (I) \u003csup\u003e17\u003c/sup\u003e. Using HeLa cells without overexpression of RAB proteins as a control, we performed co-immunoprecipitation (co-IP) followed by mass spectrometry to identify interacting partners (Fig. 1A). Since factors that bind strongly to the structurally unstable apo form may are more likely to be involved in RAB protein quality control, the abundance ratios of co-IPed proteins between WT and apo form of each RAB protein were plotted (Fig. 1A). First, we observed that GDI1, GDI2, RABGGTA, CHM, and CHML bind to the RAB WT form but not the apo form (Fig. 1A), suggesting that lipid modification may be inhibited in the apo state. Among these RAB panels, RABIF/MSS4 was found to bind specifically to the apo form of RAB8A (Fig. 1A, RAB8A panel). This finding supports a report that RABIF acts as a chaperone involved in the folding of RAB8 family proteins \u003csup\u003e18\u003c/sup\u003e. On the RAB7A panel, the subunits of RAB7A-GEF complex, MON1B, CCZ1, and RMC1 were identified as proteins that bind specifically to apo RAB7A (Fig. 1A, RAB7A panel). This is consistent with a previous report \u003csup\u003e19\u003c/sup\u003e. Interestingly, C5orf51 was identified as a factor that interacts strongly with the apo RAB7A (Fig. 1A, RAB7A panel). Among the multiple RAB proteins tested, RABIF binds specifically to the apo RAB8A, while C5orf51 binds specifically and strongly to the apo RAB7A (Fig. 1B). A similar result was observed by immunoblotting (Fig. 1C).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eC5orf51 is a protein found across metazoan species, ranging from early-branching lineages such as \u003cem\u003eTrichoplax sp.\u003c/em\u003e and \u003cem\u003eN. vectensis\u003c/em\u003e to vertebrates such as \u003cem\u003eD. rerio\u003c/em\u003e and \u003cem\u003eH. sapiens\u003c/em\u003e. In contrast, it appears to have been lost in some ecdysozoa lineages including \u003cem\u003eC. elegans\u003c/em\u003e and \u003cem\u003eD. melanogaster\u003c/em\u003e (Fig. S1). Exceptionally, an ortholog of C5orf51 is present in the cryptophyte \u003cem\u003eGuillardia theta\u003c/em\u003e, and it might have been acquired through horizontal gene transfer (Fig. S1). The human C5orf51 consists of 294 amino acid residues, and its molecular function including its cellular localization is unknown.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC5orf51 binds to RAB7A in a MON1-CCZ1-independent manner.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we investigated the intracellular interaction between C5orf51 and RAB7A using the fluoppi (fluorescent-based technology detecting protein-protein interactions) system \u003csup\u003e20\u003c/sup\u003e \u003csup\u003e21\u003c/sup\u003e. In this system, when a protein fused with a homo-tetrameric humanized azami-green (hAG) interacts with another protein fused with a homo-oligomeric assembly helper (HA-Ash) tag, they form large liquid-liquid phase separated (LLPS)-based foci in cells through multivalent interactions (Fig. 2A). When hAG-C5orf51 was co-expressed with a C-terminal Cys-truncated RAB7A (HA-Ash-RAB7A-DC WT), which was designed to maintain cytoplasmic solubility by preventing prenylation, both proteins were diffusely localized in the cytosol (Fig. 2B). A GTP-locked RAB7A mutant Q67L (HA-Ash-RAB7A-DC Q67L) also localized diffusely in the cytosol (Fig. 2B). In sharp contrast, both the GDP-bound RAB7A T22N and the apo-mimetic RAB7A N125I mutants formed fluoppi foci with hAG-C5orf51 (Figs. 2B and 2C), indicating a direct interaction. The interaction with C5orf51 was specific to RAB7A because RAB1A, which regulates vesicular transport from the ER to the Golgi apparatus \u003csup\u003e22\u003c/sup\u003e, did not interact with C5orf51 (Fig. 2B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSince 1) C5orf51 preferentially binds to apo or GDP-bound RAB7A (Fig. 2B), and 2) RMC1/C18orf8 that we previously found as an interacting protein with RAB7A T22N \u003csup\u003e23\u003c/sup\u003e was reported to be a component of the MON1-CCZ1 complex \u003csup\u003e19\u003c/sup\u003e, \u0026nbsp;C5orf51 may be an additional subunit of MON1-CCZ1-RMC1 complex. We tested this possibility using fluoppi. As expected, hAG-MON1B and HA-Ash-CCZ1 formed fluoppi foci (Fig. 2D), indicating that they interact each other. However, neither HA-Ash-MON1B nor HA-Ash-CCZ1 formed foci with hAG-C5orf51 (Fig. 2D). Furthermore, hAG-RMC1, but not hAG-C5orf51, formed fluoppi foci with MON1B-CCZ1 (Figs. 2E and 2F). The similar results were obtained by co-IP. Both HA-tagged C5orf51 (3HA-C5orf51) and HA-tagged MON1-CCZ1 complex (3HA-MON1B and 3HA-CCZ1) were efficiently co-IPed with GDP-bound and apo forms of 3FLAG-RAB7A (Figs. 2G and 2H). On the other hands, MON1-CCZ1 complex (3HA-MON1B and 3HA-CCZ1) were co-IPed by 3FLAG-RMC1, but not by 3FLAG-C5orf51 (Fig. 2I). These results indicate that, while RMC1 is a component of MON1-CCZ1 complex, C5orf51 interacts with RAB7A in a MON1-CCZ1-independent manner.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpecificity of C5orf51 interaction with mammalian RAB GTPases.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo comprehensively identify the RAB-binding specificity of C5orf51, we next performed a yeast-two hybrid (Y2H) assay using 62 different GTP-locked or apo RAB mutants \u003csup\u003e24\u003c/sup\u003e \u003csup\u003e25\u003c/sup\u003e. Based on the growth rate of the yeast colonies, we found that C5orf51 almost exclusively interacted with the apo form of RAB7A (Fig. 3A). While a marginal interaction with the apo form of RAB32 was observed in the Y2H screen, this interaction could not be confirmed in a subsequent fluoppi assay (Figs. 3B-3D). Furthermore, we could not detect an interaction via fluoppi with other RAB proteins phylogenetically close to RAB7A including RAB9A, RAB9B, RAB23 and RAB38 (Figs. 3B-3D). To further investigate the binding partner of C5orf51 in a more physiological context, we performed an IP-MS assay. 3FLAG-C5orf51 stably expressed in HeLa cells were immunoprecipitated with anti-FLAG antibody, and the interacting proteins were analyzed by mass spectrometry (Fig. 3E). The resulting volcano plot revealed that RAB7A was one of the key interacting proteins with C5orf51. On the other hand, other factors known to be involved in the RAB cycle, such as GEFs, GAPs, and effectors, were not identified in our analysis (Fig. 3E). These results collectively suggest that C5orf51 specifically interacts with RAB7A.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC5orf51 directly interacts with the apo form of RAB7A.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe above results strongly suggest that C5orf51 directly binds to RAB7A. To test this possibility, we first narrowed down the interacting region of RAB7A with C5orf51. Since The C-terminal Cys-deleted RAB7A T22N (RAB7A-DC) formed fluoppi foci with hAG-C5orf51, prenylation is not required for the interaction (Figs. 2B and S2). Furthermore, truncated RAB7A\u003csup\u003e1-176\u003c/sup\u003e (residues 1-176) still had an ability to interact with C5orf51, indicating that the hypervariable region is dispensable (Fig. S2). Next, to investigate the direct interaction, we prepared recombinant C5orf51-His\u003csub\u003e6\u003c/sub\u003e and GST-RAB7A\u003csup\u003e1-176\u003c/sup\u003e (WT, Q67L and T22N) while apo mimetic RAB7A\u003csup\u003e1-176\u003c/sup\u003e N125I could not be prepared because of severe aggregate-prone property. When GST and GST-RAB7A\u003csup\u003e1-176\u003c/sup\u003e were pulled down by glutathione Sepharose, C5orf51 was eluted only with GST-RAB7A\u003csup\u003e1-176\u003c/sup\u003e (Fig. 3F), indicating that C5orf51 directly binds to RAB7A. The elution efficiency of C5orf51 with RAB7A T22N were higher than those with RAB7A WT or Q67L (Fig. 3F). Furthermore, different eluted efficiencies of C5orf51 due to a different RAB7A nucleotide-binding state were nullified when guanine nucleotides and Mg\u003csup\u003e2+\u003c/sup\u003e ion were dissociated from RAB7A by EDTA treatment (Fig. 3G). When the apo, GDP-bound, and GppNHp (non-hydrolyzable GTP analog)-bound states were prepared using RAB7A\u003csup\u003e1-176\u003c/sup\u003e WT, the apo form strongly bounds to C5orf51 (Fig. 3H). These results indicate that C5orf51 directly interacts with RAB7A preferentially at the guanine-nucleotide free state.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLoss of C5orf51 in cells reduces RAB7A protein amount.