The Ykt6-Snap29-Syx13 SNARE complex promotes crinophagy via secretory granule fusion with Lamp1 carrier vesicles

The Ykt6-Snap29-Syx13 SNARE complex promotes crinophagy via secretory granule fusion with Lamp1 carrier vesicles | 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 Research Article The Ykt6-Snap29-Syx13 SNARE complex promotes crinophagy via secretory granule fusion with Lamp1 carrier vesicles Győző Szenci, Gábor Glatz, Szabolcs Takáts, Gábor Juhász This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3917956/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Feb, 2024 Read the published version in Scientific Reports → Version 1 posted You are reading this latest preprint version Abstract In the Drosophila larval salivary gland, developmentally programmed fusions between lysosomes and secretory granules (SGs) and their subsequent acidification promote the maturation of SGs that are secreted shortly before puparium formation. Subsequently, ongoing fusions between non-secreted SGs and lysosomes give rise to degradative crinosomes, where the superfluous secretory material is degraded. Lysosomal fusions control both the quality and quantity of SGs, however, its molecular mechanism is incompletely characterized. Here we identify the R-SNARE Ykt6 as a novel regulator of crinosome formation, but not the acidification of maturing SGs. We show that Ykt6 localizes to Lamp1 + carrier vesicles, and forms a SNARE complex with Syntaxin 13 and Snap29 to mediate fusion with SGs. These Lamp1 carriers represent a distinct vesicle population that are functionally different from canonical Arl8+, Cathepsin L + lysosomes, which also fuse with maturing SGs but are controlled by another SNARE complex composed of Syntaxin 13, Snap29 and Vamp7. Ykt6- and Vamp7-mediated vesicle fusions also determine the fate of SGs, as loss of either of these SNAREs prevents crinosomes from acquiring endosomal PI3P. Our results highlight that fusion events between SGs and different lysosome-related vesicle populations are critical for fine regulation of the maturation and crinophagic degradation of SGs. General Cell Biology & Physiology lysosome secretory granule crinophagy SNARE Ykt6 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Professional secretory cells produce large amounts of secretory material (hormones, neuropeptides, digestive enzymes, mucin, etc.) and store them in secretory granules (SGs) until a secretagogue elicits their bulk exocytosis. These cells usually produce more secretory material than is released by exocytosis 1 – 3 to provide a sufficient pool of available SGs 4 . Secretory cells continuously turn over the excess SGs by crinophagy, a specialized form of autophagy to maintain a constant releasable pool of SGs 2 , 5 – 7 . Following this route, abnormal or obsolete SGs may also be subject to crinophagic degradation 3 , 7 , 8 . In addition to degradative crinophagy, SG-lysosome fusions may also contribute to the complex maturation process of SGs and thereby determine their controlled release by exocytosis. During crinophagy, SGs directly fuse with lysosomes that gives rise to degradative crinosomes 9 . Easy genetic manipulation and highly conserved molecular mechanisms made Drosophila a powerful in-vivo model for deciphering the molecular regulation of the regulated secretory pathway and crinophagy. Salivary gland cells produce 10 , 11 and secrete 12 high amounts of Sgs (Salivary gland secretion)/glue proteins in response to peaks of the molting hormone ecdysone 10 – 12 . The released glue is then expelled from the lumen to anchor the metamorphosing prepupae to solid surfaces 12 . The nascent glue SGs emanate from the TGN 13 , 14 , increase in size by homotypic fusions 15 , 16 , and then undergo a complex maturation process during which SGs fuse with lysosomes. This promotes the acidification and profound reorganization of the inner content of SGs 3 , 17 – 19 , preparing them for secretion 17 , 19 , 20 . Excess or abnormal glue can be also degraded by crinophagy, through fusion of non-secreted SGs and lysosomes 3 , 7 , 8 , 19 , 21 , 22 . Taken together, crosstalk and fusion between SGs and the endolysosomal compartment is critical both for SG maturation and crinosome formation, however, the molecular mechanism of these processes is still incompletely understood 3 , 18 , 19 , 21 – 23 . By enabling direct fusion between SGs and lysosomes, crinophagy differs mechanistically from the canonical main autophagic pathway, which mediates the degradation of cytosolic material through autophagosome formation and their subsequent fusion with lysosomes. Accordingly, genes that are required for autophagosome formation proved dispensable to crinophagy 3 , 24 , 25 , while SG-lysosome fusion itself relies on a similar molecular machinery acting in fusions between autophagosomes and lysosomes 3 , 5 , 21 , 22 . The machinery mediating autophagosome-lysosome fusion is well characterized both in Drosophila and humans by now. Critical components include Rab2, Rab7, and Arl8 small GTPases that also contribute to defining membrane identity 21 , 26 – 29 , homotypic fusion and vacuole protein sorting (HOPS) tethering complex 30 , 31 , and a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex (SNAREpin) that executes the fusion. Based on biochemical properties, functional SNAREpins assemble from three Q- (Q abc ) and one R-SNARE domains 32 . The first discovered SNAREpin that mediates autophagosome-lysosome fusion is composed of Syntaxin 17, Snap29 and Vamp7/8 30,31,33,34 . Recently another R-SNARE: Ykt6 was also discovered to also have a role in the process, either as an R-SNARE potentially substituting for Vamp7 35 or interacting with Syntaxin 7 and Snap29 to form an alternative SNAREpin 36 , 37 . Interestingly, Drosophila crinophagic fusion of glue SGs and lysosomes depends on highly similar machinery, composed of Rab2, Rab7, Arl8, HOPS and a Syntaxin 13, Snap29 and Vamp7 SNAREpin 3 , 21 , 22 . The similarity of the molecular machinery regulating these lysosomal fusions raised the possibility that Ykt6 may also regulate SG-lysosome fusions and crinophagy. Ykt6 is a highly conserved R-SNARE that consists of an N-terminal longin domain (LD), an R-SNARE domain, and a conserved C-terminal lipidation motif with the amino acid sequence CCAIM. The latter is critical for membrane association 38 – 42 because Ykt6, unlike other R-SNAREs, lacks a canonical transmembrane domain. Moreover, the lipid anchors can hide reversibly in the hydrophobic groove of the protein, which enables Ykt6 to leave membranes and form a cytosolic pool 38 – 41 . This way, it can be rapidly incorporated into various intracellular membranes on demand and form a complex with compartment-specific Q-SNAREs to promote vesicle fusion. Membrane-associated Ykt6 regulates the anterograde ER to Golgi 43 , 44 , the intra-Golgi 45 – 47 , retrograde directed Golgi to ER, and endosome to TGN transports 48 , and the release of constitutive secretory carriers 49 or exosomes 50 , 51 along the secretory pathway. In addition, it also promotes biosynthetic transport to the yeast vacuole and lysosomes in animal cells 42 , 52 , endosomal recycling 53 , and macroautophagic degradation 35 – 37 , 54 , 55 . However, the role of Ykt6 in SG-lysosome fusion and crinophagy remained unknown. Here, we show that Ykt6 forms a canonical SNAREpin with Syntaxin 13 and Snap29, which is – similarly to the already known Syntaxin 13, Snap29, Vamp7 SNAREpin – critical for crinophagic degradation. We also demonstrate that Ykt6 localizes to small Lamp1+ (carrier) vesicles and mediates their fusion with SGs, while Vamp7 regulates the fusion of SGs and Arl8 + lysosomes. In summary, we provide evidence that SG maturation preceding exocytosis and crinophagy requires a series of fusions between SGs and two separate lysosome-related vesicle subpopulations, which are governed by different SNAREpins/SNARE complexes. Results Ykt6 is required for crinophagic degradation The crinophagic SG-lysosome and the conventional autophagosome-lysosome fusions share key regulators 7 . Since the possible role of Ykt6 as an alternative R-SNARE in SG-lysosome fusion has not been investigated so far, we analyzed the putative role of Ykt6 in crinophagic SG degradation. We carried out loss of function experiments by silencing ykt6 in prepupal (pp) salivary gland cells and assayed its effect on crinophagic flux. The acidification and lysosomal degradation of glue SGs can be monitored by simultaneous expression of the N-terminal GFP- and dsRed-tagged Sgs3 glue protein in the larval salivary glands. The differently labeled Sgs3 reporters are both in the lumen of forming SG, so these are initially positive for both fluorophores. Their fusion with acidic lysosomes results in quenching the GFP signal due to the acidic environment. Therefore, at the time of puparium formation, most of the SGs that are not secreted remain positive only for dsRed 3 . To investigate the consequence of Ykt6 loss, we used time-controlled RNA interference (RNAi)-mediated knock-down of the protein 53 , to circumvent its possible undesirable effect on the biogenesis of SGs. In control cells, most of the SGs appear dsRed-only as the Sgs3-GFP signal is quenched in acidic milieu (Fig. 1a, d). In contrast, many dsRed and GFP double-positive SGs remain in ykt6 silenced cells (Fig. 1b-d), indicating defective crinophagic SG degradation. This phenotype resembled the absence of the previously described crinophagic SNAREs, Syx13, Snap29 and Vamp7 3 . Thus, Ykt6 may mediate crinophagic SG-lysosome fusion in a similar way to Vamp7. The R-SNARE Ykt6 forms a SNARE complex with Syntaxin13 Q a and Snap29 Q bc SNAREs To test the ability of Ykt6 to form a functional SNAREpin, we examined its interactions with the previously identified crinophagic Q-SNAREs Syntaxin 13 and Snap29 3 by performing a GST pull-down assay with N-terminally GST- or MBP-tagged SNARE domains. GST-Syntaxin 13 Q a (bait) was immobilized on glutathione beads, and we observed its strong interaction with the MBP-tagged Snap29 Q bc Vamp7 as well as Ykt6 R-SNARE (prey) domains (Fig. 1e). Importantly, the binding of recombinant ykt6 to Syx13/Snap29 was much stronger than to the Syx17/Snap29 autophagic SNARE complex 35 . This finding indicates that Ykt6 regulates crinophagy by forming a SNAREpin with the crinophagic Q-SNAREs. Acidification of maturing SGs is regulated differently by Ykt6 and Vamp7 In addition to degradative crinosomes (at the prepupal stage - pp), ecdysone-induced progressive acidification also accompanies the maturation of SGs (2h before puparium formation - bpf), and is important for the remodeling of the inner structure of SGs and prepares them for exocytosis (priming) 3 , 17 – 19 , 56 , 57 . Although both Vamp7 and Ykt6 proved to be required for the acidification of degradative crinosomes at the prepupal (pp) stage, it was still elusive whether these two R-SNAREs are also equally required for proper acidification of maturing SGs before their release. To assay this, we stained the genomic Sgs3-GFP expressing salivary glands of 2h bpf larvae with LysoTracker Red (LTR), a vital dye that labels acidic structures (Fig. 2). In control cells around the time of robust secretion, mature SGs had already lost their GFP fluorescence, accompanied by a parallel accumulation of large LTR + vesicles (Fig. 2a, c). In the absence of ykt6 , no statistically significant difference was observed in the size of LTR + structures compared to the control cells (Fig. 2a, b, e) even though the Glue-GFP signal was already higher compared to the control (Fig. 2a, b), similar to the crinophagic flux experiments (Fig. 1a-c). In contrast, the lack of Vamp7 strongly reduced the size of LTR + acidic structures compared to the respective control (Fig. 2c, d, f). These findings raised the possibility that the two R-SNAREs required for crinophagy play different roles in the acidification and maturation of Glue SGs. Ykt6 and Vamp7 differently regulate the fusion of Arl8 + lysosomes with SGs The maturation and crinophagic decomposition of glue SGs both rely on a series of fusion events between SGs and lysosomes or endosomes 3 , 8 , 18 , 19 , 21 , 22 . Since Vamp7 and Ykt6 differentially affected SG maturation/acidification, we supposed that these R-SNAREs may mediate the fusion of SGs with different components of the endo-lysosomal compartment. Therefore, we tested the colocalization between glue granules and different endo-lysosomal markers in ykt6 RNAi or vamp7 RNAi salivary glands, respectively. Arl8 is a small GTPase highly specific for lysosomes and it is necessary for direct fusion of Lamp1 + lysosomes and glue SGs 21 . In control cells, endogenous Arl8 forms rings around Sgs3-dsRed + SGs (Fig. 3a, c) that indicates successful fusions between Arl8 + lysosomes and maturing SGs. The absence of ykt6 does not interfere with fusion of Arl8 + lysosomes, as Arl8 still forms rings around SGs (Fig. 3b, i). In contrast, silencing of vamp7 strongly inhibits the formation of rings around SGs: instead, Arl8 labels small vesicle aggregates (Fig. 3d, j). This reflects a strong defect in Arl8 + lysosome-SG fusions (Fig. 3c, d, j) and suggests that this fusion event is mediated by the SNAREpin containing Vamp7. Ykt6 is involved in the fusion of Lamp1 + vesicles with SGs Lamp1 is a highly glycosylated transmembrane protein, an essential component of the lysosomal membranes 58 . Although Lamp1 is often used as a lysosome marker, it is also present on a broader spectrum of vesicles belonging to the endo-lysosomal compartment 27 , 59 . Similar to our observations with Arl8, endogenous Lamp1 also forms rings along the perimeter of maturing Sgs3-dsRed SGs in control cells 2h bpf (Fig. 3e, g). However, the