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate cellular function of C5orf51, we first examined its intracellular localization. GFP-tagged RAB7A (GFP-RAB7A WT) was found primarily on late endosomes and lysosomes as confirmed by co-staining anti-LAMP2 antibody (Fig. 4A). In contrast, C5orf51-mCherry was distributed throughout the cells (Fig. 4A). When GFP-RAB7A T22N, which interacts with C5orf51, was overexpressed, the nuclear signal of C5orf51-mCherry was significantly reduced, while its cytosolic signal remained unchanged (Fig. 4A). This suggests that the interaction between C5orf51 and RAB7A T22N occurs in the cytosol, drawing C5orf51 out of the nucleus.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo further elucidate the role of C5orf51, we generated \u003cem\u003eC5orf51\u003c/em\u003e knockout (KO) HeLa cells using CRISPR/Cas9 gene editing. While the deletion of C5orf51 did not alter lysosomal morphology or pH (Fig. S3), we discovered that the loss of C5orf51 consistently reduced the amount of RAB7A protein (Figs. 4B and 4C). Specifically, the amount of RAB7A in two independent \u003cem\u003eC5orf51\u003c/em\u003e KO clones (#2-43 and #3-2) was reduced to approximately 60-70% of the levels in WT HeLa cells (Figs. 4B and 4C). Importantly, the reduced RAB7A protein levels in both clones were rescued by the exogenous expression of 3FLAG-C5orf51 (Figs. 4B and 4C). As a control, the protein levels of CCZ1, a subunit of the RAB7A GEF complex, and TOMM20, a mitochondrial protein, were not affected by C5orf51 deletion (Figs. 4B and 4C). Furthermore, we obtained similar results in \u003cem\u003eC5orf51-/-\u003c/em\u003e HCT116 cells (Figs. 4D and 4E), confirming that this phenomenon is not cell-type specific.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGiven that RAB7A protein levels are reduced in \u003cem\u003eC5orf51\u003c/em\u003e KO cells, we anticipated that RAB7A-mediated lysosomal and autophagic functions would be impaired. However, \u003cem\u003eC5orf51\u003c/em\u003e deletion alone did not affect lysosomal morphology or acidification (Fig. S3), and it did not inhibit amino acid starvation-induced autophagy as measured by a Halo-tag processing assay (Figs. 4F and 4G). Previous studies have demonstrated that RAB7A, RAB9A and RAB9B have functional redundancy, and severe defects in lysosomal function are observed only when all three are knocked out \u003csup\u003e26\u003c/sup\u003e. Therefore, we established a \u003cem\u003eRAB9A_RAB9B_C5orf51\u003c/em\u003e triple KO (9A/B/C5 TKO) cell line, and then measured amino acid starvation-induced autophagy. The TKO cells showed a significant decrease in the production of the processed Halo fragment compared to WT or \u003cem\u003eRAB9A/B\u003c/em\u003e DKO cells (Figs. 4H and 4I). Importantly, the reduced Halo fragments in the TKO cells were recovered upon exogenous expression of 3FLAG-C5orf51 (Figs. 4H and 4I). Therefore, these results indicate that loss of C5orf51 reduces RAB7A and affects an autophagy-lysosome function under a particular condition.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC5orf51 enhances thermal stability of the nucleotide-free RAB7A.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrevious studies have shown that small RAB GTPases without a bound guanine nucleotide are more susceptible to urea denaturation followed by proteolytic degradation \u003csup\u003e27\u003c/sup\u003e \u003csup\u003e28\u003c/sup\u003e \u003csup\u003e29\u003c/sup\u003e. To confirm the nucleotide-dependent stability of RAB7A, we first performed a thermal shift assay. Recombinant GST-RAB7A\u003csup\u003e1-176\u003c/sup\u003e, which was overexpressed and purified form \u003cem\u003eE.coli\u003c/em\u003e BL21(DE3) cells, was incubated with a thermofluor dye that binds to exposed hydrophobic regions of the protein. The melting temperature (Tm) was determined by monitoring fluorescence as the temperature was gradually increased. The Tm values for GST and GST-RAB7A\u003csup\u003e1-176\u003c/sup\u003e WT were 52.0\u0026deg;C and 49.5\u0026deg;C, respectively (Fig. S4A). When EDTA was added to chelate Mg\u003csup\u003e2+\u003c/sup\u003e ion and dissociate bound guanine nucleotides, the Tm of RAB7A\u003csup\u003e1-176\u003c/sup\u003e WT was lowered to 45.5\u0026deg;C, while the Tm of GST alone remained unchanged (Fig. S4A). Furthermore, the GST-RAB7A\u003csup\u003e1-176\u003c/sup\u003e T22N mutant, which has a reduced affinity for guanine nucleotides, exhibited a lower Tm of 47.5\u0026deg;C that was not affected by EDTA treatment (Fig. S4A). These results indicate that both the purified RAB7A T22N and the nucleotide-free apo form of RAB7A exhibit low resistance to thermal denaturation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNext, we investigated whether C5orf51 could suppress heat-induced aggregation of the apo form of RAB7A or the T22N mutant. For this purpose, recombinant GST or GST-RAB7A\u003csup\u003e1-176\u003c/sup\u003e T22N was incubated at various temperatures (40-64\u0026deg;C), and protein aggregates were then pelleted by centrifugation. We found that GST began to appear in the pellet fraction at 54.5\u0026deg;C, while GST-RAB7A\u003csup\u003e1-176\u003c/sup\u003e T22N started to aggregate at a lower temperature of 49.1\u0026deg;C (Figs. S4B Frac. #5 and S4C Frac. #4). The addition of recombinant C5orf51 reduced the aggregation of GST-RAB7A\u003csup\u003e1-176\u003c/sup\u003e T22N, resulting in more soluble RAB7A remaining in the supernatant fraction, while the phenomenon was not observed with GST (Figs. S4B and S4C). Further analysis at temperatures ranging from 43\u0026deg;C to 56\u0026deg;C revealed that C5orf51 exhibited chaperone activity toward the apo form of RAB7A\u003csup\u003e1-176\u003c/sup\u003e WT. This effect was not observed for RAB7A\u003csup\u003e1-176\u003c/sup\u003e bound to GppNHp, a non-hydrolyzable GTP analog (Figs. S4D-S4F). These findings suggest that C5orf51 interacts specifically with nucleotide-free RAB7A to prevent it from aggregating.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC5orf51 acts as a chaperone to facilitate correct folding of nascent RAB7A.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven that nucleotide-free (apo) RAB proteins may exist transiently during translation before GTP loading, we investigated the requirement of C5orf51 for RAB7A\u0026rsquo;s proper folding using the following two distinct approaches.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFirst, we utilized an \u003cem\u003eE.coli\u0026nbsp;\u003c/em\u003eexpression system, as procaryotes lack RAB-related factors including REPs. Although the RAB7A\u003csup\u003e1-176\u003c/sup\u003e is known to form a compact structure without intrinsically disordered region \u003csup\u003e30\u003c/sup\u003e, over half of the expressed HA-tagged RAB7A\u003csup\u003e1-176\u003c/sup\u003e was found in inclusion bodies (pellet fractions) upon induction at 28\u0026deg;C (Fig. 5A). This aggregation occurred regardless of the guanine binding state, as approximately half of the WT, Q67L, and T22N were recovered in the pellet fractions (Figs. 5A and 5B). As expected, the extent of aggregation was dependent on the culture temperature (Figs. 5A, 5B, S5A and\u0026nbsp;S5B). In sharp contrast, the co-expression of C5orf51 significantly suppressed RAB7A aggregation, with nearly all of the WT and Q67L RAB7A\u003csup\u003e1-176\u003c/sup\u003e recovered in the soluble supernatant fraction at all temperatures tested (Figs. 5A, 5B, S5A and\u0026nbsp;S5B). A similar chaperone-like activity was observed for full-length RAB7A (Figs. S5C and S5D). Importantly, once RAB7A\u003csup\u003e1-176\u003c/sup\u003e WT forms a stable structure, it no longer interacts with C5orf51; when C5orf51-His\u003csub\u003e6\u003c/sub\u003e was pulled down using a nickel affinity resin, RAB7A\u003csup\u003e1-176\u003c/sup\u003e T22N was found in the bound fraction, whereas RAB7A\u003csup\u003e1-176\u003c/sup\u003e WT was not (Fig. 5C). We also demonstrated that C5orf51 chaperone activity is specific to RAB7A, as the co-expression of C5orf51 did not prevent the aggregate formation of other RAB proteins (Figs. 5D and 5E). Furthermore, not only human C5orf51, but also its ortholog from \u003cem\u003eDanio rerio\u003c/em\u003e (Zf C5orf51) had the same ability to prevent RAB7A from aggregate formation (Fig. S5E), indicating that this specific chaperone activity is evolutionarily conserved.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor another approach to precisely monitor C5orf51 chaperone activity, we employed the PUREfrex, an in vitro coupled transcription/translation system. PUREfrex is a \u0026quot;bottom-up\u0026quot; reconstitution system that contains individually purified translational factors and ribosomes but lacks chaperones such as Hsp70 and GroEL. This allowed us to specifically monitor C5orf51-dependent chaperone activity during the translation of RAB7A.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhen GST-RAB7A\u003csup\u003e1-176\u003c/sup\u003e was translated in the presence of BSA as a control, more than half of the RAB7A WT, T22N, and Q67L proteins formed insoluble aggregates and were recovered in the pellet fraction after centrifugation (Figs. 5F and 5G). However, in the presence of recombinant C5orf51, these RAB7A proteins were translated almost completely as soluble proteins (Figs. 5F and 5G). Even the highly aggregate-prone nucleotide-free N125I mutant was rendered soluble when translated with C5orf51 (Figs. 5H and 5I). These results strongly suggest that C5orf51 acts as a specific chaperone for RAB7A and promotes its proper folding immediately after translation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCrystal structure of C5orf51 in complex with RAB7A\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo gain insight into the structural basis for the recognition of RAB7A by C5orf51, we set out to determine the C5orf51-RAB7A complex structure by crystallography. The complex was formed by mixing C5orf51 with apo RAB7A\u003csup\u003e1-176\u003c/sup\u003e (hereafter referred to as RAB7A) that had been pretreated with EDTA. The resulting C5orf51-RAB7A complex was purified and crystallized upon concentration (Fig. S6). We determined the crystal structure of the C5orf51\u0026ndash;RAB7A complex at a resolution of 3.36 \u0026Aring; (Figs. 6A\u0026shy;-6C and Table S1). The analysis of structural homologs of C5orf51 using the DALI server \u003csup\u003e31\u003c/sup\u003e revealed that C5orf51 shares similarity with tetratricopeptide repeat (TPR) proteins including LNG, CNS1, and TONSOKU (Figs. S7A and S7B). The C5orf51 consists of ten a-helices and two insertion loops (residues 179\u0026ndash;203 and 225\u0026ndash;252) (Fig. 6B). The first insertion contained a-helix (a7) while the second insertion was disordered (Fig. S7C). In other TPR proteins such as LNG and TONSOKU, the concave surfaces are crucial for recognizing interacting partners (Figs. S7D and S7E). Similarly, the concave surface of C5orf51, which is evolutionarily highly conserved (Fig. S8A), directly interacts with RAB7A (Fig. S8B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe switch I region of RAB7A adopts a chaperone-specific conformation.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRAB7A in the complex consists of a central six-stranded b-sheet (b1- b6) flanked by five a-helices (a1- a5) (Fig. 6C). Notably, the canonical a1 helix of RAB7A is unfolded in the present structure. Instead, the switch I region forms a new a-helix (referred to as aSI), which directly interacts with the concave surface of C5orf51 (Figs. 6A and 6C\u0026shy;\u0026shy;). This a1-switch I conformation in the C5orf51-RAB7A complex is distinct from other previously reported states of RAB7A (Figs. 6D-6H). The local unfolding of the a1 helix suggests an intermediate state, which is structurally different from the nucleotide-bound (GTP or GDP) forms of RAB7A (Figs. 6G and 6H). The yeast RAB7A homolog Ypt7 structure with MON1-CCZ1 also shows a distinct a1-switch I conformation due to the interaction with the GEF (Fig. 6F), suggesting the different structural dynamics of this region across the various functional states of RAB7A.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe then investigated whether similar unfolded \u0026alpha;1 and formed aSI conformation have been observed in other RAB proteins. RABIF/MSS4 was previously shown to recognize RAB8A in such a conformation, where the a1 helix is unfolded and the switch I forms a new aSI helix (Fig. S9A). While the conformation of RAB8A bound to RABIF/MSS4 closely resembles that of RAB7A bound to C5orf51 (Fig. S9B), the structures themselves (C5orf51 and RABIF/MSS4) are substantially different (Figs. S9C and S9D). Despite the similar switch I conformations, RAB7A and RAB8A are recognized at the different binding sites on their primary sequence (Fig. S9E). As a result, RAB7A has a more bent conformation with C5orf51 compared to RAB8A bound to RABIF/MSS4.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe interface between C5orf51 and RAB7A.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe RAB7A-binding interface of C5orf51 is composed of three distinct hydrophobic pockets located on its concave surface (Figs. 6I-6M and S10A). The first pocket extensively accommodates and captures the\u0026nbsp;aSI region of RAB7A (Figs. 6J, 6K and S10). This interaction is mediated by bulky hydrophobic residues of RAB7A (Y28, F33, and Y37). M25 and V29 of RAB7A are also oriented toward C5orf51. C5orf51 provides a conserved hydrophobic concavity formed by Y76, C130, Y140, M141, L209, M212, Y213, and W277, which protects the hydrophobic side of RAB7A from solvent exposure.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe second hydrophobic pocket of C5orf51, formed by W277, L197, L208, and L272, covers invariant residues (F45 and W62) in the switch I and inter-switch regions of RAB7A (Figs. 6L and S10). The third pocket of C5orf51 is comprised of I181, V187, F189, L197, and L198, which are located within the first insertion loop (Figs. 6M and S10). This pocket is buried by L8 and T47 from the b1 strand of RAB7A (Figs. 6M and S10). The hydrophilic interaction is also observed between K38 of RAB7A and D205 of C5orf51, with a proximity of 3.0 \u0026Aring; although the electron density for K38 is relatively weak (Fig. 6L).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGTP loading affects the interaction of C5orf51 with RAB7A.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo complement the biochemical and crystallographic analyses, we next conducted molecular dynamics (MD) simulations of both\u0026nbsp;the C5orf51-apo RAB7A complex\u0026nbsp;and the C5orf51-GTP-bound RAB7A complex. We prepared the structure of the\u0026nbsp;GTP-bound form\u0026nbsp;based on\u0026nbsp;the crystal structure of the apo form (see \u0026lsquo;Methods\u0026rsquo;) and noted that\u0026nbsp;the GTP-binding site is the opposite side from the interface between C5orf51 and RAB7A and that the C5orf51-RAB7A complex can accommodate GTP though\u0026nbsp;the switch I region adopts its distinctive conformation which is structurally different from the nucleotide-bound (GTP or GDP) forms of RAB7A monomer.\u003c/p\u003e\n\u003cp\u003eThe results of the simulations showed that, in GTP-bound RAB7A, the C-terminal region (a4- a5 and b5- b6) containing guanine base recognition (G4-G5) motifs bent towards the N-terminal region (a1- a2) around the phosphate-binding (G1-G3) motifs, compared to the apo form (Fig. 6N). Moreover, torsion of the N-terminal region opened a pathway to the binding site, enabling access for GTP. Over the 1 ms simulation, the guanosine moiety of GTP was moving in and out of the site located between the G1 (P-loop) and G4\u0026ndash;G5 regions, while the triphosphate groups of GTP was anchored to the P-loop (Fig. S11A). These results suggest that the C5orf51-RAB7A complex can load GTP onto apo RAB7A while maintaining chaperone-specific conformations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRegarding interactions within the complex, the number of hydrogen bonds between C5orf51 and RAB7A, as well as the buried surface area of the complex, tended to decrease in the GTP-bound form compared to the apo form (Figs. 6O and 6P), indicating a reduction in the interaction between C5orf51 and RAB7A. Especially, the number of hydrogen bonds to C5orf51 in the unfolded a1 region of RAB7A (M25, Y28, and V29), which is critical for C5orf51 recognition as described below, decreased in the GTP-bound form (Table S2). This is mainly because the changes in the P-loop conformation during GTP binding lead to more extensive unfolding of the helix around the a1 region (Fig. S11B). These results collectively suggest that the C5orf51-RAB7A complex can load GTP into the binding site of apo RAB7A and that C5orf51 and RAB7A dissociate coupled with loading GTP.