formation of these rings was strongly perturbed both in ykt6 (Fig. 3f) and vamp7 (Fig. 3h) silenced salivary gland cells (Fig. 3e-h, k, l). Thus, these lysosome markers are delivered to maturing SGs through independent fusion events governed by different R-SNAREs. Our data point to the involvement of a heterogeneous population of Arl8 + and Lamp1 + lysosomes and related vesicles in maturation and crinophagic degradation of glue SGs. Ykt6 and Vamp7 are required for endosomal fusions of SGs following secretion It was previously described that the maturation of SGs requires contribution from the endosomal system 60 – 63 , but it remains unclear whether the fusion of endosomes with SGs is important for crinosome formation. To explore this, we labeled endosomes harboring phosphatidylinositol 3-phosphate (PI3P) with GFP-Myc-2xFYVE probe specific for PI3P and tested its overlap with Glue-dsRed. We observed that GFP-FYVE marks usually small endosome clusters among the SGs at 2h bpf (Fig. 4a.) 60 – 62 . Later on, non-secreted SGs trapped in cytosol are most likely removed by crinophagy by transforming into crinosomes 3 , 19 which appear as large Sgs3-dsRed + granules encircled by GFP-FYVE + membranes (Fig. 4b, d). This suggests that crinosomes receive extensive membrane input from PI3P-positive endosomes. However, it remained unclear whether this endosomal input was dependent on the preceding fusion events between lysosomes and maturing SGs. We observed that in the lack of Ykt6, these PI3P + endosomes are clustered between SGs, rather than forming a ring around them (Fig. 4b, c, f). Similarly, the absence of Vamp7 strongly inhibited the fusion of PI3P + endosomes and SGs compared to the respective control (Fig. 4d, e, g). These results show that the Ykt6- and Vamp7-mediated lysosomal fusions determine the subsequent fate and fusion capacity of the SGs because they fail to fuse with PI3P + endosomes in the absence of either SNARE. Ykt6 localizes to small Lamp1 + vesicles Since we found that Ykt6 and Vamp7 mediate the fusion of maturing SGs with different lysosome-related vesicle populations, we also aimed to elucidate the subcellular localization of Ykt6. By carrying out immunolabeling with antibodies specific for Ykt6 and various lysosomal markers, we found that endogenous Ykt6 shows a punctate pattern which overlaps significantly with small Lamp1 + vesicles, while it is absent from the large Lamp1 + rings that presumably formed around maturing SGs (Fig. 5a, d). Ykt6 does not colocalize with other lysosome markers, such as Arl8 (Fig. 5b, d) or the lysosomal hydrolase Cathepsin L (Fig. 5c, d). These data are in line with our results that Ykt6 is mainly involved in the fusion of SGs and Lamp1 + vesicles, but not Arl8 + lysosomes (Fig. 3). Ykt6 does not affect the localization of Vamp7 We also wondered whether the two SNAREpins that mediate SG-lysosome fusions indeed function independently. Therefore, we investigated the localization of Vamp7 by using N-terminal GFP-tagged Vamp7 in the absence of the other R-SNARE, Ykt6. The loss of Ykt6 has not altered the localization pattern of Vamp7, as it is still able to form rings around larger SGs (Fig. 6a, b). This further suggests that the Ykt6- and Vamp7-containing SNAREpins independently regulate the maturation and crinophagic degradation of SGs by mediating fusion between SGs and Arl8+ (by Vamp7) or Arl8- but Lamp1+ (Vamp7, Ykt6) lysosome subpopulations. Discussion In this work, we revealed that Ykt6 acts together with Syntaxin 13 and Snap29 to form a functional SNAREpin. This SNARE complex is required for efficient SG fusion with Lamp1 carrier vesicles, thereby promoting the maturation and crinophagic elimination of SGs. The first vesicle fusions occur just before the bulk secretion of SGs. These early fusions may drive the acidification and inner reorganization of SGs to promote SG maturation 17 – 19 . In line with this, SG-lysosome fusion is claimed to cause enhanced secretion in Trpml1-/- mutant pancreatic acinar cells 20 . Our data indicate that the two R-SNAREs Ykt6 and Vamp7 play different roles in regulating the maturation of SGs, because SGs fail to acidify properly without Vamp7, while the silencing of ykt6 did not prevent this. Moreover, we found that Vamp7 is required for the localization multiple lysosomal markers to maturing glue granules, while Ykt6 only affects the fusion of SGs with Lamp1 + vesicles and it is dispensable for fusion with Arl8 + lysosomes. We hypothesize that maturing SGs first undergo Vamp7-mediated fusion with Arl8 + lysosomes, which is required for their maturation. The Ykt6-mediated fusions between SGs and Lamp1 + vesicles likely represent a later step of SG maturation. Although our findings suggest the existence of at least two separate vesicle subpopulations carrying these lysosomal markers (Arl8 + ones and Lamp1+/Arl8- ones), Arl8 + lysosomes possibly also contain Lamp1. The coexistence and sequential contribution of multiple lysosomal subpopulations/Lamp1 carriers in distinct steps of SG maturation could be an advantage for secretory cells. Different vesicle subpopulations can act as carriers that deliver different lysosomal membrane proteins and enzymes that are required for lysosome biogenesis. The volume of SGs is enormous compared to these small vesicles, hence the desired concentration of lysosomal proteins in matured SGs or crinosomes could be fine-tuned by a series of membrane fusions with different lysosomal populations. This model is further supported by findings by others, showing that Vamp7 is required for the transport of lysosomal membrane proteins (LMPs, including Lamp1) 64 or the potential role of Ykt6 in lysosomal enzyme transport 42 , 52 . Since we found that the vesicles to which Ykt6 localizes are positive only for Lamp1, but negative for Arl8 and the lysosomal protease Cathepsin L, Ykt6 appears to be required for the delivery of lysosomal membrane proteins such as Lamp1 itself to glue granules. As the highly glycosylated Lamp1 is essential for protecting the lysosomal membrane from acidic internal pH and enzymatic degradation 58 , the Ykt6-mediated delivery of these Lamp1 carrier vesicles to mature SGs could prepare them for the degradative crinosomal fate. We have also demonstrated the importance of endosomal contribution to crinosome formation. PI3P + endosomes are much smaller than SGs and initially form clusters among SGs before secretion 60 – 62 , eventually fusing with the residual, non-secreted mature SGs. One can assume that these endosomal fusions prepare the obsolete SGs for crinophagic breakdown, possibly through the recruitment of Rab7, which is implicated in crinophagic SG-lysosome fusion 3 , 5 . Importantly, we found that these fusions are equally hampered in the absence of either Ykt6 or Vamp7. Thus, the early Ykt6- and Vamp7-mediated vesicle fusions determine the subsequent fate and fusion potential of maturing SGs. Small PI3P + endosomes that fuse with residual SGs are likely derived from endocytic activity that follows the programmed secretion of SGs. The convergence of secretory, endosomal, and even autophagic routes in lysosomes was also demonstrated in larval Drosophila fat tissue 65 . Overall, our results refine the model of glue granule maturation and lysosome fusions: SGs probably first acquire the lysosomal small GTPase Arl8 and begin to acidify via fusion by the canonical Vamp7 containing SNAREpin. This primary fusion event engages maturing SGs for subsequent volume-increasing lysosomal fusions that already involve Lamp1 + lysosomes. Ykt6 reaches SGs by forming a SNAREpin with Syntaxin 13 Q a - and Snap29 Q bc -SNAREs to mediate SG-Lamp1 carrier vesicle fusion, and these separate fusion events together promote the maturation and crinophagic degradation of residual glue granules after secretion (Fig. 6c). Methods Drosophila genetics Fly stocks were maintained on standard yeast-cornmeal-agar medium at 25°C temperature. To avoid undesirable effects of Ykt6 on SG biogenesis, temperature sensitive tubP-Gal80 construct was used to temporally control the expression of ykt6 RNAi transgenes 53 . These crosses were shifted from the 18°C restrictive temperature to 29°C for 36 hours at the late (wandering) L3 stage. Accordingly, separate controls were used for room temperature vamp7 and temperature-induced ykt6 RNAi experiments. The w 1118 (#3605), fkh-Gal4 (#78060), tubP-Gal80 ts (#7017 and #7108), Sgs3-GFP (#5884) and the UAS-GFP-myc-2xFYVE (#42712) lines were obtained from Bloomington Drosophila Stock Center. The UAS-Ykt6 NIG.1515R (#1515R-1) (ykt6 RNAi/1 in the text) RNAi line was obtained from NIG-Fly (National Institute of Genetics) 35 . The UAS-Ykt6 KK101343 (#v105648) (ykt6 RNAi/2 in the text) and UAS-Vamp7 KK107576 (#v108733) RNAi stocks were purchased from Vienna Drosophila Resource Center. The Sgs3-dsRed line was kindly provided by Andrew Andres (University of Nevada, US). Sgs3-GFP; fkh-Gal4, the Sgs3-dsRed; fkh-Gal4, the Sgs3-dsRed, UAS-GFP-myc-2xFYVE; fkh-Gal4 and the Sgs3-dsRed, Sgs3-GFP; fkh-Gal4 lines were used to study the endo-lysosomal transport to secretory granules or secretory granule acidification. For our experiments we used late L3 staged larvae that had already completed their wandering (considered as 2 hours before puparium formation (2h bpf)) or white prepupae (pp). LysoTracker Red (LTR) staining The larval salivary glands were dissected in cold PBS (pH = 7.4) and permeabilized for 30 s (2h bpf) or 15 s (pp) in 0.05% Triton X-100-PBS (PBTX) solution. The samples were rinsed in PBS (3x30 sec) and incubated for 2 min in 0.5 nM LTR (in PBS, Invitrogen) staining solution, then washed in PBS and mounted with 9:1 PBS: glycerol solution that contains 1 µg/mL DAPI (4′,6-diamidino-2-phenylindole, Sigma Aldrich) to stain the nuclei of cells. Immunohistochemistry The larval salivary glands were dissected in cold PBS, gently permeabilized with 0.05% PBTX solution either for 30 s (2h bpf) or 15s (prepupae) and fixed in 4% formaldehyde-PBS (40 min, RT). Then, the samples were rinsed with PBS (3x5 min, RT), incubated in a blocking solution (5% fetal calf serum in 0.1% PBTX, 30 min, RT), and incubated with the first antibodies dissolved in the blocking solution (ON, 4°C). After washing (3x15 min PBTX), salivary glands were incubated in blocking solution (30 min, RT), then with the secondary antibodies diluted in blocking solution (3 hr, RT). Thereafter samples were incubated in 4% NaCl solution (15min, RT) that was supplemented with Hoechst (1:200, Sigma-Aldrich) nuclear dye and washed (2x15 min in 0.1% PBTX, 3x15 min in PBS). The specimens were dissected and mounted in Vectashield (Vector Laboratories). For the salivary gland immunostainings rabbit anti-Arl8 (1:100, DSHB) 21 , rabbit anti-dLamp1 (1:1000, kind gift of Andreas Jenny) 58 , rat anti-Ykt6 (1:30) 35 , rat anti-mCherry (1:300) and rabbit anti-CathL/MEP (1:100, Abcam, #ab58991) 31 , 35 primary and the AlexaFluor488-conjugated anti-mouse, anti-rabbit, anti-goat and AlexaFluor568-conjugated anti-mouse, anti-rabbit and anti-rat secondary antibodies (all 1:1000, Invitrogen) were used. Fluorescent imaging Fluorescent images were taken at room temperature with an AxioImager M2 microscope (Zeiss) equipped with an ApoTome.2 structured illumination unit, Orca-Flash 4.0 LT3 digital sCMOS camera (Hamamatsu Photonics), EC Plan-Neofluar 20x/0.50, Plan-Apochromat 40x/0.95 and Plan-Apochromat 63x/1.4 Oil objectives (Zeiss). Raw images were processed with ZEN2.3 lite Microscopy Software and Photoshop CS4 (Adobe Systems). To improve clarity in Fig. 2, Fig. 3, Fig. 4 and Fig. 6 consecutive optical slices spanning a depth of 3 µm were projected onto single images. Single focal planes were presented in other figures, including colocalization tests in Fig. 1 and Fig. 5. GST pulldown assay SNARE fragments were cloned into pETARA or/and pETMBP vectors, which contain C-terminal Glutathione S-transferase/Maltose Binding Protein tag and C-terminal hexahistidine-tag, respectively, using BamHI and XhoI restriction sites. Syx13 was amplified from the EST LD27581 (DGRC Stock 4205 ; https://dgrc.bio.indiana.edu//stock/4205 ; RRID:DGRC_4205) with primers 5’-ATCGGATCCCACGACATGCTCGAC-3’ and 5’-ATCCTCGAGCGCCTTGGCCAGTTC-3’. The remaining constructs were already reported in an earlier study 35 . For pulldown experiments, recombinant SNARE constructs were expressed overnight at 18°C in E. coli Rosetta(DE3) pLysS (Novagen) cells induced with 0.1 mM IPTG at OD 0.6–0.7. Cells were then centrifuged and suspended in lysis buffer (pH 8.0, 50 mM Na 2 HPO 4 , 300 mM NaCl, 20mM imidazole, 0.1% Triton-X, 5 mM-β-mercaptoethanol, protease inhibitors). Lysed samples were centrifuged (48.000g, 30 min). Ni-NTA resin was added to the supernatant and incubated for 30 minutes at 4°C. Beads were washed with washing buffer (pH 8.0, 50 mM Na 2 HPO 4 , 1 M NaCl, 40mM imidazole, 0,1% Triton-X, 5 mM β-mercaptoethanol) and then eluated in Elution buffer (pH 8.0, 20 mM Tris, 200 mM NaCl, 400 mM imidazole, 10% glicerol, 0,1% Triton-X, 5 mM-β-mercaptoethanol) used for pulldown assays. Prey proteins for pulldown experiments were purified with further MBP affinity chromatography using standard protocols. All resins were from GE Healthcare. For GST pulldown assays, the glutathione resin (New England BioLabs) was first equilibrated with binding buffer (20 mM Tris, 50 mM NaCl, 0.1% Triton-X, 2 mM β–β-mercaptoethanol), then 0.5 mg GST fused SNARE proteins (and GST as negative control) were immobilized on it. In the binding experiments, 40 µl of resin saturated with baits were incubated in the presence of 20 µM preys in binding buffer (200 µl total volume, 30 min at 4°C). Glutathione beads were pelleted with centrifugation (200 g, 2 min) and washed 3x with 20 mM Tris, 