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe recognition of RAB7A by the concave surface of C5orf51 is required for chaperone activity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the recognition mechanism revealed by the structural analysis of C5orf51-RAB7A, we introduced structure-based mutations into residues of C5orf51 that face RAB7A and tested their interactions by fluoppi assay (Fig. 7A). Alanine substitutions of Y140, L209, Y213, and W277 in C5orf51 abolished fluoppi foci formation with RAB7A, indicating a loss of interaction. The small side-chain mutation C130A in C5orf51 had no effect on the interaction, whereas the replacement with a bulky side chain (C130W) impaired the interaction. The substitution of Y76 and M212 of C5orf51 with a small hydrophilic serine residue reduced the interaction with RAB7A. The L272 of C5orf51 was substituted with an asparagine residue based on its proximity to RAB7A and its hydrophobic character. However, the L272N mutation in C5orf51 did not impair RAB7A recognition. Similarly, the D205A mutation, which was designed to disrupt the electrostatic interaction between D205 of C5orf51 and K38 of RAB7A, did not affect the interaction. Alanine substitution of the five hydrophobic residues (I181, V187, F189, L197, and L198) in the insertion1 of C5orf51 also had no effect on the interaction. Moreover, deletion of the entire hydrophobic cluster (residues 181\u0026ndash;199) did not impair the interaction. We next examined the effect of C5orf51 mutants on RAB7A solubility. The C5orf51 mutants Y76S, C130W, L209A, M212S, Y213A, and W277A were prepared as a recombinant protein and used for in vitro PUREfrex assays (Fig. 7B). The L209A and W277A mutants of C5orf51 almost completely lost their chaperone activity toward RAB7A (Figs. 7B and 7C). The Y76S, C130W, M212S, and Y213A mutants of C5orf51 retained partial activity, but they showed lower activity than wild-type C5orf51 (Figs. 7B and 7C). These results indicate that residues composing the concave surface of C5orf51 are important for RAB7A recognition (Fig. 7D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003cstrong\u003eSI region of RAB7A is essential for recognition and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003esolubilization\u0026nbsp;by C5orf51.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo identify the residues of RAB7A critical for the interaction with C5orf51, we examined both the in vitro solubilization efficiency and intracellular interaction using RAB7A mutants. Substitutions of residues within the\u0026nbsp;aSI region (Y28A, V29A, F33A, and Y37A), which bind to the hydrophobic pocket 1 of C5orf51, significantly abrogated C5orf51-dependent solubilization (Figs. 7E and 7F). Moreover, the Y28A and Y37A mutants showed no detectable interaction with C5orf51 in the fluoppi assay, and the V29A and F33A mutants exhibited reduced foci formation (Figs. 7G and 7H). On the other hands, alanine mutations at L8, F45, and W62 of RAB7A, which contact the hydrophobic pockets 2 and 3 of C5orf51, showed in vitro solubility comparable to that of wild-type RAB7A (Figs. 7E and 7F) and formed fluoppi foci with C5orf51 (Figs. 7G and 7H). Taken together, these results indicate that the aSI region of RAB7A is a primary recognition site for C5orf51 (Figs. 7I and S10).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAmong the four RAB7A residues (Y28A, V29A, F33A, and Y37A) critical for C5orf51 recognition, V29 and Y37 are less conserved among RAB family proteins (Fig. 7J). This sequence comparison supports the idea that non-conserved residues in RAB7A contribute to the substrate specificity of C5orf51. Given that mutations in the structural core of RAB7A did not affect its interaction with and solubilization by C5orf51, we hypothesized that the N-terminal region of RAB7A might be sufficient for recognition by C5orf51. Indeed, using fluoppi assays, we found that the N-terminal 38 residues of RAB7A is sufficient for the interaction with C5orf51 (Figs. 7G and 7H). This finding supports the idea that C5orf51 primarily recognizes the N-terminal region of RAB7A. To further test the sufficiency of the RAB7A aSI region, we constructed chimeric proteins in which the a1-switch I segments (residues 25-38 in each case) of RAB8A and RAB9A were replaced with the corresponding region of RAB7A (Fig. 7K). While C5orf51 interacted with neither RAB8A nor RAB9A, these chimeric proteins were recognized by C5orf51 in cells as shown by fluoppi assay (Figs. 7L and 7M). These results demonstrate that C5orf51 discriminates RAB7A from the other RABs by recognizing the a1-switch I sequence.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWhile the structural stability of Rab GTPases was previously known to be influenced by their guanine-nucleotide binding state \u003csup\u003e27\u003c/sup\u003e, our understanding of the cellular quality control mechanisms that support Rab protein stability has been limited. In this study, we identified C5orf51 as a factor that binds strongly to the nucleotide-free state of RAB7A, which is critical for lysosome and autophagy regulation.\u003c/p\u003e \u003cp\u003eBecause C5orf51 also binds to the GDP-bound mutant RAB7A T22N, it may function as a guanine nucleotide exchange factor (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). However, C5orf51 is localized diffusely in the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), unlike the endomembrane-associated RAB7A GEF, the MON1\u0026ndash;CCZ1\u0026ndash;RMC1 complex \u003csup\u003e32\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eC5orf51 was previously suggested to be a new subunit of the MON1-CCZ1 complex \u003csup\u003e33\u003c/sup\u003e and was designated RIMOC1 (RAB7A-interacting MON1-CCZ1 complex subunit 1). Our fluoppi assay and co-immunoprecipitation experiments, however, did not detect an interaction between C5orf51 and the MON1-CCZ1 complex (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). Furthermore, structural superposition of the RAB7-MON1-CCZ1 complex and our RAB7A-C5orf51 complex revealed a substantial overlap between the binding surfaces of MON1-CCZ1 and C5orf51 on RAB7A (Fig. S12). These observations strongly suggest that C5orf51 and the MON1-CCZ1 complex do not function cooperatively but rather compete for binding to RAB7A. Therefore, the name RIMOC1 is not appropriate as it does not reflect the actual function of C5orf51. In this study, we discovered that C5orf51 acts as a chaperone-like protein, binding to the structurally unstable, nucleotide-free form of RAB7A to increase its solubility. The term \u0026ldquo;holdase\u0026rdquo; is used for a molecular chaperone that binds to an unfolded or partially folded substrate to prevent aggregation without using ATP hydrolysis energy. Therefore, we propose to name C5orf51 as RAB7HD, for \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eRAB7A\u003c/span\u003e-specific \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003eol\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eD\u003c/span\u003ease.\u003c/p\u003e \u003cp\u003eRAB7HD/C5orf51 increased the solubility of RAB7A (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Interestingly, even the wild-type and GTP-locked forms of RAB7A, which do not stably interact with C5orf51, also require C5orf51 to facilitate their folding to become stable structure. Our results strongly suggest that RAB7HD/C5orf51-RAB7A complex forms immediately after RAB7A translation, and they dissociate when GTP is loaded onto apo-RAB7A. C5orf51 recognizes the N-terminal region of RAB7A (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH), which is consistent with the idea that C5orf51 binds to RAB7A during the early stages of translation, interacting with the nascent polypeptide chain to prevent aggregation.\u003c/p\u003e \u003cp\u003eInterestingly, the RAB7A-RAB7HD/C5orf51 complex shows structural similarities to the RAB8A-RABIF/MSS4 complex (Fig. S9). While RABIF/MSS4 was initially reported as a GEF for RAB8A \u003csup\u003e34\u003c/sup\u003e, a recent study showed that it also functions as a chaperone that contributes to RAB8A stabilization in cells \u003csup\u003e18\u003c/sup\u003e. Although there is no structural similarity between the individual RAB7HD/C5orf51 and RABIF/MSS4, it is intriguing that they both induce a functional and structural similarity upon binding to their respective RAB proteins: the unfolding of the RAB a1 helix and the formation of a new a-helix in the Switch I region. The bulky hydrophobic residues F45 and W62 of RAB7A are located at the interaction interfaces not only in a complex with RAB7HD/C5orf51 but also in the complexes with the GEF MON1-CCZ1 and effector proteins like RILP and PDZD8 \u003csup\u003e30 35\u003c/sup\u003e. This suggests that RAB7HD/C5orf51 may transiently cover the interaction surface with other binding partners, protecting the hydrophobic surface of RAB7A from non-specific interactions or premature binding. In this way, RAB7HD/C5orf51 functions consistent with the classical definition of a molecular chaperone, assisting the proper folding of RAB7A by preventing misfolding and aggregation without becoming a part of the final structure or influencing its final conformation.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eCell lines, antibodies, plasmid DNAs and reagents used in this study are listed in Table S3, S4, S5 and S6, respectively.\u003c/p\u003e\n\u003ch3\u003ePlasmid Construction\u003c/h3\u003e\n\u003cp\u003eHuman \u003cem\u003eC5orf51\u003c/em\u003e, \u003cem\u003eRMC1\u003c/em\u003e, \u003cem\u003eRAB1A\u003c/em\u003e, \u003cem\u003eRAB4A\u003c/em\u003e, \u003cem\u003eRAB6A\u003c/em\u003e, \u003cem\u003eRAB8A\u003c/em\u003e, \u003cem\u003eRAB11A\u003c/em\u003e and \u003cem\u003eRAB18\u003c/em\u003e coding sequences were amplified by PCR from HeLa cDNA library. Mouse \u003cem\u003eRAB9A\u003c/em\u003e, \u003cem\u003eRAB9B\u003c/em\u003e, \u003cem\u003eRAB23\u003c/em\u003e, \u003cem\u003eRAB32\u003c/em\u003e and \u003cem\u003eRAB38\u003c/em\u003e coding sequences were purchased from RIKEN BRC DNA bank. For preparation of recombinant proteins, human \u003cem\u003eC5orf51\u003c/em\u003e, zebrafish \u003cem\u003eC5orf51\u003c/em\u003e and human \u003cem\u003eRAB7A\u003c/em\u003e genes, which are codon-optimized for \u003cem\u003eEscherichia coli\u003c/em\u003e K12, were purchased from Eurofins. For biochemical assay, codon-optimized \u003cem\u003eC5orf51\u003c/em\u003e gene was inserted into BamHI/NotI sites of a pET21a vector. Codon-optimized human \u003cem\u003eRAB7A\u003c/em\u003e\u003csup\u003e\u003cem\u003e1\u0026ndash;176\u003c/em\u003e\u003c/sup\u003e (WT, Q67L, T22N, and N125I) genes were inserted into BamHI/EcoRI sites of a pGEX6P1 vector. For structure determination, \u003cem\u003eRAB7A\u003c/em\u003e\u003csup\u003e\u003cem\u003e1\u0026ndash;176\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eC5orf51\u003c/em\u003e genes were subcloned into pET21a and pET16b vectors to produce the C-terminal His\u003csub\u003e6\u003c/sub\u003e-tagged RAB7A\u003csup\u003e1\u0026ndash;176\u003c/sup\u003e and N-terminal His\u003csub\u003e10\u003c/sub\u003e-tagged C5orf51, respectively. For yeast two-hybrid assay, \u003cem\u003eC5orf51\u003c/em\u003e gene was inserted into pAct2 plasmid (TaKaRa Bio) and pGBD-C1-Rab1-43(GTP-locked or Apo)-DCys (RIKEN) were prepared as described previously\u003csup\u003e24 25\u003c/sup\u003e. Mutations were introduced by PCR-based DNA mutagenesis. DNA sequences inserted into vectors were confirmed by Sanger sequencing.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and transfection\u003c/h2\u003e \u003cp\u003eHeLa and HEK293T cells were cultured in DMEM supplemented with 10% (v/v) FBS, 1 mM sodium pyruvate, nonessential amino acids, and Penicillin-Streptomycin-Glutamine. HCT116 cells were cultured in McCoy\u0026rsquo;s 5A medium supplemented with 10% (v/v) FBS, nonessential amino acids, and 2 mM GlutaMax. Cell lines used in this study were authenticated and tested for mycoplasma contamination.\u003c/p\u003e \u003cp\u003eStable cell lines were made by recombinant retrovirus infection \u003csup\u003e21\u003c/sup\u003e. FuGENE6 and FuGENE HD reagents were used for plasmid transfection according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnti-C5orf51 Antibody\u003c/h3\u003e\n\u003cp\u003eAnti-C5orf51 antiserum was acquired by immunizing rabbits with the purified C5orf51-His\u003csub\u003e6\u003c/sub\u003e and the anti-C5orf51 antibody was purified using CNBr-activated Sepharose 4B coupled to recombinant C5orf51 (Eurofins Genomics).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCRISPR/Cas9-edited gene knockout.\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eC5orf51-/-\u003c/em\u003e HCT116 cell lines were established using a CRISPR/Cas9-based genome editing with an antibiotic selection strategy described previously \u003csup\u003e36\u003c/sup\u003e. Three different guide-RNAs (gRNAs) targeted to the exon 1 in \u003cem\u003eC5orf51\u003c/em\u003e gene (5\u0026rsquo;- ACT AGA GAC TGC GGC CGC CA -3\u0026rsquo;, 5\u0026rsquo;- GGC GGC CGC AGT CTC TAG TG -3\u0026rsquo;, and 5\u0026rsquo;- CTT CGC TAA GTT GCT GTA TG -3\u0026rsquo;) were designed using an online CRISPR design tool: CRISPOR (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://crispor.tefor.net/\u003c/span\u003e\u003cspan address=\"http://crispor.tefor.net/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Three pairs of DNA oligonucleotides: C5orf51-ex1-PX459-F1/R1 (5\u0026rsquo;- cac cgA CTA GAG ACT GCG GCC GCC A -3\u0026rsquo; and 5\u0026rsquo;-aaa cTG GCG GCC GCA GTC TCT AGT c -3\u0026rsquo;), C5orf51-ex1-PX459-F2/R2 (5\u0026rsquo;- cac cgG GCG GCC GCA GTC TCT AGT G -3\u0026rsquo; and 5\u0026rsquo;-aaa cCA CTA GAG ACT GCG GCC GCC c -3\u0026rsquo;), and C5orf51-ex1-PX459-F3/R3 (5\u0026rsquo;- cac cgC TTC GCT AAG TTG CTG TAT G -3\u0026rsquo; and 5\u0026rsquo;- aaa cCA TAC AGC AAC TTA GCG AAG c -3\u0026rsquo;) were annealed and inserted into BpiI sites of PX459 vector. To construct donor plasmids, 500 bp sequences, which include 247 bp of the 5\u0026rsquo; and 3\u0026rsquo; of the gRNA targeting regions as homologous arms and a BamHI site in the middle instead of the gRNA sequence were synthesized in a pEX-A2J2 vector (Eurofins Genomics). The neomycin-resistant gene (NeoR) and the hygromycin-resistant gene (HygroR), including the appropriate promoter and terminator\u003csup\u003e37\u003c/sup\u003e were inserted into the BamHI site of the pEX-A2J2 plasmids. The resultant PX459_C5orf51-ex1 plasmids and pEX-A2J2 donor plasmids containing NeoR or HygroR were transfected into HCT116 cells using FuGENE HD. After 48 hours transfection, cells were grown in McCoy\u0026rsquo;s 5A media containing 700 \u0026micro;g/ml G418 and 100 \u0026micro;g/ml hygromycin B. Single colonies were isolated into a 24-well plate. Genomic DNA was extracted using a Microprep Kit Quick-gDNA and neomycin-resistant and hygromycin-resistant gene insertions into the target region were verified by PCR.\u003c/p\u003e \u003cp\u003e \u003cem\u003eC5orf51\u003c/em\u003e KO, \u003cem\u003eRAB9A_RAB9B\u003c/em\u003e double knockout (DKO), \u003cem\u003eC5orf51_RAB9A_RAB9B\u003c/em\u003e triple knockout (TKO) HeLa cell lines were established as follows. The gRNA target sequences (5\u0026rsquo;- GAT TGA CAT AAG CGA ACG GC -3\u0026rsquo; and 5\u0026rsquo;- GAG TTC TTG CTA CCG AGG AT -3\u0026rsquo; ) for RAB9A exon 3, (5\u0026rsquo;- CTG CCG ATC ATC CAC GCT GA -3\u0026rsquo; and 5\u0026rsquo;- CAA ATT TGT TGG TTA CGT AA -3\u0026rsquo;) for RAB9B exon 3 were designed using CRISPOR. These DNA oligonucleotides were inserted into BpiI sites of PX459 vector. The same PX459 plasmids described above were used for knocking out \u003cem\u003eC5orf51\u003c/em\u003e genes in HeLa cells. The PX459 plasmids were transfected into HeLa cells, and puromycin-resistant cells were seeded onto 96-well plates. The knockout of \u003cem\u003eC5orf51\u003c/em\u003e was analyzed by immunoblotting and the knockout of \u003cem\u003eRAB9A\u003c/em\u003e and \u003cem\u003eRAB9B\u003c/em\u003e genes were analyzed by sanger sequencing of the genome extracted from single clones.\u003c/p\u003e\n\u003ch3\u003eImmunoblotting\u003c/h3\u003e\n\u003cp\u003eCells grown in 6-well plates were washed twice with Phosphate Buffered Saline (PBS) and solubilized with 2% CHAPS buffer (25 mM HEPES-KOH pH7.5, 300 mM NaCl, 2% [w/v] CHAPS, a protease inhibitor cocktail) on ice for 10 min. After centrifugation at 12,000 \u0026times;g for 2 min at 4\u0026deg;C, the supernatants were collected, and protein concentrations were determined on a DS-11\u0026thinsp;+\u0026thinsp;spectrophotometer (DeNovix). SDS-PAGE sample buffer with DTT was added to the supernatants, which were then incubated at 42\u0026deg;C for 5 min. To detect hAG constructs, the samples were boiled at 95\u0026deg;C for 5 min. Total cell lysates were loaded on NuPAGE 4\u0026ndash;12% Bis-Tris gels and electrophoresed using MES or MOPS running buffer. Proteins were transferred to PVDF membranes that were blocked with 2% (w/v) skim-milk/TBS-T and then incubated with primary and HRP-conjugated secondary antibodies. Proteins were detected using a Western Lighting Plus-ECL Kit on an ImageQuant LAS4000 (GE Healthcare) or a FUSION SOLO S system (VILBER). ImageJ was used to quantify protein bands.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmunostaining and immunofluorescence microscopy\u003c/h2\u003e \u003cp\u003eCells grown on 35-mm glass-bottom dishes were fixed with 4% paraformaldehyde (PFA) in PBS for 20 min at room temperature. The cells were then permeabilized with 0.15% (v/v) TritonX-100 for 15 min at room temperature, and incubated with 0.1% (w/v) gelatin in PBS for 30 min. The cells were incubated with primary antibodies diluted in 0.1% gelatin for 2 hours at room temperature. After washing with PBS-T (PBS with Tween 20), the cells were incubated with Alexa Fluor-conjugated secondary antibodies diluted in 0.1% gelatin for 1 hour. Primary antibodies and Alexa Fluor-conjugated secondary antibodies used in this study are listed in Table S3. 