300 mM NaCl, 0.1% Triton-X, 2 mM β–mercaptoethanol. Retained proteins were eluted from the resin with an SDS loading buffer. Samples were subjected to SDS-PAGE and interactions were detected by Coomassie protein dye. The original gel image is provided in the Supplementary Information (Supplementary Fig. S1). Statistics ImageJ software (National Institutes of Health, Bethesda, Maryland, US) was used for quantitative analysis of fluorescent structures. Overlap of the markers was assessed by Pearson’s correlation analysis using the Coloc2 plugin (Fig. 1) or in the case of membrane markers encircling granular structures (Fig. 5), the signal was calculated manually. For manual colocalization assessment, 200 immunolabeled structures were selected. The threshold for LTR quantification was set by the same person in a dark room in all images and structures were counted. The structure diameter range was set to 1–99 µm 2 to exclude background noise and unrealistic clumped structures of several SG sizes (Fig. 2). The ring-like fluorescent structures in Fig. 3 and Fig. 4 were selected manually by the same person and the percentage of them located around SGs was examined. For pairwise comparisons of datasets that followed Gaussian distribution unpaired t-test (Fig. 3i, j, Fig. 4f, g) or where at least one of the datasets followed non-Gaussian distribution, Mann-Whitney U test (Fig. 2, Fig. 3k, l) were performed. To analyze multiple datasets with Gaussian distribution, one-way ANOVA with Tukey’s post hoc test was performed (Fig. 1d). The distribution tests of datasets and statistical analyses were carried out using GraphPadPrism 9.0.0 software (Boston, Massachusetts, US). All source data about the quantifications related to the presented experiments are available as Supplementary Information (Supplementary Table S1). Declarations Acknowledgments We thank Sarolta Pálfia, Dávid Hargitai, Dorottya Károlyi and Fanni Ősz for their assistance in the maintenance of the fly lines and Zsófia Gyetvai for helpful discussions and Győző Szenci for drawing the summary figure. Funding This research was funded by the National Research, Development and Innovation Office of Hungary (Elvonal KKP129797 to GJ, HunProtExc 2018-1.2.1-NKP-2018–00005 to GJ, OTKA FK_142508 for ST), the Hungarian Academy of Sciences (BO/00400/23 for ST), the Excellence Fund of Eötvös Lorand University (EKA_2022/045-P302-1 for ST) and the UNKP New National Excellence Program of the Ministry of Human Capacities of Hungary (ÚNKP-23-5-ELTE-1257 for ST). Author contributions GS, ST and GJ designed the research. GS and GG performed the experiments and analyzed the data. GS, GG, ST and GJ wrote the manuscript. The research was equally supervised by ST and GJ. Conflict of interest The authors declare that no conflict of interest exists. Data availability All data needed to evaluate the conclusions in this paper are present in the paper and its Supplementary Information. Other data associated with the article, such as raw data, are available upon request. Materials availability All materials, Drosophila stocks and related information are available from the corresponding authors upon reasonable request. References Halban, P. A. & Wollheim, C. B. Intracellular degradation of insulin stores by rat pancreatic islets in vitro. An alternative pathway for homeostasis of pancreatic insulin content. Journal of Biological Chemistry 255 , 6003-6006 (1980). https://doi.org/10.1016/s0021-9258(18)43686-1 Schnell, A. H., Swenne, I. & Borg, L. A. H. Lysosomes and pancreatic islet function - A quantitative estimation of crinophagy in the mouse pancreatic B-cell. Cell and Tissue Research 252 , 9-15 (1988). https://doi.org/10.1007/BF00213820 Csizmadia, T. et al. Molecular mechanisms of developmentally programmed crinophagy in Drosophila . J Cell Biol 217 , 361-374 (2018). https://doi.org/10.1083/jcb.201702145 Kim, T., Gondré-Lewis, M. C., Arnaoutova, I. & Loh, Y. P. Dense-core secretory granule biogenesis. Physiology (Bethesda) 21 , 124-133 (2006). https://doi.org/10.1152/physiol.00043.2005 Zhou, Y. et al. RILP restricts insulin secretion through mediating lysosomal degradation of proinsulin. Diabetes 69 , 67-82 (2020). https://doi.org/10.2337/db19-0086 Li, M. et al. VAMP4 regulates insulin levels by targeting secretory granules to lysosomes. Journal of Cell Biology 221 , e202110164 (2022). https://doi.org/10.1083/jcb.202110164 Szenci, G., Csizmadia, T. & Juhász, G. The role of crinophagy in quality control of the regulated secretory pathway. J Cell Sci 136 , jcs260741 (2023). https://doi.org/10.1242/jcs.260741 Csizmadia, T. et al. Developmental program-independent secretory granule degradation in larval salivary gland cells of Drosophila. Traffic 13 , 568-586 (2022). https://doi.org/10.1111/tra.12871 Glaumann, H. et al. Isolation and characterization of crinosomes--a subclass of secondary lysosomes. Exp Mol Pathol 50 , 167-182 (1989). https://doi.org/10.1016/0014-4800(89)90028-2 Costantino, B. F. et al. A novel ecdysone receptor mediates steroid-regulated developmental events during the mid-third instar of Drosophila. PLoS Genet 4 , e1000102 (2008). https://doi.org/10.1371/journal.pgen.1000102 Kaieda, Y. et al. Glue protein production can be triggered by steroid hormone signaling independent of the developmental program in Drosophila melanogaster. Dev Biol 430 , 166-176 (2017). https://doi.org/10.1016/j.ydbio.2017.08.002 Biyasheva, A., Do, T. V., Lu, Y., Vaskova, M. & Andres, A. J. Glue secretion in the Drosophila salivary gland: a model for steroid-regulated exocytosis. Dev Biol 231 , 234-251 (2001). https://doi.org/10.1006/dbio.2000.0126 Torres, I. L., Rosa-Ferreira, C. & Munro, S. The Arf family G protein Arl1 is required for secretory granule biogenesis in Drosophila. J Cell Sci 127 , 2151-2160 (2014). https://doi.org/10.1242/jcs.122028 Burgess, J. et al. AP-1 and clathrin are essential for secretory granule biogenesis in Drosophila. Mol Biol Cell 22 , 2094-2105 (2011). https://doi.org/10.1091/mbc.E11-01-0054 Neuman, S. D., Lee, A. R., Selegue, J. E., Cavanagh, A. T. & Bashirullah, A. A novel function for Rab1 and Rab11 during secretory granule maturation. J Cell Sci 134 (2021). https://doi.org/10.1242/jcs.259037 Niemeyer, B. A. & Schwarz, T. L. SNAP-24, a Drosophila SNAP-25 homologue on granule membranes, is a putative mediator of secretion and granule-granule fusion in salivary glands. J Cell Sci 113 , 4055-4064 (2000). https://doi.org/10.1242/jcs.113.22.4055 Syed, Z. A., Zhang, L., Tran, D. T., Bleck, C. K. E. & Ten Hagen, K. G. Regulated Restructuring of Mucins During Secretory Granule Maturation In Vivo. Proc Natl Acad Sci U S A 119 , e2209750119 (2022). https://doi.org/10.1073/pnas.2209750119 Nagy, A. et al. Ecdysone receptor isoform specific regulation of secretory granule acidification in the larval Drosophila salivary gland. Eur J Cell Biol 101 , 151279 (2022). https://doi.org/10.1016/j.ejcb.2022.151279 Boda, A. et al. Rab26 controls secretory granule maturation and breakdown in Drosophila. Cell Mol Life Sci 80 , 24 (2023). https://doi.org/10.1007/s00018-022-04674-8 Park, S. et al. Fusion of lysosomes with secretory organelles leads to uncontrolled exocytosis in the lysosomal storage disease mucolipidosis type IV. EMBO Rep 17 , 266-278 (2016). https://doi.org/10.15252/embr.201541542 Boda, A. et al. Drosophila Arl8 is a general positive regulator of lysosomal fusion events. Biochimica et Biophysica Acta - Molecular Cell Research 1866 , 533-544 (2019). https://doi.org/10.1016/j.bbamcr.2018.12.011 Lőrincz, P. et al. Vps8 overexpression inhibits HOPS-dependent trafficking routes by outcompeting Vps41/Lt. eLife 8 , 1-25 (2019). https://doi.org/10.7554/eLife.45631.001 Ma, C. I. J., Burgess, J. & Brill, J. A. Maturing secretory granules: Where secretory and endocytic pathways converge. Advances in Biological Regulation 80 , 100807-100807 (2021). https://doi.org/10.1016/j.jbior.2021.100807 Pasquier, A. et al. Lysosomal degradation of newly formed insulin granules contributes to β cell failure in diabetes. Nature Communications 10 , 1-14 (2019). https://doi.org/10.1038/s41467-019-11170-4 Kanai, A. et al. Genome-wide screening for regulators of degradation of insulin secretory granules with a fluorescent reporter. Biochem Biophys Res Commun 676 , 132-140 (2023). https://doi.org/10.1016/j.bbrc.2023.07.040 Gutierrez, M. G., Munafó, D. B., Berón, W. & Colombo, M. I. Rab7 is required for the normal progression of the autophagic pathway in mammalian cells. J Cell Sci 117 , 2687-2697 (2004). https://doi.org/10.1242/jcs.01114 Hegedűs, K. et al. The Ccz1-Mon1-Rab7 module and Rab5 control distinct steps of autophagy. Mol Biol Cell 27 , 3132-3142 (2016). https://doi.org/10.1091/mbc.E16-03-0205 Fujita, N. et al. Genetic screen in Drosophila muscle identifies autophagy-mediated T-tubule remodeling and a Rab2 role in autophagy. Elife 6 , e23367 (2017). https://doi.org/10.7554/eLife.23367 Lőrincz, P. et al. Rab2 promotes autophagic and endocytic lysosomal degradation. J Cell Biol 216 , 1937-1947 (2017). https://doi.org/10.1083/jcb.201611027 Jiang, P. et al. The HOPS complex mediates autophagosome-lysosome fusion through interaction with syntaxin 17. Mol Biol Cell 25 , 1327-1337 (2014). https://doi.org/10.1091/mbc.E13-08-0447 Takáts, S. et al. Interaction of the HOPS complex with Syntaxin 17 mediates autophagosome clearance in Drosophila. Mol Biol Cell 25 , 1338-1354 (2014). https://doi.org/10.1091/mbc.E13-08-0449 Mion, D., Bunel, L., Heo, P. & Pincet, F. The beginning and the end of SNARE-induced membrane fusion. FEBS Open Bio 12 , 1958-1979 (2022). https://doi.org/10.1002/2211-5463.13447 Itakura, E., Kishi-Itakura, C. & Mizushima, N. The hairpin-type tail-anchored SNARE syntaxin 17 targets to autophagosomes for fusion with endosomes/lysosomes. Cell 151 , 1256-1269 (2012). https://doi.org/10.1016/j.cell.2012.11.001 Takáts, S. et al. Autophagosomal Syntaxin17-dependent lysosomal degradation maintains neuronal function in Drosophila. J Cell Biol 201 , 531-539 (2013). https://doi.org/10.1083/jcb.201211160 Takáts, S. et al. Non-canonical role of the SNARE protein Ykt6 in autophagosome-lysosome fusion. PLoS Genet 14 , e1007359 (2018). https://doi.org/10.1371/journal.pgen.1007359 Matsui, T. et al. Autophagosomal YKT6 is required for fusion with lysosomes independently of syntaxin 17. J Cell Biol 217 , 2633-2645 (2018). https://doi.org/10.1083/jcb.201712058 Sánchez-Martín, P. et al. ULK1-mediated phosphorylation regulates the conserved role of YKT6 in autophagy. J Cell Sci 136 , jcs260546 (2023). https://doi.org/10.1242/jcs.260546 Fukasawa, M., Varlamov, O., Eng, W. S., Söllner, T. H. & Rothman, J. E. Localization and activity of the SNARE Ykt6 determined by its regulatory domain and palmitoylation. Proc Natl Acad Sci U S A 101 , 4815-4820 (2004). https://doi.org/10.1073/pnas.0401183101 Hasegawa, H., Yang, Z., Oltedal, L., Davanger, S. & Hay, J. C. Intramolecular protein-protein and protein-lipid interactions control the conformation and subcellular targeting of neuronal Ykt6. J Cell Sci 117 , 4495-4508 (2004). https://doi.org/10.1242/jcs.01314 Pylypenko, O. et al. Farnesylation of the SNARE protein Ykt6 increases its stability and helical folding. J Mol Biol 377 , 1334-1345 (2008). https://doi.org/10.1016/j.jmb.2008.01.099 Shirakawa, R. et al. A SNARE geranylgeranyltransferase essential for the organization of the Golgi apparatus. EMBO J 39 , e104120 (2020). https://doi.org/10.15252/embj.2019104120 Sakata, N., Shirakawa, R., Goto, K., Trinh, D. A. & Horiuchi, H. Double prenylation of SNARE protein Ykt6 is required for lysosomal hydrolase trafficking. J Biochem 169 , 363-370 (2021). https://doi.org/10.1093/jb/mvaa111 McNew, J. A. et al. Ykt6p, a prenylated SNARE essential for endoplasmic reticulum-Golgi transport. J Biol Chem 272 , 17776-17783 (1997). https://doi.org/10.1074/jbc.272.28.17776 Zhang, T. & Hong, W. Ykt6 forms a SNARE complex with syntaxin 5, GS28, and Bet1 and participates in a late stage in endoplasmic reticulum-Golgi transport. J Biol Chem 276 , 27480-27487 (2001). https://doi.org/10.1074/jbc.M102786200 Xu, Y., Martin, S., James, D. E. & Hong, W. GS15 forms a SNARE complex with syntaxin 5, GS28, and Ykt6 and is implicated in traffic in the early cisternae of the Golgi apparatus. Mol Biol Cell 13 , 3493-3507 (2002). https://doi.org/10.1091/mbc.e02-01-0004 Parlati, F. et al. Distinct SNARE complexes mediating membrane fusion in Golgi transport based on combinatorial specificity. Proc Natl Acad Sci U S A 99 , 5424-5429 (2002). https://doi.org/10.1073/pnas.082100899 Volchuk, A. et al. Countercurrent distribution of two distinct SNARE complexes mediating transport within the Golgi stack. Mol Biol Cell 15 , 1506-1518 (2004). https://doi.org/10.1091/mbc.e03-08-0625 Tai, G. et al. Participation of the syntaxin 5/Ykt6/GS28/GS15 SNARE complex in transport from the early/recycling endosome to the trans-Golgi network. Mol Biol Cell 15 , 4011-4022 (2004). https://doi.org/10.1091/mbc.e03-12-0876 Gordon, D. E. et al. VAMP3/Syb and YKT6 are required for the fusion of constitutive secretory carriers with the plasma membrane. PLoS Genet 13 , e1006698 (2017). https://doi.org/10.1371/journal.pgen.1006698 Sun, C. et al. LncRNA PVT1 promotes exosome secretion through YKT6, RAB7, and VAMP3 in pancreatic cancer. Aging (Albany NY) 12 , 10427-10440 (2020). https://doi.org/10.18632/aging.103268 Gross, J. C., Chaudhary, V., Bartscherer, K. & Boutros, M. Active Wnt proteins are secreted on exosomes. Nat Cell Biol 14 , 1036-1045 (2012). https://doi.org/10.1038/ncb2574 Cuddy, L. K. et al. Stress-Induced Cellular Clearance Is Mediated by the SNARE Protein ykt6 and Disrupted by α-Synuclein. Neuron 104 , 869-884.e811 (2019). https://doi.org/10.1016/j.neuron.2019.09.001 Linnemannstöns, K. et