4',6-diamidino-2-phenylindole (DAPI) was used for nuclear staining. The images were acquired using an inverted confocal microscope LSM780 (Carl Zeiss) with a Plan-Apochromat 63\u0026times;/1.4 oil lens, an FV3000 (EVIDENT) with a PlanApo N 60x/1.4 oil objective lens, or EVOS M5000 imaging system (Thermo Fisher Scientific). For image analysis, ZEN microscope software and Photoshop (Adobe) were used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCo-Immunoprecipitation\u003c/h2\u003e \u003cp\u003eHeLa cells expressed with FLAG-tagged proteins were grown in a 6-well plate. The cells were washed with PBS twice and solubilized on ice for 15 min in IP buffer (50 mM Tris-HCl pH7.5, 150 mM NaCl, 0.5% (w/v) sodium deoxycholate, 1% (v/v) Triton X-100, a protease inhibitor cocktail). Insolubilized debris was removed by centrifugation at 16,000 \u0026times;g, 4\u0026deg;C for 10 min. The supernatants were incubated with anti-DDDDK-tag mAb magnetic beads or anti-FLAG M2 magnetic beads for 1 hour at 4\u0026deg;C under gentle rotation. 5\u0026ndash;30% of the supernatants was collected as an input. Beads were washed with IP buffer three times and bound proteins were eluted with SDS-PAGE sample buffer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMass spectrometry (MS) analysis\u003c/h2\u003e \u003cp\u003eTo identify nucleotide-dependent RAB interacting proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), samples for MS analysis were prepared as follows. HeLa cells transiently expressing 3FLAG-TEV-RAB proteins grown in a well of 6-well plate were washed with PBS and solubilized on ice for 10 min in TX-100 buffer (50 mM Tris\u0026ndash;HCl pH7.5, 150 mM NaCl, 1% (v/v) Triton X-100, a protease inhibitor cocktail). To identify C5orf51 binding proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), HeLa cells (control) and those stably expressing 3FLAG-TEV-C5orf51 were grown in a 10 cm dish. The cells were washed with PBS with three times and solubilized on ice for 10 min in TX-100 buffer.\u003c/p\u003e \u003cp\u003eAfter centrifugation at 14,000 \u0026times;g, 4\u0026deg;C for 10 min, the supernatants were incubated with equilibrated anti-FLAG M2 magnetic beads for 1.5 hours at 4\u0026deg;C under gentle rotation. The beads were collected using a magnetic stand, washed three times with TX-100 buffer, and another three times with 50 mM ammonium bicarbonate. Proteins bound to the beads were digested with 200 ng of trypsin/Lys-C mix for 16 hours at 37\u0026deg;C. The digests were reduced, alkylated, acidified with trifluoroacetic acid, and desalted using GL-Tip SDB. The eluates were evaporated in a SpeedVac concentrator and dissolved in 0.1% TFA and 3% acetonitrile (ACN).\u003c/p\u003e \u003cp\u003eLC-MS/MS analysis of the resultant peptides was performed using an EASY-nLC 1200 UHPLC connected to an Orbitrap Fusion mass spectrometer equipped with a nanoelectrospray ion source (Thermo Fisher Scientific). The peptides were separated on a 75 \u0026micro;m inner diameter \u0026times; 150 mm C18 reversed-phase column using a linear 4\u0026ndash;32% ACN gradient for 0-100 min followed by an increase to 80% ACN for 10 min and a final hold at 80% ACN for 10 min. The mass spectrometer was operated in data-dependent acquisition mode with a maximum duty cycle of 3 s. MS1 spectra were measured at a resolution of 120,000, an automatic gain control (AGC) target of 4 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e, and a mass range from 375 to 1,500 \u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e. HCD MS/MS spectra were acquired in the linear ion trap with an AGC target of 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e, an isolation window of 1.6 \u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e, a maximum injection time of 35 ms, and a normalized collision energy of 30. Dynamic exclusion was set to 20 s. Raw data were directly analyzed against the SwissProt database restricted to \u003cem\u003eH. sapiens\u003c/em\u003e using Proteome Discoverer version 2.5 (Thermo Fisher Scientific) with Sequest HT search engine for identification and label-free precursor ion quantification. The search parameters were as follows: (i) trypsin as an enzyme with up to two missed cleavages; (ii) precursor mass tolerance of 10 ppm; (iii) fragment mass tolerance of 0.6 Da; (iv) carbamidomethylation of cysteine as a fixed modification; and (v) acetylation of the protein N-terminus and oxidation of methionine as variable modifications. Peptides and proteins were filtered at a false discovery rate (FDR) of 1% using the percolator node and the protein FDR validator node, respectively. Normalization was performed such that the total sum of abundance values for each sample over all peptides was the same.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eYeast two hybrid\u003c/h2\u003e \u003cp\u003eThe yeast strain (PJ69-4A), medium, culture conditions, and transformation protocol used were described previously \u003csup\u003e38\u003c/sup\u003e. Yeast cells containing pGBD-C1-Rab(GTP-locked or Apo)-DCys and pAct2-C5orf51 were streaked on the synthetic complete medium lacking adenine, histidine, leucine and tryptophan were incubated at 30\u0026deg;C for 4\u0026ndash;6 days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of recombinant proteins for biochemical assay\u003c/h2\u003e \u003cp\u003eRecombinant C5orf51 was prepared as follows. pET21a plasmid harboring C5orf51 was introduced into BL21-CodonPlus(DE3)-RIL. The transformants were cultured in LB media at 37\u0026deg;C until the logarithmic growth phase. Protein expression was induced with 100\u0026ndash;200 \u0026micro;M Isopropyl b-D-1-thiogalactopyranoside (IPTG) overnight at 18\u0026deg;C. The cells were collected by centrifugation, lysed with TBS buffer (50 mM Tris-HCl pH7.5, 120 mM NaCl, 50 \u0026micro;g/ml lysozyme, 1 \u0026micro;g/ml DNaseI, 1 mM DTT, 1 mM MgCl\u003csub\u003e2\u003c/sub\u003e, a protease inhibitor cocktail) and sonicated. After centrifugation at 10,000 \u0026times;g, 4\u0026deg;C for 10 min, the supernatants were incubated with Ni-NTA agarose for 30 min at 4\u0026deg;C under gentle rotation. The agarose was washed three times with TBS\u0026thinsp;+\u0026thinsp;TCEP (TBS with 1mM tris(2-carboxyethl)phosphine) buffer, and bound proteins were eluted with TBS\u0026thinsp;+\u0026thinsp;TCEP buffer containing 40, 80, and 200 mM imidazole in a stepwise fashion. Recombinant His\u003csub\u003e6\u003c/sub\u003e-tagged C5orf51 (C5orf51-His) was then concentrated using an Amicon-Ultra-15, and imidazole was removed using PD midiTrap G-25.\u003c/p\u003e \u003cp\u003eRecombinant GST-tagged RAB7A was prepared as follows. pGEX6P1 plasmids were introduced into Chaperone competent cells pGro7/BL21. The transformants were cultured in LB media at 37\u0026deg;C until the logarithmic growth phase. Expressions of GroES-GroEL and GST-RAB7A were induced with 2 mg/ml L-(+)-arabinose and 100 \u0026micro;M IPTG overnight at 18\u0026deg;C. The cells were collected by centrifugation, lysed with TBS buffer (50 mM Tris-HCl pH7.5, 120 mM NaCl, 50 \u0026micro;g/ml lysozyme, 1 \u0026micro;g/ml DNaseI, 1 mM DTT, 1 mM MgCl\u003csub\u003e2\u003c/sub\u003e, a protease inhibitor cocktail) and sonicated. After centrifugation at 10,000 \u0026times;g, 4\u0026deg;C for 10 min, the supernatants were incubated with glutathione Sepharose 4B for 30 min at 4\u0026deg;C under gentle rotation. The Sepharose was washed three times with TBS\u0026thinsp;+\u0026thinsp;TCEP buffer, and bound proteins were eluted with TBS\u0026thinsp;+\u0026thinsp;TCEP buffer containing 20 mM L-glutathione reduced (GSH). Recombinant GST-RAB7A was then concentrated using an Amicon-Ultra and GSH was removed using PD midiTrap G-25.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of recombinant proteins for structure determination\u003c/h2\u003e \u003cp\u003e \u003cem\u003eEscherichia coli\u003c/em\u003e strain Rosetta (DE3) cells were transformed with pET21a and pET16b expression vector containing the gene encoding RAB7A\u003csup\u003e1\u0026ndash;176\u003c/sup\u003e-His\u003csub\u003e6\u003c/sub\u003e and His\u003csub\u003e10\u003c/sub\u003e-C5orf51, and cultured in LB medium containing 100 mg/L ampicillin at 37\u0026deg;C until optical density at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e) reached\u0026thinsp;~\u0026thinsp;0.8. The expression of RAB7A\u003csup\u003e1\u0026ndash;176\u003c/sup\u003e-His\u003csub\u003e6\u003c/sub\u003e and His\u003csub\u003e10\u003c/sub\u003e-C5orf51 was induced by adding IPTG at the final concentration of 0.1 mM The culture was further continued for 18 h at 15\u0026deg;C. The cells were collected by centrifugation at 7,000 \u0026times;g for 10 min and then disrupted by sonication in 50 mM Tris-HCl buffer (pH 8.0) containing 150 mM NaCl, 0.5%(v/v) Triton X-100 and 20 mM imidazole (buffer A). The lysates were centrifuged at 17,000 rpm for 60 min, and the supernatants were loaded onto a Ni-NTA agarose column. After the column was washed with buffer A and buffer A without Triton X-100, the proteins were eluted with 50 mM Tris-HCl buffer (pH 8.0) containing 150 mM NaCl, 1 mM DTT and 200 mM imidazole. RAB7A\u003csup\u003e1\u0026ndash;176\u003c/sup\u003e-His\u003csub\u003e6\u003c/sub\u003e incubates with 5 mM EDTA for 30 min on ice, and then mix with His\u003csub\u003e10\u003c/sub\u003e-C5orf51 in an equimolar ratio. The mixture of RAB7A\u003csup\u003e1\u0026ndash;176\u003c/sup\u003e-His\u003csub\u003e6\u003c/sub\u003e and His\u003csub\u003e10\u003c/sub\u003e-C5orf51 were further purified by size exclusion chromatography using Hiload 16/600 Superdex200 prep grade (GE healthcare) with 10 mM Tris-HCl (pH 7.2) buffer containing 50 mM NaCl and 5 mM b-mercaptoethanol (buffer B). The fractions abundant in RAB7A\u003csup\u003e1\u0026ndash;176\u003c/sup\u003e-His\u003csub\u003e6\u003c/sub\u003e and His\u003csub\u003e6\u003c/sub\u003e-C5orf51 proteins were collected and concentrated to 2 g/L using an Amicon Ultra-15 (Millipore). For data collection, the crystals of the RAB7A\u003csup\u003e1\u0026ndash;176\u003c/sup\u003e-His\u003csub\u003e6\u003c/sub\u003e and His\u003csub\u003e10\u003c/sub\u003e-C5orf51 complex were soaked in the buffer B containing 30% glycerol and then flash frozen in liquid N\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStructure determination and refinement.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eDiffraction data sets of the RAB7A\u003csup\u003e1\u0026ndash;176\u003c/sup\u003e and C5orf51 were collected at beamline BL45XU in SPring-8 (Hyogo, Japan) and processed with XDS \u003csup\u003e39\u003c/sup\u003e and the CCP4i2 program suite\u003csup\u003e40\u003c/sup\u003e. The complex structure was determined by molecular replacement using MolRep \u003csup\u003e41\u003c/sup\u003e. The structures of C5orf51 and RAB7A\u003csup\u003e1\u0026ndash;176\u003c/sup\u003e predicted by AlphaFold2 were used as the search models, respectively. Atomic models were corrected using Coot \u003csup\u003e42\u003c/sup\u003e with careful inspection. Structure refinement was carried out using the program Phenix \u003csup\u003e43\u003c/sup\u003e with iterative correction and refinement of the atomic models. Data collection and refinement statistics are shown in Table S1. All molecular graphics were prepared with PyMOL (DeLano Scientific; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.pymol.org\u003c/span\u003e\u003cspan address=\"http://www.pymol.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of apo or nucleotide-bound RAB7A in vitro\u003c/h2\u003e \u003cp\u003eGST-RAB7A WT was incubated with glutathione Sepharose in TBS-T-E buffer (50 mM Tris-HCl pH7.5, 120 mM NaCl, 1 mM TCEP, 5 mM EDTA) for the apo form, in TBS-T-E containing 40 \u0026micro;M GppNHp for the GTP-bound form, or in TBS-T-E containing 40 \u0026micro;M GDP for the GDP-bound form, for 2 hours at room temperature. After adding 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e, the resin was washed 3 times with GEF-TX buffer (50 mM Tris-HCl pH7.5, 120 mM NaCl, 1 mM TECP, 0.5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 0.1%[v/v] TX-100) for the apo form, with GEF-TX buffer containing 100 \u0026micro;M GppNHp for the GTP-bound form, and with GEF-TX buffer containing 100 \u0026micro;M GDP for the GDP-bound form.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eThermal shift assay\u003c/h2\u003e \u003cp\u003eDetermination of thermal stability of recombinant RAB7A was undertaken using Protein Thermal Shift Dye (Thermo Fisher Scientific). GST or GST-RAB7A\u003csup\u003e1\u0026ndash;176\u003c/sup\u003e (WT or T22N) (30 \u0026micro;M) were added to the reaction buffer containing 0.01% thermal shift dye with or without 5 mM EDTA. Fluorescence was measured using the ROX channel of a BioRad CFX96 Real Time PCR machine, with a 0.5\u0026deg;C/15 s per step (20\u0026ndash;95\u0026deg;C) melting curve. Peaks were determined using BioRad CFX Manager software. Fluorescence intensity was measured using the ROX reporter of a StepOnePlus\u0026trade; Real-Time PCR system, with a ramp rate of 0.05\u0026deg;C/s per step (25\u0026ndash;99\u0026deg;C). Melting temperature were determined using Protein Thermal Shift\u0026trade; Software (Thermo Fisher Scientific).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eHeat-stress chaperone assay\u003c/h2\u003e \u003cp\u003eHoldase activity of C5orf51 for RAB7A against heat-stress was performed as follows. Recombinant GST (11 \u0026micro;M) or GST-RAB7A\u003csup\u003e1\u0026ndash;176\u003c/sup\u003e (7 \u0026micro;M) with or without recombinant C5orf51-His\u003csub\u003e6\u003c/sub\u003e (11 \u0026micro;M) in TBS buffer were divided equally into 8 samples and incubated at 40\u0026ndash;64\u0026deg;C or 43\u0026ndash;56\u0026deg;C gradient for 5min using a BioRad T100 thermal cycler. The samples were subsequently separated into supernatants and pellets by centrifugation at 20,000 \u0026times;g for 10min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003ePUREfrex chaperone assay\u003c/h2\u003e \u003cp\u003ePUREfrex\u0026reg;1.0 was used for \u003cem\u003ein vitro\u003c/em\u003e transcription/translation reactions to test chaperone-like activity. \u003cem\u003eGST-RAB7A\u003c/em\u003e\u003csup\u003e\u003cem\u003e1\u0026ndash;176\u003c/em\u003e\u003c/sup\u003e-coding DNA fragment was amplified via 3-step PCR reactions. For the 1st PCR, pGEX6P1_Opt_RAB7A(1-176) plasmid was used for a template, and FOR-GST-F (5\u0026rsquo;- AAG GAG ATA TAC CAA TGT CCC CTA TAC TAG GTT A -3\u0026rsquo;) and pGEX-PURE-R (5\u0026rsquo;- CGC TCG AGT CGA CCC GGT TA -3\u0026rsquo;) were used for primers. INTR-PURE-F (5\u0026rsquo;- GGT TTC CCT CTA GAA ATA ATT TTG TTT AAC TTT AAG AAG GAG ATA TAC CA -3\u0026rsquo;) and pGEX-PURE-R was used for the 2nd PCR, and T7pro-PURE-F (5\u0026rsquo;- GAA ATT AAT ACG ACT CAC TAT AGG GAG ACC ACA ACG GTT TCC CTC TAG AAA \u0026minus;\u0026thinsp;3\u0026rsquo;) and pGEX-PURE-R was used for the 3rd PCR to fuse T7 promoter and ribosome binding site sequences to the \u003cem\u003eGST-RAB7A\u003c/em\u003e\u003csup\u003e\u003cem\u003e1\u0026ndash;176\u003c/em\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e transcription/translation reaction carried out according to the manufacture\u0026rsquo;s instruction in the presence of 3 ng/\u0026micro;L DNA fragments and 0.5 mg/mL BSA or recombinant C5orf51-His\u003csub\u003e6\u003c/sub\u003e. After 2 hours of 37\u0026deg;C incubation, supernatants and pellets were separated by centrifugation at 20,000 \u0026times;g, 10min, 4\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eCo-expression of RAB GTPases and C5orf51 in bacterial cells\u003c/h2\u003e \u003cp\u003epETDuet-1 plasmids were introduced into BL21-CodonPlus(DE3)-RIL Competent Cells and ampicillin/chloramphenicol resistant cells were isolated. The 2 mL saturation culture was first prepared by growing the cells in LB media containing ampicillin/chloramphenicol (LB/amp\u0026thinsp;+\u0026thinsp;Cm) overnight. 100 \u0026micro;L saturation culture was then diluted in 2 mL of fresh LB/amp\u0026thinsp;+\u0026thinsp;Cm media and grown at 37\u0026deg;C for 2 hours. IPTG (final 200 \u0026micro;M) was added to the culture and the cells were grown for 5 hours at 28\u0026deg;C and 3 hours at 32\u0026deg;C or 37\u0026deg;C. 400 \u0026micro;L of culture was transferred to 1.5 mL tube and the cells collected by centrifugation at 12,000 \u0026times;g for 2 min were resuspended in 200 \u0026micro;L TBS containing 1 \u0026micro;g/mL DNaseI, 50 \u0026micro;g/mL lysozyme, 1 mM DTT, 1mM MgCl\u003csub\u003e2\u003c/sub\u003e and protease inhibitor cocktail, frozen with liquid N\u003csub\u003e2\u003c/sub\u003e and kept in -20\u0026deg;C. The frozen cells were thawed on ice and subjected to sonication (ultrasonic disrupter UR-21P; TOMY). The supernatants and pellets were separated by centrifugation at 14,000 \u0026times;g for 5 min, and solubilized with SDS-PAGE sample buffer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eHalo-tag processing assay\u003c/h2\u003e \u003cp\u003eCells stably expressing Halo-GFP were pre-treated with 100 nM TMR-conjugated Halo ligand for 20 min. After washing twice with PBS, the cells were incubated in amino-acid free DMEM for 9 hours.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eMeasurement of Lysosomal activity\u003c/h2\u003e \u003cp\u003eCells grown on 35-mm glass-bottom dishes were treated with 75 nM LysoTracker Green DND-26 for 1 hour. DMEM medium was replaced with fresh medium, and the cells were observed using an inverted confocal microscope (LSM780; Carl Zeiss) equipped with a Plan-Apochromat 63\u0026times;/1.4 oil lens. For signal quantification, images were analyzed using ImageJ software. A threshold value of 20/255 was applied to the images. The total fluorescence-positive area (Area value) was measured and then normalized by the number of cells to obtain the area value per cell. 10 microscopic images were quantified for each experimental condition, with each image containing 10\u0026ndash;30 cells.