al. Ykt6-dependent endosomal recycling is required for Wnt secretion in the Drosophila wing epithelium. Development 147 , dev185421 (2020). https://doi.org/10.1242/dev.185421 Bas, L. et al. Reconstitution reveals Ykt6 as the autophagosomal SNARE in autophagosome-vacuole fusion. J Cell Biol 217 , 3656-3669 (2018). https://doi.org/10.1083/jcb.201804028 Gao, J., Reggiori, F. & Ungermann, C. A novel in vitro assay reveals SNARE topology and the role of Ykt6 in autophagosome fusion with vacuoles. J Cell Biol 217 , 3670-3682 (2018). https://doi.org/10.1083/jcb.201804039 Davidson, H. W., Rhodes, C. J. & Hutton, J. C. Intraorganellar calcium and pH control proinsulin cleavage in the pancreatic beta cell via two distinct site-specific endopeptidases. Nature 333 , 93-96 (1988). https://doi.org/10.1038/333093a0 Barg, S. et al. Priming of insulin granules for exocytosis by granular Cl(-) uptake and acidification. J Cell Sci 114 , 2145-2154 (2001). https://doi.org/10.1242/jcs.114.11.2145 Chaudhry, N. et al. Lamp1 mediates lipid transport, but is dispensable for autophagy in Drosophila . Autophagy 18 , 2443-2458 (2022). https://doi.org/10.1080/15548627.2022.2038999 Cook, N. R., Row, P. E. & Davidson, H. W. Lysosome associated membrane protein 1 (Lamp1) traffics directly from the TGN to early endosomes. Traffic 5 , 685-699 (2004). https://doi.org/10.1111/j.1600-0854.2004.00212.x Burgess, J. et al. Type II phosphatidylinositol 4-kinase regulates trafficking of secretory granule proteins in Drosophila. Development (Cambridge) 139 , 3040-3050 (2012). https://doi.org/10.1242/dev.077644 Ma, C. I. J. et al. An early endosome-derived retrograde trafficking pathway promotes secretory granule maturation. Journal of Cell Biology 219 , e201808017 (2020). https://doi.org/10.1083/jcb.201808017 Neuman, S. D., Terry, E. L., Selegue, J. E., Cavanagh, A. T. & Bashirullah, A. Mistargeting of secretory cargo in retromer-deficient cells. Dis Model Mech 14 , dmm046417 (2021). https://doi.org/10.1242/dmm.046417 Ma, C. J. & Brill, J. A. Endosomal Rab GTPases regulate secretory granule maturation in Drosophila larval salivary glands. Commun Integr Biol 14 , 15-20 (2021). https://doi.org/10.1080/19420889.2021.1874663 Pols, M. S. et al. hVps41 and VAMP7 function in direct TGN to late endosome transport of lysosomal membrane proteins. Nat Commun 4 , 1361 (2013). https://doi.org/10.1038/ncomms2360 Zhou, L. et al. Convergence of secretory, endosomal, and autophagic routes in trans-Golgi-associated lysosomes. J Cell Biol 222 , e202203045 (2023). https://doi.org/10.1083/jcb.202203045 Additional Declarations The authors declare no competing interests. Supplementary Files SupplementaryFigureS1.pdf Supplementary Figure S1. The original gel image of the GST-pulldown experiment presented in Fig. 1e. SupplementaryTableS1.xlsx Supplementary Table S1. Source data for the statistics presented on Figures Cite Share Download PDF Status: Published Journal Publication published 07 Feb, 2024 Read the published version in Scientific Reports → 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3917956","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":270496089,"identity":"9f3d2b3f-7898-4465-b17e-76f8b1e16c1f","order_by":0,"name":"Győző Szenci","email":"","orcid":"","institution":"Eötvös Loránd University","correspondingAuthor":false,"prefix":"","firstName":"Győző","middleName":"","lastName":"Szenci","suffix":""},{"id":270496090,"identity":"7f3d6f11-a90d-401e-859b-efbbc17134d3","order_by":1,"name":"Gábor Glatz","email":"","orcid":"","institution":"Eötvös Loránd University","correspondingAuthor":false,"prefix":"","firstName":"Gábor","middleName":"","lastName":"Glatz","suffix":""},{"id":270496091,"identity":"346ef58a-2db7-44bf-9f86-eea05d1a8545","order_by":2,"name":"Szabolcs Takáts","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6klEQVRIie2RMQoCMRBFJwibZsA2smKuELEVvYqy4BkEQbMI2gi2LnqIPUIksDY5wILNiq2gdhaCRhsLIYqVRR4EhjCP/2EAPJ6/RdlHJRAJULVj8GnfLipggJrEVkH8XmHRlwqfK1pcTGvIk+Nmt4AmtqkM9uhQhFIknuURE1ubkkIPERVtOBUiicRTiYnQKgVoRNYJQpfCx0Di62nEeKIfyg2RF24FMiBjzDWDvPQopmwKuBVhSLysmk0lNVE9WYgI0XQnlZWr2Fyr8yEblPl0XZxn/VaNTnXGDq5iz5O/Qt9+PB6Px/MLd7+tSHI2P5a1AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-2139-7740","institution":"Eötvös Loránd University","correspondingAuthor":true,"prefix":"","firstName":"Szabolcs","middleName":"","lastName":"Takáts","suffix":""},{"id":270496092,"identity":"f5baf9bf-a01c-4eb7-a2bb-eeccfb3ec39c","order_by":3,"name":"Gábor Juhász","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYBACxgYeKEsCiD82QBlEa2GcSYwWBgYkLcy8xGhhnpF78OOPPwx2/bObj3223XFPnkG6xwC/w2bkJUvz8DAkz7hzLHl27pliwwaZM4S05BhIA92SzHAjx5g5ty0hgUEih6AW458/DBiS5UFaLInUYibBk8BgZwDSwkiUlp43ZtY8ByQSDIF+Yew9k2DYJpFWgFeLYXuO8c0ff2zs5W43H2b4uSNBnl8ieQN+LQ1gSiKxASbChlc9EMhDaXtCCkfBKBgFo2AEAwBr/ECJY5kcxwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-8548-8874","institution":"HUN-REN Biological Research Centre Szeged","correspondingAuthor":true,"prefix":"","firstName":"Gábor","middleName":"","lastName":"Juhász","suffix":""}],"badges":[],"createdAt":"2024-02-01 16:01:40","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-3917956/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3917956/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-53607-x","type":"published","date":"2024-02-08T00:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":50544180,"identity":"53e96d5b-f609-44a2-837a-ff08434faeca","added_by":"auto","created_at":"2024-02-02 08:38:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1802816,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eYkt6 forms a SNARE complex with Syntaxin 13 Q\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e- and SNAP29 Q\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003ebc\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e SNAREs to regulate the crinophagic degradation of SGs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e In the salivary gland cells from control prepupae (pp), most Sgs3-GFP signals are quenched due to the fusion of SGs with acidic lysosomes, resulting in the appearance of dsRed-only degradative crinosomes. \u003cstrong\u003e(b-d)\u003c/strong\u003e In contrast, RNAi-mediated silencing of \u003cem\u003eykt6\u003c/em\u003e by two independent RNAi transgenes leads to defective crinophagic degradation based on retained Sgs3-GFP signal. \u003cstrong\u003e(d) \u003c/strong\u003eQuantification of the overlap between Sgs3-GFP and Sgs3-dsRed markers from \u003cstrong\u003e(a-c)\u003c/strong\u003e, n=40 cells from 8 different larvae. Dashed lines mark the median and the dotted lines are the upper and lower quartiles of violin plots. ** p\u0026lt;0.01, **** p\u0026lt;0.0001, ns p\u0026gt;0.05. \u003cstrong\u003e(e)\u003c/strong\u003e GST pull-down experiment with N-terminally GST- or MBP-tagged recombinant SNARE domains purified from \u003cem\u003eE. coli\u003c/em\u003e. GST alone served as a negative control. The immobilized GST-Syntaxin13 Q\u003csub\u003ea\u003c/sub\u003e bait strongly interacts with MBP-tagged Snap29 Q\u003csub\u003ebc\u003c/sub\u003e-, and Vamp7 or Ykt6 R-SNARE prey motifs. This suggests that Ykt6 can assemble with the Syntaxin 13 Q\u003csub\u003ea\u003c/sub\u003e and Snap29 Q\u003csub\u003ebc\u003c/sub\u003e SNARE proteins to form a functional SNAREpin, similar to the previously identified crinophagic Syntaxin 13, Snap29 and Vamp7 SNAREpin.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-3917956/v1/6aa816f6a1f43fe5a55bd6a6.png"},{"id":50544183,"identity":"381e05ee-73aa-4f08-ba1a-d9a16c935430","added_by":"auto","created_at":"2024-02-02 08:38:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4295697,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eYkt6 and Vamp7 R-SNAREs differentially regulate the acidification of maturing secretory granules.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e In control salivary gland cells, maturing SGs become positive for LysoTracker Red, and their Sgs3-GFP fluorescence is quenched due to the acidic milieu. Loss of either of the two R-SNAREs impacted the proper acidification of maturing SGs differently. The absence of \u003cem\u003eykt6\u003c/em\u003e \u003cstrong\u003e(b)\u003c/strong\u003e did not have a statistically significant effect on the size of LTR+ structures. In contrast, the loss of \u003cem\u003evamp7\u003c/em\u003e \u003cstrong\u003e(d) \u003c/strong\u003estrongly inhibited the acidification of maturing SGs compared to the corresponding control \u003cstrong\u003e(c)\u003c/strong\u003e, resulting in the accumulation of smaller (presumably fusion incompetent) LTR+ lysosomes. \u003cstrong\u003e(e,f) \u003c/strong\u003eQuantification of the size of LTR+ structures shown in \u003cstrong\u003e(a-d)\u003c/strong\u003e, n=250 LTR+ structures from 5 cells of 5 different larvae, **** p\u0026lt;0.0001, ns p\u0026gt;0.05.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-3917956/v1/cf961c244a06f450aa982818.png"},{"id":50544554,"identity":"9624c28c-f8f4-4634-bc78-25f728366f95","added_by":"auto","created_at":"2024-02-02 08:46:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":9548618,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eYkt6 is required for fusion of SGs with Lamp1+ but not Arl8+ vesicles, while Vamp7 is required for both\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a,c)\u003c/strong\u003e In control larvae, maturing SGs fuse with Arl8+ lysosomes leading to the ring-like appearance of Arl8 around the Sgs3-dsRed+ SGs (turquoise arrowheads). Knockdown of \u003cem\u003eykt6\u003c/em\u003e \u003cstrong\u003e(b)\u003c/strong\u003edoes not affect these types of lysosomal fusions as Arl8 rings still appear around most SGs. \u003cstrong\u003e(c,d)\u003c/strong\u003eHowever, these fusions are inhibited in \u003cem\u003evamp7 \u003c/em\u003esilenced salivary glands\u003cstrong\u003e(d)\u003c/strong\u003e compared to the control \u003cstrong\u003e(c)\u003c/strong\u003e, causing aggregation of the fusion incompetent Arl8+ lysosomes (yellow arrowheads) between the SGs \u003cstrong\u003e(d)\u003c/strong\u003e. \u003cstrong\u003e(e-h) \u003c/strong\u003eFusion of Lamp1+ vesicles supports the maturation of SGs and Lamp1 forms rings around SGs in control cells in a similar fashion to Arl8 \u003cstrong\u003e(e)\u003c/strong\u003e. \u003cem\u003eYkt6 \u003c/em\u003edeficiency \u003cstrong\u003e(f)\u003c/strong\u003e strongly inhibits the fusion of SGs with Lamp1+ lysosomes/carrier vesicles, leading to the clustering of unfused dLamp1+ vesicles between SGs. \u003cstrong\u003e(g-h)\u003c/strong\u003e Silencing of \u003cem\u003evamp7\u003c/em\u003e also inhibits these kinds of fusions, resulting in the accumulation of small-size Lamp1+ lysosomes around SGs that are unable to fuse \u003cstrong\u003e(h)\u003c/strong\u003e, unlike in the corresponding control \u003cstrong\u003e(g)\u003c/strong\u003e. \u003cstrong\u003e(i-l) \u003c/strong\u003eQuantification of the data shown in \u003cstrong\u003e(a-h)\u003c/strong\u003e, n=875 \u003cstrong\u003e(a)\u003c/strong\u003e, n=772 \u003cstrong\u003e(b)\u003c/strong\u003e, n=956 \u003cstrong\u003e(c)\u003c/strong\u003e, n=611 \u003cstrong\u003e(d)\u003c/strong\u003eArl8+ structures and n=513 \u003cstrong\u003e(e)\u003c/strong\u003e, n=724 \u003cstrong\u003e(f)\u003c/strong\u003e, n=516 \u003cstrong\u003e(g)\u003c/strong\u003e, n=1186 \u003cstrong\u003e(h)\u003c/strong\u003e Lamp1+ structures from 3 cells of 5 different larvae, **** p\u0026lt;0.0001, ns p\u0026gt;0.05. Insets show the outlined areas magnified (2X) and split into channels in panels \u003cstrong\u003e(a-h)\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-3917956/v1/7fa738de95ef77e74dd7296c.png"},{"id":50544556,"identity":"710bdca4-7f09-4de5-a8ad-b3da5225b1dd","added_by":"auto","created_at":"2024-02-02 08:46:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5507252,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVamp7 and Ykt6 mediated fusions are both required for PI3P positivity of residual SGs after secretion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParallel with the onset of the massive release of SGs, PI3P+ endosomes are formed to restore the balance between the apical and basolateral membranes. These endosomes are visualized by the GFP-Myc-2xFYVE reporter and appear as small aggregates or rings among the SGs at this stage \u003cstrong\u003e(a)\u003c/strong\u003e. Later on, these endosomes subsequently fuse with the SGs at the prepupal (pp) stage in control cells, forming rings around the SGs (turquoise arrowheads), promoting the transformation of mature SGs \u003cstrong\u003e(b)\u003c/strong\u003e. In the absence of \u003cem\u003eykt6 \u003c/em\u003e\u003cstrong\u003e(c)\u003c/strong\u003e, GFP-FYVE+ endosomes form clusters rather than rings around SGs. \u003cstrong\u003e(d-e)\u003c/strong\u003e Compared to their respective control \u003cstrong\u003e(d)\u003c/strong\u003e, \u003cem\u003evamp7 \u003c/em\u003esilenced cells \u003cstrong\u003e(e)\u003c/strong\u003e also exhibit a failure in fusion of GFP-FYVE+ endosomes with mature SGs. \u003cstrong\u003e(f-g)\u003c/strong\u003e Quantification of the data shown in panels \u003cstrong\u003e(b-e)\u003c/strong\u003e, n=1205 \u003cstrong\u003e(b)\u003c/strong\u003e, n=1408 \u003cstrong\u003e(c)\u003c/strong\u003e, n=606 \u003cstrong\u003e(d)\u003c/strong\u003e, n=1154 \u003cstrong\u003e(e) \u003c/strong\u003eGFP-FYVE+ structures from 3 cells of 5 different larvae, **** p\u0026lt;0.0001. Insets show the outlined areas magnified (2X) and split into channels in panels \u003cstrong\u003e(a-e)\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-3917956/v1/d52cc63e3d31846e0d368702.png"},{"id":50544185,"identity":"9d2faf8d-30b5-4091-9654-c13355f8092f","added_by":"auto","created_at":"2024-02-02 08:38:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3501449,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eYkt6 localizes to small Lamp1+ (carrier) vesicles.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a-c)\u003c/strong\u003e Ykt6 localizes to small punctate structures that are evenly distributed in cells. \u003cstrong\u003e(a) \u003c/strong\u003eThese vesicles often overlap with small Lamp1+ vesicles (turquoise arrowheads) while the larger Lamp1+ rings (yellow arrowheads) that form around maturing SGs are devoid of Ykt6. Ykt6 does not overlap with established lysosomal markers Arl8 \u003cstrong\u003e(b) \u003c/strong\u003eand Cathepsin L \u003cstrong\u003e(c)\u003c/strong\u003e, consistent with the different roles of Vamp7 and Ykt6 in SG maturation-promoting lysosomal fusions. \u003cstrong\u003e(d) \u003c/strong\u003eQuantification of the overlap between markers in \u003cstrong\u003e(a-c)\u003c/strong\u003e, n=200 structures from 5 cells of 5 different larvae. Error bars mark ± SEMs. Insets show the outlined areas magnified (2X) and split into channels in panels \u003cstrong\u003e(a-c)\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-3917956/v1/c8766467497059267e991b5c.png"},{"id":50544186,"identity":"e56a8584-48f6-445a-8627-ba4d5482e19c","added_by":"auto","created_at":"2024-02-02 08:38:47","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2380023,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe absence of Ykt6 does not affect the localization of the other crinophagic R-SNARE, Vamp7.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e In control prepupal salivary gland cells, GFP-Vamp7 forms rings around mature SGs and is also found on smaller structures that are presumably lysosomes or endosomes. \u003cstrong\u003e(b)\u003c/strong\u003e The absence of ykt6 does not affect the GFP-Vamp7 localization pattern as it still forms rings around SGs. Insets show the outlined areas magnified (2X) and split into channels in panels \u003cstrong\u003e(a-b)\u003c/strong\u003e. \u003cstrong\u003e(c) \u003c/strong\u003eOur proposed model for lysosomal fusions that regulate the maturation and crinophagic breakdown of SGs. Immature SGs (iSGs) bud from the trans-Golgi network (TGN), increase in size by homotypic fusions, then undergo a complex maturation process involving a series of lysosomal fusions. First, Arl8+ lysosomes fuse with the maturing SGs by the canonical Syx13-Snap29-Vamp7 SNARE complex, enabling subsequent fusions with the Lamp1 carrier vesicles, which in turn is mediated by the Syx13-Snap29-Ykt6 SNAREpin. These consecutive lysosomal fusions promote the progressive acidification and inner reorganization (decondensation) of SG contents. Mature SGs (mSGs) are eventually released by regulated exocytosis, while the residual SGs (rSGs) that evade secretion are selectively degraded by crinophagy, which requires further fusions between the endolysosomal compartment and rSGs.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-3917956/v1/3a3d97b77de5b457aa3ba4f9.png"},{"id":51122589,"identity":"7a58f16c-47fe-496e-b005-039adcc21325","added_by":"auto","created_at":"2024-02-14 14:22:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9939794,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3917956/v1/6278c45f-7537-4ae3-bf8b-6828ac6fe941.pdf"},{"id":50544553,"identity":"28129c5b-cc47-4e9a-bd69-9761b2c087ca","added_by":"auto","created_at":"2024-02-02 08:46:47","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":141659,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figure S1. The original gel image of the GST-pulldown experiment presented in Fig. 1e.\u003c/p\u003e","description":"","filename":"SupplementaryFigureS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3917956/v1/b80c2b8c68942c15695ca45e.pdf"},{"id":50544555,"identity":"d5ba47c7-67fe-42db-af7e-21d5d36cfabf","added_by":"auto","created_at":"2024-02-02 08:46:47","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":30770,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table S1. Source data for the statistics presented on Figures\u0026nbsp;\u003c/p\u003e","description":"","filename":"SupplementaryTableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3917956/v1/88487e4acf197a1eddffa4be.xlsx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eThe Ykt6-Snap29-Syx13 SNARE complex promotes crinophagy via secretory granule fusion with Lamp1 carrier vesicles\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eProfessional secretory cells produce large amounts of secretory material (hormones, neuropeptides, digestive enzymes, mucin, etc.) and store them in secretory granules (SGs) until a secretagogue elicits their bulk exocytosis. These cells usually produce more secretory material than is released by exocytosis\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e to provide a sufficient pool of available SGs\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Secretory cells continuously turn over the excess SGs by crinophagy, a specialized form of autophagy to maintain a constant releasable pool of SGs\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Following this route, abnormal or obsolete SGs may also be subject to crinophagic degradation\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. In addition to degradative crinophagy, SG-lysosome fusions may also contribute to the complex maturation process of SGs and thereby determine their controlled release by exocytosis. During crinophagy, SGs directly fuse with lysosomes that gives rise to degradative crinosomes\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eEasy genetic manipulation and highly conserved molecular mechanisms made \u003cem\u003eDrosophila\u003c/em\u003e a powerful in-vivo model for deciphering the molecular regulation of the regulated secretory pathway and crinophagy. Salivary gland cells produce\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e and secrete\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e high amounts of Sgs (Salivary gland secretion)/glue proteins in response to peaks of the molting hormone ecdysone\u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. The released glue is then expelled from the lumen to anchor the metamorphosing prepupae to solid surfaces\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. The nascent glue SGs emanate from the TGN \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, increase in size by homotypic fusions\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, and then undergo a complex maturation process during which SGs fuse with lysosomes. This promotes the acidification and profound reorganization of the inner content of SGs\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, preparing them for secretion\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Excess or abnormal glue can be also degraded by crinophagy, through fusion of non-secreted SGs and lysosomes\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Taken together, crosstalk and fusion between SGs and the endolysosomal compartment is critical both for SG maturation and crinosome formation, however, the molecular mechanism of these processes is still incompletely understood\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBy enabling direct fusion between SGs and lysosomes, crinophagy differs mechanistically from the canonical main autophagic pathway, which mediates the degradation of cytosolic material through autophagosome formation and their subsequent fusion with lysosomes. Accordingly, genes that are required for autophagosome formation proved dispensable to crinophagy\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, while SG-lysosome fusion itself relies on a similar molecular machinery acting in fusions between autophagosomes and lysosomes\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The machinery mediating autophagosome-lysosome fusion is well characterized both in Drosophila and humans by now. Critical components include Rab2, Rab7, and Arl8 small GTPases that also contribute to defining membrane identity\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan additionalcitationids=\"CR27 CR28\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, homotypic fusion and vacuole protein sorting (HOPS) tethering complex\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, and a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex (SNAREpin) that executes the fusion. Based on biochemical properties, functional SNAREpins assemble from three Q- (Q\u003csub\u003eabc\u003c/sub\u003e) and one R-SNARE domains\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The first discovered SNAREpin that mediates autophagosome-lysosome fusion is composed of Syntaxin 17, Snap29 and Vamp7/8\u003csup\u003e30,31,33,34\u003c/sup\u003e. Recently another R-SNARE: Ykt6 was also discovered to also have a role in the process, either as an R-SNARE potentially substituting for Vamp7\u003csup\u003e35\u003c/sup\u003e or interacting with Syntaxin 7 and Snap29 to form an alternative SNAREpin\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Interestingly, Drosophila crinophagic fusion of glue SGs and lysosomes depends on highly similar machinery, composed of Rab2, Rab7, Arl8, HOPS and a Syntaxin 13, Snap29 and Vamp7 SNAREpin\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The similarity of the molecular machinery regulating these lysosomal fusions raised the possibility that Ykt6 may also regulate SG-lysosome fusions and crinophagy.\u003c/p\u003e \u003cp\u003eYkt6 is a highly conserved R-SNARE that consists of an N-terminal longin domain (LD), an R-SNARE domain, and a conserved C-terminal lipidation motif with the amino acid sequence CCAIM. The latter is critical for membrane association\u003csup\u003e\u003cspan additionalcitationids=\"CR39 CR40 CR41\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e because Ykt6, unlike other R-SNAREs, lacks a canonical transmembrane domain. Moreover, the lipid anchors can hide reversibly in the hydrophobic groove of the protein, which enables Ykt6 to leave membranes and form a cytosolic pool\u003csup\u003e\u003cspan additionalcitationids=\"CR39 CR40\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. This way, it can be rapidly incorporated into various intracellular membranes on demand and form a complex with compartment-specific Q-SNAREs to promote vesicle fusion. Membrane-associated Ykt6 regulates the anterograde ER to Golgi\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, the intra-Golgi\u003csup\u003e\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, retrograde directed Golgi to ER, and endosome to TGN transports\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, and the release of constitutive secretory carriers\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e or exosomes\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e along the secretory pathway. In addition, it also promotes biosynthetic transport to the yeast vacuole and lysosomes in animal cells\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e, endosomal recycling\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e, and macroautophagic degradation\u003csup\u003e\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. However, the role of Ykt6 in SG-lysosome fusion and crinophagy remained unknown.\u003c/p\u003e \u003cp\u003eHere, we show that Ykt6 forms a canonical SNAREpin with Syntaxin 13 and Snap29, which is \u0026ndash; similarly to the already known Syntaxin 13, Snap29, Vamp7 SNAREpin \u0026ndash; critical for crinophagic degradation. We also demonstrate that Ykt6 localizes to small Lamp1+ (carrier) vesicles and mediates their fusion with SGs, while Vamp7 regulates the fusion of SGs and Arl8\u0026thinsp;+\u0026thinsp;lysosomes. In summary, we provide evidence that SG maturation preceding exocytosis and crinophagy requires a series of fusions between SGs and two separate lysosome-related vesicle subpopulations, which are governed by different SNAREpins/SNARE complexes.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eYkt6 is required for crinophagic degradation\u003c/h2\u003e\n\u003cp\u003eThe crinophagic SG-lysosome and the conventional autophagosome-lysosome fusions share key regulators\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Since the possible role of Ykt6 as an alternative R-SNARE in SG-lysosome fusion has not been investigated so far, we analyzed the putative role of Ykt6 in crinophagic SG degradation. We carried out loss of function experiments by silencing \u003cem\u003eykt6\u003c/em\u003e in prepupal (pp) salivary gland cells and assayed its effect on crinophagic flux. The acidification and lysosomal degradation of glue SGs can be monitored by simultaneous expression of the N-terminal GFP- and dsRed-tagged Sgs3 glue protein in the larval salivary glands. The differently labeled Sgs3 reporters are both in the lumen of forming SG, so these are initially positive for both fluorophores. Their fusion with acidic lysosomes results in quenching the GFP signal due to the acidic environment. Therefore, at the time of puparium formation, most of the SGs that are not secreted remain positive only for dsRed\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. To investigate the consequence of Ykt6 loss, we used time-controlled RNA interference (RNAi)-mediated knock-down of the protein\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e, to circumvent its possible undesirable effect on the biogenesis of SGs. In control cells, most of the SGs appear dsRed-only as the Sgs3-GFP signal is quenched in acidic milieu (Fig.\u0026nbsp;1a, d). In contrast, many dsRed and GFP double-positive SGs remain in \u003cem\u003eykt6\u003c/em\u003e silenced cells (Fig.