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eEvolutionary profile\u003c/h2\u003e \u003cp\u003eWe generated the evolutionary profile based on presence or absence of ortholog of C5orf51. Orthologs of C5orf51 were obtained from Ortholog Group, OG6_113094 in OrthoMCL DB version 6.5 \u003csup\u003e44\u003c/sup\u003e. We searched homologs of C5orf51 using jackhammer in HMMER 3.3.2\u003csup\u003e45\u003c/sup\u003e against reference proteomes of UniProt \u003csup\u003e46\u003c/sup\u003e if organism is not included in OrthoMCL DB. We then performed orthology inference using reciprocal best hit methodology \u003csup\u003e47\u003c/sup\u003e.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eMolecular dynamics simulation\u003c/h2\u003e \u003cp\u003eMolecular dynamics (MD) simulations were carried out for the apo and GTP-bound forms of the RAB7A-C5orf51 complex structure, whose undetermined regions were completed using MODELLER \u003csup\u003e48\u003c/sup\u003e. The systems consisted of one molecule of RAB7A and one molecule of C5orf51, and more than 33,000 TIP3P water molecules \u003csup\u003e49\u003c/sup\u003e. Approximately 100 Na⁺ and 100 Cl⁻ were added to neutralize the system at a physiological concentration of 0.15 mol/L. In the GTP-bound form, they additionally contained one GTP whose position was calculated using DiffDock \u003csup\u003e50\u003c/sup\u003e and one Mg\u003csup\u003e2+\u003c/sup\u003e, superimposed onto the RAB7A conformation with a crystallographic structure of RAB7A in the GTP bound state (PDB: 1T91) \u003csup\u003e30\u003c/sup\u003e as a reference. All these molecules were placed in an isotropic box with the size of 108 \u0026Aring; \u0026times;108 \u0026Aring; \u0026times;108 \u0026Aring;, to ensure a minimum distance of more than 10 \u0026Aring; from non-solvent molecules. As the force field of the system, AMBER ff14SB \u003csup\u003e51\u003c/sup\u003e for protein atoms, Carlson 2003 model \u003csup\u003e52\u003c/sup\u003e in AMBER parameter database for GTP, Sorensen 2012 model \u003csup\u003e53\u003c/sup\u003e in AMBER parameter database for Mg\u003csup\u003e2+\u003c/sup\u003e, and Joung-Cheatham model for Na\u003csup\u003e+\u003c/sup\u003e/Cl\u003csup\u003e\u0026minus;\u0026thinsp;54\u003c/sup\u003e were used.\u003c/p\u003e \u003cp\u003eThe systems (Table S7) were subjected to energy minimization with the positional restraints on non-hydrogen atoms of protein molecules, GTP, and Mg\u003csup\u003e2+\u003c/sup\u003e, gradually relaxed to the unrestrained condition. Subsequently, temperature annealing protocol was applied to the system from 0 K to 300 K for 10,000 steps with a time step of 2 fs under NVT ensemble with the velocity-rescale thermostat \u003csup\u003e55\u003c/sup\u003e as temperature coupling with a coupling constant (t\u003csub\u003et\u003c/sub\u003e) of 2 ps. After that, NVT equilibration was performed for total 20,000 steps with a time step of 2 fs under the same condition, gradually relaxing restraints on heavy atoms. After reaching NVT equilibrium, NPT equilibration was conducted for 500,000 steps with a time step of 2 fs without any restraints. The subsequent production runs were carried out for 1 \u0026micro;s simulation with a time step of 2 fs. Simulations, energy minimization and equilibration of the system using the GROMACS software \u003csup\u003e56\u003c/sup\u003e. The electrostatic interactions were computed by the particle mesh Ewald method under the periodic boundary conditions.\u003c/p\u003e \u003cp\u003eIn addition, analysIes, including monitoring the number of hydration bonds and the Buried Surface Area (\u003cem\u003eBSA\u003c/em\u003e) between RAB7A and C5orf51 over the simulation time, were performed using GROMACS tools. The \u003cem\u003eBSA\u003c/em\u003e was defined as follows:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:BSA=SASA\\left(\\text{R}\\text{A}\\text{B}7\\text{A}\\right)+SASA\\left(\\text{C}5\\text{o}\\text{r}\\text{f}51\\right)-SASA(\\text{R}\\text{A}\\text{B}7\\text{A}+\\text{C}5\\text{o}\\text{r}\\text{f}51),$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cem\u003eSASA\u003c/em\u003e represents solvent accessible surface area.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe coordinates and structure factors of C5orf51 in complex with RAB7A have been deposited in the Protein Data Bank under the accession code 9X2L. Other data are available from the corresponding author upon reasonable request.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using data obtained from three or more biologically independent experimental replicates. Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test was used for comparisons between two groups and Dunnett\u0026rsquo;s test was used for multiple comparisons using GraphPad Prism (n.s., not significant; *, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDeclaration of interests\u003c/h2\u003e \u003cp\u003eWe declare no competing financial interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eWe thank the beamline staff of BL45XU in SPring-8 (Hyogo, Japan) for technical help during data collection. We are also grateful to Dr. Kohei Nishino (Tokushima University) for his assistance with mass spectrometry analysis and to Dr. Tomohide Saio for valuable discussion. This work was supported by JSPS KAKENHI (21K15084, 24H01894 to K.O., 18H05501 to S.F., 22K15045 to W.K., 23K23841, 23H04923 to K.Y., 25K02267 to M. F., 21H03551, 25K03224 to K.I., 19H05712 to N.M.), Medical Research Center Initiative for High Depth Omics to H.K., Research Support Project for Life Science and Drug Discovery (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under Grant Number JP25ama121028 (support number 5738) to K.T., and JP25ama121029j0004 to K.I., AMED CREST Grant JP24gm1410004 and Nanken-Kyoten Foundation in Science Tokyo, and Medical Research Center Initiative for High Depth Omics to N.M., and AMED PRIME Grant JP25gm6910030h0001 to K.Y.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHomma, Y., Hiragi, S., and Fukuda, M. (2021). Rab family of small GTPases: an updated view on their regulation and functions. 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GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX \u003cem\u003e1-2\u003c/em\u003e, 19\u0026ndash;25. https://doi.org/10.1016/j.softx.2015.06.001.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8547387/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8547387/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRAB GTPases are essential molecular switches governing eukaryotic vesicular trafficking by cycling between active GTP-bound and inactive GDP-bound states. While conformational changes facilitate their function, exposure of the hydrophobic switch regions presents a risk, potentially leading to toxic aggregation and degradation, a process increasingly recognized as RAB quality control. In this study, we identified RAB7HD/C5orf51 as a novel RAB7A partner that plays a crucial role in RAB quality control. C5orf51 strongly binds to newly translated, guanine nucleotide-free (apo) RAB7A and acts as a specialized chaperone to maintain its solubility, thereby preventing aggregation. Furthermore, we determined the crystal structure of the C5orf51-apo RAB7A complex at 3.36 \u0026Aring; resolution, revealing a specific RAB7A intermediate. This structural and functional evidence provides new molecular insights into the initial regulation and quality control of RAB proteins, ensuring their proper folding and availability for vesicular trafficking.\u003c/p\u003e","manuscriptTitle":"RAB7HD/C5orf51 stabilizes newly synthesized RAB7A and facilitates its GTP loading","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-22 11:12:45","doi":"10.21203/rs.3.rs-8547387/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d8745846-62ab-478c-be2b-fa2441c87d27","owner":[],"postedDate":"January 22nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":61516675,"name":"Biological sciences/Cell biology/Membrane trafficking/Small GTPases"},{"id":61516676,"name":"Biological sciences/Molecular biology/Protein folding/Chaperones"}],"tags":[],"updatedAt":"2026-01-27T19:51:01+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-22 11:12:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8547387","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8547387","identity":"rs-8547387","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}