\u0026nbsp;1b-d), indicating defective crinophagic SG degradation. This phenotype resembled the absence of the previously described crinophagic SNAREs, Syx13, Snap29 and Vamp7\u003csup\u003e3\u003c/sup\u003e. Thus, Ykt6 may mediate crinophagic SG-lysosome fusion in a similar way to Vamp7.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003eThe R-SNARE Ykt6 forms a SNARE complex with Syntaxin13 Q\u003csub\u003ea\u003c/sub\u003e and Snap29 Q\u003csub\u003ebc\u003c/sub\u003e SNAREs\u003c/h2\u003e\n\u003cp\u003eTo test the ability of Ykt6 to form a functional SNAREpin, we examined its interactions with the previously identified crinophagic Q-SNAREs Syntaxin 13 and Snap29\u003csup\u003e3\u003c/sup\u003e by performing a GST pull-down assay with N-terminally GST- or MBP-tagged SNARE domains. GST-Syntaxin 13 Q\u003csub\u003ea\u003c/sub\u003e (bait) was immobilized on glutathione beads, and we observed its strong interaction with the MBP-tagged Snap29 Q\u003csub\u003ebc\u003c/sub\u003e Vamp7 as well as Ykt6 R-SNARE (prey) domains (Fig.\u0026nbsp;1e). Importantly, the binding of recombinant ykt6 to Syx13/Snap29 was much stronger than to the Syx17/Snap29 autophagic SNARE complex\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. This finding indicates that Ykt6 regulates crinophagy by forming a SNAREpin with the crinophagic Q-SNAREs.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003eAcidification of maturing SGs is regulated differently by Ykt6 and Vamp7\u003c/h2\u003e\n\u003cp\u003eIn addition to degradative crinosomes (at the prepupal stage - pp), ecdysone-induced progressive acidification also accompanies the maturation of SGs (2h before puparium formation - bpf), and is important for the remodeling of the inner structure of SGs and prepares them for exocytosis (priming)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Although both Vamp7 and Ykt6 proved to be required for the acidification of degradative crinosomes at the prepupal (pp) stage, it was still elusive whether these two R-SNAREs are also equally required for proper acidification of maturing SGs before their release. To assay this, we stained the genomic Sgs3-GFP expressing salivary glands of 2h bpf larvae with LysoTracker Red (LTR), a vital dye that labels acidic structures (Fig.\u0026nbsp;2). In control cells around the time of robust secretion, mature SGs had already lost their GFP fluorescence, accompanied by a parallel accumulation of large LTR\u0026thinsp;+\u0026thinsp;vesicles (Fig.\u0026nbsp;2a, c). In the absence of \u003cem\u003eykt6\u003c/em\u003e, no statistically significant difference was observed in the size of LTR\u0026thinsp;+\u0026thinsp;structures compared to the control cells (Fig.\u0026nbsp;2a, b, e) even though the Glue-GFP signal was already higher compared to the control (Fig.\u0026nbsp;2a, b), similar to the crinophagic flux experiments (Fig.\u0026nbsp;1a-c). In contrast, the lack of Vamp7 strongly reduced the size of LTR\u0026thinsp;+\u0026thinsp;acidic structures compared to the respective control (Fig.\u0026nbsp;2c, d, f). These findings raised the possibility that the two R-SNAREs required for crinophagy play different roles in the acidification and maturation of Glue SGs.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003ch2\u003eYkt6 and Vamp7 differently regulate the fusion of Arl8\u0026thinsp;+\u0026thinsp;lysosomes with SGs\u003c/h2\u003e\n\u003cp\u003eThe maturation and crinophagic decomposition of glue SGs both rely on a series of fusion events between SGs and lysosomes or endosomes\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Since Vamp7 and Ykt6 differentially affected SG maturation/acidification, we supposed that these R-SNAREs may mediate the fusion of SGs with different components of the endo-lysosomal compartment. Therefore, we tested the colocalization between glue granules and different endo-lysosomal markers in \u003cem\u003eykt6\u003c/em\u003e RNAi or \u003cem\u003evamp7\u003c/em\u003e RNAi salivary glands, respectively. Arl8 is a small GTPase highly specific for lysosomes and it is necessary for direct fusion of Lamp1\u0026thinsp;+\u0026thinsp;lysosomes and glue SGs\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. In control cells, endogenous Arl8 forms rings around Sgs3-dsRed\u0026thinsp;+\u0026thinsp;SGs (Fig.\u0026nbsp;3a, c) that indicates successful fusions between Arl8\u0026thinsp;+\u0026thinsp;lysosomes and maturing SGs. The absence of \u003cem\u003eykt6\u003c/em\u003e does not interfere with fusion of Arl8\u0026thinsp;+\u0026thinsp;lysosomes, as Arl8 still forms rings around SGs (Fig.\u0026nbsp;3b, i). In contrast, silencing of \u003cem\u003evamp7\u003c/em\u003e strongly inhibits the formation of rings around SGs: instead, Arl8 labels small vesicle aggregates (Fig.\u0026nbsp;3d, j). This reflects a strong defect in Arl8\u0026thinsp;+\u0026thinsp;lysosome-SG fusions (Fig.\u0026nbsp;3c, d, j) and suggests that this fusion event is mediated by the SNAREpin containing Vamp7.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch2\u003eYkt6 is involved in the fusion of Lamp1\u0026thinsp;+\u0026thinsp;vesicles with SGs\u003c/h2\u003e\n\u003cp\u003eLamp1 is a highly glycosylated transmembrane protein, an essential component of the lysosomal membranes\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Although Lamp1 is often used as a lysosome marker, it is also present on a broader spectrum of vesicles belonging to the endo-lysosomal compartment\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Similar to our observations with Arl8, endogenous Lamp1 also forms rings along the perimeter of maturing Sgs3-dsRed SGs in control cells 2h bpf (Fig.\u0026nbsp;3e, g). However, the formation of these rings was strongly perturbed both in \u003cem\u003eykt6\u003c/em\u003e (Fig.\u0026nbsp;3f) and \u003cem\u003evamp7\u003c/em\u003e (Fig.\u0026nbsp;3h) silenced salivary gland cells (Fig.\u0026nbsp;3e-h, k, l). Thus, these lysosome markers are delivered to maturing SGs through independent fusion events governed by different R-SNAREs. Our data point to the involvement of a heterogeneous population of Arl8\u0026thinsp;+\u0026thinsp;and Lamp1\u0026thinsp;+\u0026thinsp;lysosomes and related vesicles in maturation and crinophagic degradation of glue SGs.\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003ch2\u003eYkt6 and Vamp7 are required for endosomal fusions of SGs following secretion\u003c/h2\u003e\n\u003cp\u003eIt was previously described that the maturation of SGs requires contribution from the endosomal system\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e, but it remains unclear whether the fusion of endosomes with SGs is important for crinosome formation. To explore this, we labeled endosomes harboring phosphatidylinositol 3-phosphate (PI3P) with GFP-Myc-2xFYVE probe specific for PI3P and tested its overlap with Glue-dsRed. We observed that GFP-FYVE marks usually small endosome clusters among the SGs at 2h bpf (Fig.\u0026nbsp;4a.)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. Later on, non-secreted SGs trapped in cytosol are most likely removed by crinophagy by transforming into crinosomes\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e which appear as large Sgs3-dsRed\u0026thinsp;+\u0026thinsp;granules encircled by GFP-FYVE\u0026thinsp;+\u0026thinsp;membranes (Fig.\u0026nbsp;4b, d). This suggests that crinosomes receive extensive membrane input from PI3P-positive endosomes. However, it remained unclear whether this endosomal input was dependent on the preceding fusion events between lysosomes and maturing SGs. We observed that in the lack of Ykt6, these PI3P\u0026thinsp;+\u0026thinsp;endosomes are clustered between SGs, rather than forming a ring around them (Fig.\u0026nbsp;4b, c, f). Similarly, the absence of Vamp7 strongly inhibited the fusion of PI3P\u0026thinsp;+\u0026thinsp;endosomes and SGs compared to the respective control (Fig.\u0026nbsp;4d, e, g). These results show that the Ykt6- and Vamp7-mediated lysosomal fusions determine the subsequent fate and fusion capacity of the SGs because they fail to fuse with PI3P\u0026thinsp;+\u0026thinsp;endosomes in the absence of either SNARE.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch2\u003eYkt6 localizes to small Lamp1\u0026thinsp;+\u0026thinsp;vesicles\u003c/h2\u003e\n\u003cp\u003eSince we found that Ykt6 and Vamp7 mediate the fusion of maturing SGs with different lysosome-related vesicle populations, we also aimed to elucidate the subcellular localization of Ykt6. By carrying out immunolabeling with antibodies specific for Ykt6 and various lysosomal markers, we found that endogenous Ykt6 shows a punctate pattern which overlaps significantly with small Lamp1\u0026thinsp;+\u0026thinsp;vesicles, while it is absent from the large Lamp1\u0026thinsp;+\u0026thinsp;rings that presumably formed around maturing SGs (Fig.\u0026nbsp;5a, d). Ykt6 does not colocalize with other lysosome markers, such as Arl8 (Fig.\u0026nbsp;5b, d) or the lysosomal hydrolase Cathepsin L (Fig.\u0026nbsp;5c, d). These data are in line with our results that Ykt6 is mainly involved in the fusion of SGs and Lamp1\u0026thinsp;+\u0026thinsp;vesicles, but not Arl8\u0026thinsp;+\u0026thinsp;lysosomes (Fig.\u0026nbsp;3).\u003c/p\u003e\n\u003ch2\u003eYkt6 does not affect the localization of Vamp7\u003c/h2\u003e\n\u003cp\u003eWe also wondered whether the two SNAREpins that mediate SG-lysosome fusions indeed function independently. Therefore, we investigated the localization of Vamp7 by using N-terminal GFP-tagged Vamp7 in the absence of the other R-SNARE, Ykt6. The loss of Ykt6 has not altered the localization pattern of Vamp7, as it is still able to form rings around larger SGs (Fig.\u0026nbsp;6a, b). This further suggests that the Ykt6- and Vamp7-containing SNAREpins independently regulate the maturation and crinophagic degradation of SGs by mediating fusion between SGs and Arl8+ (by Vamp7) or Arl8- but Lamp1+ (Vamp7, Ykt6) lysosome subpopulations.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this work, we revealed that Ykt6 acts together with Syntaxin 13 and Snap29 to form a functional SNAREpin. This SNARE complex is required for efficient SG fusion with Lamp1 carrier vesicles, thereby promoting the maturation and crinophagic elimination of SGs. The first vesicle fusions occur just before the bulk secretion of SGs. These early fusions may drive the acidification and inner reorganization of SGs to promote SG maturation\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. In line with this, SG-lysosome fusion is claimed to cause enhanced secretion in Trpml1-/- mutant pancreatic acinar cells\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Our data indicate that the two R-SNAREs Ykt6 and Vamp7 play different roles in regulating the maturation of SGs, because SGs fail to acidify properly without Vamp7, while the silencing of \u003cem\u003eykt6\u003c/em\u003e did not prevent this.\u003c/p\u003e\n\u003cp\u003eMoreover, we found that Vamp7 is required for the localization multiple lysosomal markers to maturing glue granules, while Ykt6 only affects the fusion of SGs with Lamp1\u0026thinsp;+\u0026thinsp;vesicles and it is dispensable for fusion with Arl8\u0026thinsp;+\u0026thinsp;lysosomes. We hypothesize that maturing SGs first undergo Vamp7-mediated fusion with Arl8\u0026thinsp;+\u0026thinsp;lysosomes, which is required for their maturation. The Ykt6-mediated fusions between SGs and Lamp1\u0026thinsp;+\u0026thinsp;vesicles likely represent a later step of SG maturation. Although our findings suggest the existence of at least two separate vesicle subpopulations carrying these lysosomal markers (Arl8\u0026thinsp;+\u0026thinsp;ones and Lamp1+/Arl8- ones), Arl8\u0026thinsp;+\u0026thinsp;lysosomes possibly also contain Lamp1.\u003c/p\u003e\n\u003cp\u003eThe coexistence and sequential contribution of multiple lysosomal subpopulations/Lamp1 carriers in distinct steps of SG maturation could be an advantage for secretory cells. Different vesicle subpopulations can act as carriers that deliver different lysosomal membrane proteins and enzymes that are required for lysosome biogenesis. The volume of SGs is enormous compared to these small vesicles, hence the desired concentration of lysosomal proteins in matured SGs or crinosomes could be fine-tuned by a series of membrane fusions with different lysosomal populations. This model is further supported by findings by others, showing that Vamp7 is required for the transport of lysosomal membrane proteins (LMPs, including Lamp1)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e or the potential role of Ykt6 in lysosomal enzyme transport\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Since we found that the vesicles to which Ykt6 localizes are positive only for Lamp1, but negative for Arl8 and the lysosomal protease Cathepsin L, Ykt6 appears to be required for the delivery of lysosomal membrane proteins such as Lamp1 itself to glue granules. As the highly glycosylated Lamp1 is essential for protecting the lysosomal membrane from acidic internal pH and enzymatic degradation\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e, the Ykt6-mediated delivery of these Lamp1 carrier vesicles to mature SGs could prepare them for the degradative crinosomal fate.\u003c/p\u003e\n\u003cp\u003eWe have also demonstrated the importance of endosomal contribution to crinosome formation. PI3P\u0026thinsp;+\u0026thinsp;endosomes are much smaller than SGs and initially form clusters among SGs before secretion\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e, eventually fusing with the residual, non-secreted mature SGs. One can assume that these endosomal fusions prepare the obsolete SGs for crinophagic breakdown, possibly through the recruitment of Rab7, which is implicated in crinophagic SG-lysosome fusion\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Importantly, we found that these fusions are equally hampered in the absence of either Ykt6 or Vamp7. Thus, the early Ykt6- and Vamp7-mediated vesicle fusions determine the subsequent fate and fusion potential of maturing SGs. Small PI3P\u0026thinsp;+\u0026thinsp;endosomes that fuse with residual SGs are likely derived from endocytic activity that follows the programmed secretion of SGs. The convergence of secretory, endosomal, and even autophagic routes in lysosomes was also demonstrated in larval Drosophila fat tissue\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eOverall, our results refine the model of glue granule maturation and lysosome fusions: SGs probably first acquire the lysosomal small GTPase Arl8 and begin to acidify via fusion by the canonical Vamp7 containing SNAREpin. This primary fusion event engages maturing SGs for subsequent volume-increasing lysosomal fusions that already involve Lamp1\u0026thinsp;+\u0026thinsp;lysosomes. Ykt6 reaches SGs by forming a SNAREpin with Syntaxin 13 Q\u003csub\u003ea\u003c/sub\u003e- and Snap29 Q\u003csub\u003ebc\u003c/sub\u003e-SNAREs to mediate SG-Lamp1 carrier vesicle fusion, and these separate fusion events together promote the maturation and crinophagic degradation of residual glue granules after secretion (Fig.\u0026nbsp;6c).\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\n\u003ch2\u003eDrosophila genetics\u003c/h2\u003e\n\u003cp\u003eFly stocks were maintained on standard yeast-cornmeal-agar medium at 25\u0026deg;C temperature. To avoid undesirable effects of Ykt6 on SG biogenesis, temperature sensitive tubP-Gal80 construct was used to temporally control the expression of ykt6 RNAi transgenes\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. These crosses were shifted from the 18\u0026deg;C restrictive temperature to 29\u0026deg;C for 36 hours at the late (wandering) L3 stage. Accordingly, separate controls were used for room temperature vamp7 and temperature-induced ykt6 RNAi experiments. The w\u003csup\u003e1118\u003c/sup\u003e (#3605), fkh-Gal4 (#78060), tubP-Gal80\u003csup\u003ets\u003c/sup\u003e (#7017 and #7108), Sgs3-GFP (#5884) and the UAS-GFP-myc-2xFYVE (#42712) lines were obtained from Bloomington Drosophila Stock Center. The UAS-Ykt6\u003csup\u003eNIG.1515R\u003c/sup\u003e (#1515R-1) (ykt6 RNAi/1 in the text) RNAi line was obtained from NIG-Fly (National Institute of Genetics)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. The UAS-Ykt6\u003csup\u003eKK101343\u003c/sup\u003e (#v105648) (ykt6 RNAi/2 in the text) and UAS-Vamp7\u003csup\u003eKK107576\u003c/sup\u003e (#v108733) RNAi stocks were purchased from Vienna Drosophila Resource Center. The Sgs3-dsRed line was kindly provided by Andrew Andres (University of Nevada, US). Sgs3-GFP; fkh-Gal4, the Sgs3-dsRed; fkh-Gal4, the Sgs3-dsRed, UAS-GFP-myc-2xFYVE; fkh-Gal4 and the Sgs3-dsRed, Sgs3-GFP; fkh-Gal4 lines were used to study the endo-lysosomal transport to secretory granules or secretory granule acidification. For our experiments we used late L3 staged larvae that had already completed their wandering (considered as 2 hours before puparium formation (2h bpf)) or white prepupae (pp).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003ch2\u003eLysoTracker Red (LTR) staining\u003c/h2\u003e\n\u003cp\u003eThe larval salivary glands were dissected in cold PBS (pH\u0026thinsp;=\u0026thinsp;7.4) and permeabilized for 30 s (2h bpf) or 15 s (pp) in 0.05% Triton X-100-PBS (PBTX) solution. The samples were rinsed in PBS (3x30 sec) and incubated for 2 min in 0.5 nM LTR (in PBS, Invitrogen) staining solution, then washed in PBS and mounted with 9:1 PBS: glycerol solution that contains 1 \u0026micro;g/mL DAPI (4\u0026prime;,6-diamidino-2-phenylindole, Sigma Aldrich) to stain the nuclei of cells.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003eImmunohistochemistry\u003c/h2\u003e\n\u003cp\u003eThe larval salivary glands were dissected in cold PBS, gently permeabilized with 0.05% PBTX solution either for 30 s (2h bpf) or 15s (prepupae) and fixed in 4% formaldehyde-PBS (40 min, RT). Then, the samples were rinsed with PBS (3x5 min, RT), incubated in a blocking solution (5% fetal calf serum in 0.1% PBTX, 30 min, RT), and incubated with the first antibodies dissolved in the blocking solution (ON, 4\u0026deg;C). After washing (3x15 min PBTX), salivary glands were incubated in blocking solution (30 min, RT), then with the secondary antibodies diluted in blocking solution (3 hr, RT). Thereafter samples were incubated in 4% NaCl solution (15min, RT) that was supplemented with Hoechst (1:200, Sigma-Aldrich) nuclear dye and washed (2x15 min in 0.1% PBTX, 3x15 min in PBS). The specimens were dissected and mounted in Vectashield (Vector Laboratories).\u003c/p\u003e\n\u003cp\u003eFor the salivary gland immunostainings rabbit anti-Arl8 (1:100, DSHB)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, rabbit anti-dLamp1 (1:1000, kind gift of Andreas Jenny)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e, rat anti-Ykt6 (1:30)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, rat anti-mCherry (1:300) and rabbit anti-CathL/MEP (1:100, Abcam, #ab58991)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e primary and the AlexaFluor488-conjugated anti-mouse, anti-rabbit, anti-goat and AlexaFluor568-conjugated anti-mouse, anti-rabbit and anti-rat secondary antibodies (all 1:1000, Invitrogen) were used.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003ch2\u003eFluorescent imaging\u003c/h2\u003e\n\u003cp\u003eFluorescent images were taken at room temperature with an AxioImager M2 microscope (Zeiss) equipped with an ApoTome.2 structured illumination unit, Orca-Flash 4.0 LT3 digital sCMOS camera (Hamamatsu Photonics), EC Plan-Neofluar 20x/0.50, Plan-Apochromat 40x/0.95 and Plan-Apochromat 63x/1.4 Oil objectives (Zeiss). Raw images were processed with ZEN2.3 lite Microscopy Software and Photoshop CS4 (Adobe Systems). To improve clarity in Fig.\u0026nbsp;2, Fig.\u0026nbsp;3, Fig.\u0026nbsp;4 and Fig.\u0026nbsp;6 consecutive optical slices spanning a depth of 3 \u0026micro;m were projected onto single images. Single focal planes were presented in other figures, including colocalization tests in Fig.\u0026nbsp;1 and Fig.\u0026nbsp;5.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n\u003ch2\u003eGST pulldown assay\u003c/h2\u003e\n\u003cp\u003eSNARE fragments were cloned into pETARA or/and pETMBP vectors, which contain C-terminal Glutathione S-transferase/Maltose Binding Protein tag and C-terminal hexahistidine-tag, respectively, using BamHI and XhoI restriction sites. Syx13 was amplified from the EST LD27581 (DGRC Stock 4205 ; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://dgrc.bio.indiana.edu//stock/4205\u003c/span\u003e\u003c/span\u003e ; RRID:DGRC_4205) with primers 5\u0026rsquo;-ATCGGATCCCACGACATGCTCGAC-3\u0026rsquo; and 5\u0026rsquo;-ATCCTCGAGCGCCTTGGCCAGTTC-3\u0026rsquo;. The remaining constructs were already reported in an earlier study\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eFor pulldown experiments, recombinant SNARE constructs were expressed overnight at 18\u0026deg;C in E. coli Rosetta(DE3) pLysS (Novagen) cells induced with 0.1 mM IPTG at OD 0.6\u0026ndash;0.7. Cells were then centrifuged and suspended in lysis buffer (pH 8.0, 50 mM Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 300 mM NaCl, 20mM imidazole, 0.1% Triton-X, 5 mM-\u0026beta;-mercaptoethanol, protease inhibitors). Lysed samples were centrifuged (48.000g, 30 min). Ni-NTA resin was added to the supernatant and incubated for 30 minutes at 4\u0026deg;C. Beads were washed with washing buffer (pH 8.0, 50 mM Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 1 M NaCl, 40mM imidazole, 0,1% Triton-X, 5 mM \u0026beta;-mercaptoethanol) and then eluated in Elution buffer (pH 8.0, 20 mM Tris, 200 mM NaCl, 400 mM imidazole, 10% glicerol, 0,1% Triton-X, 5 mM-\u0026beta;-mercaptoethanol) used for pulldown assays. Prey proteins for pulldown experiments were purified with further MBP affinity chromatography using standard protocols. All resins were from GE Healthcare.\u003c/p\u003e\n\u003cp\u003eFor GST pulldown assays, the glutathione resin (New England BioLabs) was first equilibrated with binding buffer (20 mM Tris, 50 mM NaCl, 0.1% Triton-X, 2 mM \u0026beta;\u0026ndash;\u0026beta;-mercaptoethanol), then 0.5 mg GST fused SNARE proteins (and GST as negative control) were immobilized on it. In the binding experiments, 40 \u0026micro;l of resin saturated with baits were incubated in the presence of 20 \u0026micro;M preys in binding buffer (200 \u0026micro;l total volume, 30 min at 4\u0026deg;C). Glutathione beads were pelleted with centrifugation (200 g, 2 min) and washed 3x with 20 mM Tris, 300 mM NaCl, 0.1% Triton-X, 2 mM \u0026beta;\u0026ndash;mercaptoethanol. Retained proteins were eluted from the resin with an SDS loading buffer. Samples were subjected to SDS-PAGE and interactions were detected by Coomassie protein dye. The original gel image is provided in the Supplementary Information (Supplementary Fig. S1).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n\u003ch2\u003eStatistics\u003c/h2\u003e\n\u003cp\u003eImageJ software (National Institutes of Health, Bethesda, Maryland, US) was used for quantitative analysis of fluorescent structures. Overlap of the markers was assessed by Pearson\u0026rsquo;s correlation analysis using the Coloc2 plugin (Fig.\u0026nbsp;1) or in the case of membrane markers encircling granular structures (Fig.\u0026nbsp;5), the signal was calculated manually. For manual colocalization assessment, 200 immunolabeled structures were selected. The threshold for LTR quantification was set by the same person in a dark room in all images and structures were counted. The structure diameter range was set to 1\u0026ndash;99 \u0026micro;m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e to exclude background noise and unrealistic clumped structures of several SG sizes (Fig.\u0026nbsp;2). The ring-like fluorescent structures in Fig.\u0026nbsp;3 and Fig.\u0026nbsp;4 were selected manually by the same person and the percentage of them located around SGs was examined. For pairwise comparisons of datasets that followed Gaussian distribution unpaired t-test (Fig.\u0026nbsp;3i, j, Fig.\u0026nbsp;4f, g) or where at least one of the datasets followed non-Gaussian distribution, Mann-Whitney U test (Fig.\u0026nbsp;2, Fig.\u0026nbsp;3k, l) were performed. To analyze multiple datasets with Gaussian distribution, one-way ANOVA with Tukey\u0026rsquo;s post hoc test was performed (Fig.\u0026nbsp;1d). The distribution tests of datasets and statistical analyses were carried out using GraphPadPrism 9.0.0 software (Boston, Massachusetts, US). All source data about the quantifications related to the presented experiments are available as Supplementary Information (Supplementary Table S1).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Sarolta P\u0026aacute;lfia, D\u0026aacute;vid Hargitai, Dorottya K\u0026aacute;rolyi and Fanni Ősz for their assistance in the maintenance of the fly lines and Zs\u0026oacute;fia Gyetvai for helpful discussions and Győző Szenci for drawing the summary figure.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the National Research, Development and Innovation Office of Hungary (Elvonal KKP129797 to GJ, HunProtExc 2018-1.2.1-NKP-2018\u0026ndash;00005 to GJ, OTKA FK_142508 for ST), the Hungarian Academy of Sciences (BO/00400/23 for ST), the Excellence Fund of E\u0026ouml;tv\u0026ouml;s Lorand University (EKA_2022/045-P302-1 for ST) and the UNKP New National Excellence Program of the Ministry of Human Capacities of Hungary (\u0026Uacute;NKP-23-5-ELTE-1257 for ST).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGS, ST and GJ designed the research. GS and GG performed the experiments and analyzed the data. GS, GG, ST and GJ wrote the manuscript. The research was equally supervised by ST and GJ.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that no conflict of interest exists.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data needed to evaluate the conclusions in this paper are present in the paper and its Supplementary Information. Other data associated with the article, such as raw data, are available upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll materials, Drosophila stocks and related information are available from the corresponding authors upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHalban, P. A. \u0026amp; Wollheim, C. B. Intracellular degradation of insulin stores by rat pancreatic islets in vitro. An alternative pathway for homeostasis of pancreatic insulin content. \u003cem\u003eJournal of Biological Chemistry\u003c/em\u003e \u003cstrong\u003e255\u003c/strong\u003e, 6003-6006 (1980). https://doi.org/10.1016/s0021-9258(18)43686-1\u003c/li\u003e\n\u003cli\u003eSchnell, A. H., Swenne, I. \u0026amp; Borg, L. A. H. Lysosomes and pancreatic islet function - A quantitative estimation of crinophagy in the mouse pancreatic B-cell. \u003cem\u003eCell and Tissue Research\u003c/em\u003e \u003cstrong\u003e252\u003c/strong\u003e, 9-15 (1988). https://doi.org/10.1007/BF00213820\u003c/li\u003e\n\u003cli\u003eCsizmadia, T.\u003cem\u003e et al.\u003c/em\u003e Molecular mechanisms of developmentally programmed crinophagy in \u003cem\u003eDrosophila\u003c/em\u003e. \u003cem\u003eJ Cell Biol\u003c/em\u003e \u003cstrong\u003e217\u003c/strong\u003e, 361-374 (2018). https://doi.org/10.1083/jcb.201702145\u003c/li\u003e\n\u003cli\u003eKim, T., Gondr\u0026eacute;-Lewis, M. C., Arnaoutova, I. \u0026amp; Loh, Y. P. Dense-core secretory granule biogenesis. \u003cem\u003ePhysiology (Bethesda)\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 124-133 (2006). https://doi.org/10.1152/physiol.00043.2005\u003c/li\u003e\n\u003cli\u003eZhou, Y.\u003cem\u003e et al.\u003c/em\u003e RILP restricts insulin secretion through mediating lysosomal degradation of proinsulin. \u003cem\u003eDiabetes\u003c/em\u003e \u003cstrong\u003e69\u003c/strong\u003e, 67-82 (2020). https://doi.org/10.2337/db19-0086\u003c/li\u003e\n\u003cli\u003eLi, M.\u003cem\u003e et al.\u003c/em\u003e VAMP4 regulates insulin levels by targeting secretory granules to lysosomes. \u003cem\u003eJournal of Cell Biology\u003c/em\u003e \u003cstrong\u003e221\u003c/strong\u003e, e202110164 (2022). https://doi.org/10.1083/jcb.202110164\u003c/li\u003e\n\u003cli\u003eSzenci, G., Csizmadia, T. \u0026amp; Juh\u0026aacute;sz, G. The role of crinophagy in quality control of the regulated secretory pathway. \u003cem\u003eJ Cell Sci\u003c/em\u003e \u003cstrong\u003e136\u003c/strong\u003e, jcs260741 (2023). https://doi.org/10.1242/jcs.260741\u003c/li\u003e\n\u003cli\u003eCsizmadia, T.\u003cem\u003e et al.\u003c/em\u003e Developmental program-independent secretory granule degradation in larval salivary gland cells of Drosophila. \u003cem\u003eTraffic\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 568-586 (2022). https://doi.org/10.1111/tra.12871\u003c/li\u003e\n\u003cli\u003eGlaumann, H.\u003cem\u003e et al.\u003c/em\u003e Isolation and characterization of crinosomes--a subclass of secondary lysosomes. \u003cem\u003eExp Mol Pathol\u003c/em\u003e \u003cstrong\u003e50\u003c/strong\u003e, 167-182 (1989). https://doi.org/10.1016/0014-4800(89)90028-2\u003c/li\u003e\n\u003cli\u003eCostantino, B. F.\u003cem\u003e et al.\u003c/em\u003e A novel ecdysone receptor mediates steroid-regulated developmental events during the mid-third instar of Drosophila. \u003cem\u003ePLoS Genet\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, e1000102 (2008). https://doi.org/10.1371/journal.pgen.1000102\u003c/li\u003e\n\u003cli\u003eKaieda, Y.\u003cem\u003e et al.\u003c/em\u003e Glue protein production can be triggered by steroid hormone signaling independent of the developmental program in Drosophila melanogaster. \u003cem\u003eDev Biol\u003c/em\u003e \u003cstrong\u003e430\u003c/strong\u003e, 166-176 (2017). https://doi.org/10.1016/j.ydbio.2017.08.002\u003c/li\u003e\n\u003cli\u003eBiyasheva, A., Do, T. V., Lu, Y., Vaskova, M. \u0026amp; Andres, A. J. Glue secretion in the Drosophila salivary gland: a model for steroid-regulated exocytosis. \u003cem\u003eDev Biol\u003c/em\u003e \u003cstrong\u003e231\u003c/strong\u003e, 234-251 (2001). https://doi.org/10.1006/dbio.2000.0126\u003c/li\u003e\n\u003cli\u003eTorres, I. L., Rosa-Ferreira, C. \u0026amp; Munro, S. The Arf family G protein Arl1 is required for secretory granule biogenesis in Drosophila. \u003cem\u003eJ Cell Sci\u003c/em\u003e \u003cstrong\u003e127\u003c/strong\u003e, 2151-2160 (2014). https://doi.org/10.1242/jcs.122028\u003c/li\u003e\n\u003cli\u003eBurgess, J.\u003cem\u003e et al.\u003c/em\u003e AP-1 and clathrin are essential for secretory granule biogenesis in Drosophila. \u003cem\u003eMol Biol Cell\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 2094-2105 (2011). https://doi.org/10.1091/mbc.E11-01-0054\u003c/li\u003e\n\u003cli\u003eNeuman, S. D., Lee, A. R., Selegue, J. E., Cavanagh, A. T. \u0026amp; Bashirullah, A. A novel function for Rab1 and Rab11 during secretory granule maturation. \u003cem\u003eJ Cell Sci\u003c/em\u003e \u003cstrong\u003e134\u003c/strong\u003e (2021). https://doi.org/10.1242/jcs.259037\u003c/li\u003e\n\u003cli\u003eNiemeyer, B. A. \u0026amp; Schwarz, T. L. SNAP-24, a Drosophila SNAP-25 homologue on granule membranes, is a putative mediator of secretion and granule-granule fusion in salivary glands. \u003cem\u003eJ Cell Sci\u003c/em\u003e \u003cstrong\u003e113\u003c/strong\u003e, 4055-4064 (2000). https://doi.org/10.1242/jcs.113.22.4055\u003c/li\u003e\n\u003cli\u003eSyed, Z. A., Zhang, L., Tran, D. T., Bleck, C. K. E. \u0026amp; Ten Hagen, K. G. Regulated Restructuring of Mucins During Secretory Granule Maturation In Vivo. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e119\u003c/strong\u003e, e2209750119 (2022). https://doi.org/10.1073/pnas.2209750119\u003c/li\u003e\n\u003cli\u003eNagy, A.\u003cem\u003e et al.\u003c/em\u003e Ecdysone receptor isoform specific regulation of secretory granule acidification in the larval Drosophila salivary gland. \u003cem\u003eEur J Cell Biol\u003c/em\u003e \u003cstrong\u003e101\u003c/strong\u003e, 151279 (2022). https://doi.org/10.1016/j.ejcb.2022.151279\u003c/li\u003e\n\u003cli\u003eBoda, A.\u003cem\u003e et al.\u003c/em\u003e Rab26 controls secretory granule maturation and breakdown in Drosophila. \u003cem\u003eCell Mol Life Sci\u003c/em\u003e \u003cstrong\u003e80\u003c/strong\u003e, 24 (2023). https://doi.org/10.1007/s00018-022-04674-8\u003c/li\u003e\n\u003cli\u003ePark, S.\u003cem\u003e et al.\u003c/em\u003e Fusion of lysosomes with secretory organelles leads to uncontrolled exocytosis in the lysosomal storage disease mucolipidosis type IV. \u003cem\u003eEMBO Rep\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 266-278 (2016). https://doi.org/10.15252/embr.201541542\u003c/li\u003e\n\u003cli\u003eBoda, A.\u003cem\u003e et al.\u003c/em\u003e Drosophila Arl8 is a general positive regulator of lysosomal fusion events. \u003cem\u003eBiochimica et Biophysica Acta - Molecular Cell Research\u003c/em\u003e \u003cstrong\u003e1866\u003c/strong\u003e, 533-544 (2019). https://doi.org/10.1016/j.bbamcr.2018.12.011\u003c/li\u003e\n\u003cli\u003eLőrincz, P.\u003cem\u003e et al.\u003c/em\u003e Vps8 overexpression inhibits HOPS-dependent trafficking routes by outcompeting Vps41/Lt. \u003cem\u003eeLife\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 1-25 (2019). https://doi.org/10.7554/eLife.45631.001\u003c/li\u003e\n\u003cli\u003eMa, C. I. J., Burgess, J. \u0026amp; Brill, J. A. Maturing secretory granules: Where secretory and endocytic pathways converge. \u003cem\u003eAdvances in Biological Regulation\u003c/em\u003e \u003cstrong\u003e80\u003c/strong\u003e, 100807-100807 (2021). https://doi.org/10.1016/j.jbior.2021.100807\u003c/li\u003e\n\u003cli\u003ePasquier, A.\u003cem\u003e et al.\u003c/em\u003e Lysosomal degradation of newly formed insulin granules contributes to \u0026beta; cell failure in diabetes. \u003cem\u003eNature Communications\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 1-14 (2019). https://doi.org/10.1038/s41467-019-11170-4\u003c/li\u003e\n\u003cli\u003eKanai, A.\u003cem\u003e et al.\u003c/em\u003e Genome-wide screening for regulators of degradation of insulin secretory granules with a fluorescent reporter. \u003cem\u003eBiochem Biophys Res Commun\u003c/em\u003e \u003cstrong\u003e676\u003c/strong\u003e, 132-140 (2023). https://doi.org/10.1016/j.bbrc.2023.07.040\u003c/li\u003e\n\u003cli\u003eGutierrez, M. G., Munaf\u0026oacute;, D. B., Ber\u0026oacute;n, W. \u0026amp; Colombo, M. I. Rab7 is required for the normal progression of the autophagic pathway in mammalian cells. \u003cem\u003eJ Cell Sci\u003c/em\u003e \u003cstrong\u003e117\u003c/strong\u003e, 2687-2697 (2004). https://doi.org/10.1242/jcs.01114\u003c/li\u003e\n\u003cli\u003eHegedűs, K.\u003cem\u003e et al.\u003c/em\u003e The Ccz1-Mon1-Rab7 module and Rab5 control distinct steps of autophagy. \u003cem\u003eMol Biol Cell\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 3132-3142 (2016). https://doi.org/10.1091/mbc.E16-03-0205\u003c/li\u003e\n\u003cli\u003eFujita, N.\u003cem\u003e et al.\u003c/em\u003e Genetic screen in Drosophila muscle identifies autophagy-mediated T-tubule remodeling and a Rab2 role in autophagy. \u003cem\u003eElife\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, e23367 (2017). https://doi.org/10.7554/eLife.23367\u003c/li\u003e\n\u003cli\u003eLőrincz, P.\u003cem\u003e et al.\u003c/em\u003e Rab2 promotes autophagic and endocytic lysosomal degradation. \u003cem\u003eJ Cell Biol\u003c/em\u003e \u003cstrong\u003e216\u003c/strong\u003e, 1937-1947 (2017). https://doi.org/10.1083/jcb.201611027\u003c/li\u003e\n\u003cli\u003eJiang, P.\u003cem\u003e et al.\u003c/em\u003e The HOPS complex mediates autophagosome-lysosome fusion through interaction with syntaxin 17. \u003cem\u003eMol Biol Cell\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 1327-1337 (2014). https://doi.org/10.1091/mbc.E13-08-0447\u003c/li\u003e\n\u003cli\u003eTak\u0026aacute;ts, S.\u003cem\u003e et al.\u003c/em\u003e Interaction of the HOPS complex with Syntaxin 17 mediates autophagosome clearance in Drosophila. \u003cem\u003eMol Biol Cell\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 1338-1354 (2014). https://doi.org/10.1091/mbc.E13-08-0449\u003c/li\u003e\n\u003cli\u003eMion, D., Bunel, L., Heo, P. \u0026amp; Pincet, F. The beginning and the end of SNARE-induced membrane fusion. \u003cem\u003eFEBS Open Bio\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 1958-1979 (2022). https://doi.org/10.1002/2211-5463.13447\u003c/li\u003e\n\u003cli\u003eItakura, E., Kishi-Itakura, C. \u0026amp; Mizushima, N. The hairpin-type tail-anchored SNARE syntaxin 17 targets to autophagosomes for fusion with endosomes/lysosomes. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e151\u003c/strong\u003e, 1256-1269 (2012). https://doi.org/10.1016/j.cell.2012.11.001\u003c/li\u003e\n\u003cli\u003eTak\u0026aacute;ts, S.\u003cem\u003e et al.\u003c/em\u003e Autophagosomal Syntaxin17-dependent lysosomal degradation maintains neuronal function in Drosophila. \u003cem\u003eJ Cell Biol\u003c/em\u003e \u003cstrong\u003e201\u003c/strong\u003e, 531-539 (2013). https://doi.org/10.1083/jcb.201211160\u003c/li\u003e\n\u003cli\u003eTak\u0026aacute;ts, S.\u003cem\u003e et al.\u003c/em\u003e Non-canonical role of the SNARE protein Ykt6 in autophagosome-lysosome fusion. \u003cem\u003ePLoS Genet\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, e1007359 (2018). https://doi.org/10.1371/journal.pgen.1007359\u003c/li\u003e\n\u003cli\u003eMatsui, T.\u003cem\u003e et al.\u003c/em\u003e Autophagosomal YKT6 is required for fusion with lysosomes independently of syntaxin 17. \u003cem\u003eJ Cell Biol\u003c/em\u003e \u003cstrong\u003e217\u003c/strong\u003e, 2633-2645 (2018). https://doi.org/10.1083/jcb.201712058\u003c/li\u003e\n\u003cli\u003eS\u0026aacute;nchez-Mart\u0026iacute;n, P.\u003cem\u003e et al.\u003c/em\u003e ULK1-mediated phosphorylation regulates the conserved role of YKT6 in autophagy. \u003cem\u003eJ Cell Sci\u003c/em\u003e \u003cstrong\u003e136\u003c/strong\u003e, jcs260546 (2023). https://doi.org/10.1242/jcs.260546\u003c/li\u003e\n\u003cli\u003eFukasawa, M., Varlamov, O., Eng, W. S., S\u0026ouml;llner, T. H. \u0026amp; Rothman, J. E. Localization and activity of the SNARE Ykt6 determined by its regulatory domain and palmitoylation. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e101\u003c/strong\u003e, 4815-4820 (2004). https://doi.org/10.1073/pnas.0401183101\u003c/li\u003e\n\u003cli\u003eHasegawa, H., Yang, Z., Oltedal, L., Davanger, S. \u0026amp; Hay, J. C. Intramolecular protein-protein and protein-lipid interactions control the conformation and subcellular targeting of neuronal Ykt6. \u003cem\u003eJ Cell Sci\u003c/em\u003e \u003cstrong\u003e117\u003c/strong\u003e, 4495-4508 (2004). https://doi.org/10.1242/jcs.01314\u003c/li\u003e\n\u003cli\u003ePylypenko, O.\u003cem\u003e et al.\u003c/em\u003e Farnesylation of the SNARE protein Ykt6 increases its stability and helical folding. \u003cem\u003eJ Mol Biol\u003c/em\u003e \u003cstrong\u003e377\u003c/strong\u003e, 1334-1345 (2008). https://doi.org/10.1016/j.jmb.2008.01.099\u003c/li\u003e\n\u003cli\u003eShirakawa, R.\u003cem\u003e et al.\u003c/em\u003e A SNARE geranylgeranyltransferase essential for the organization of the Golgi apparatus. \u003cem\u003eEMBO J\u003c/em\u003e \u003cstrong\u003e39\u003c/strong\u003e, e104120 (2020). https://doi.org/10.15252/embj.2019104120\u003c/li\u003e\n\u003cli\u003eSakata, N., Shirakawa, R., Goto, K., Trinh, D. A. \u0026amp; Horiuchi, H. Double prenylation of SNARE protein Ykt6 is required for lysosomal hydrolase trafficking. \u003cem\u003eJ Biochem\u003c/em\u003e \u003cstrong\u003e169\u003c/strong\u003e, 363-370 (2021). https://doi.org/10.1093/jb/mvaa111\u003c/li\u003e\n\u003cli\u003eMcNew, J. A.\u003cem\u003e et al.\u003c/em\u003e Ykt6p, a prenylated SNARE essential for endoplasmic reticulum-Golgi transport. \u003cem\u003eJ Biol Chem\u003c/em\u003e \u003cstrong\u003e272\u003c/strong\u003e, 17776-17783 (1997). https://doi.org/10.1074/jbc.272.28.17776\u003c/li\u003e\n\u003cli\u003eZhang, T. \u0026amp; Hong, W. Ykt6 forms a SNARE complex with syntaxin 5, GS28, and Bet1 and participates in a late stage in endoplasmic reticulum-Golgi transport. \u003cem\u003eJ Biol Chem\u003c/em\u003e \u003cstrong\u003e276\u003c/strong\u003e, 27480-27487 (2001). https://doi.org/10.1074/jbc.M102786200\u003c/li\u003e\n\u003cli\u003eXu, Y., Martin, S., James, D. E. \u0026amp; Hong, W. GS15 forms a SNARE complex with syntaxin 5, GS28, and Ykt6 and is implicated in traffic in the early cisternae of the Golgi apparatus. \u003cem\u003eMol Biol Cell\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 3493-3507 (2002). https://doi.org/10.1091/mbc.e02-01-0004\u003c/li\u003e\n\u003cli\u003eParlati, F.\u003cem\u003e et al.\u003c/em\u003e Distinct SNARE complexes mediating membrane fusion in Golgi transport based on combinatorial specificity. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e99\u003c/strong\u003e, 5424-5429 (2002). https://doi.org/10.1073/pnas.082100899\u003c/li\u003e\n\u003cli\u003eVolchuk, A.\u003cem\u003e et al.\u003c/em\u003e Countercurrent distribution of two distinct SNARE complexes mediating transport within the Golgi stack. \u003cem\u003eMol Biol Cell\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 1506-1518 (2004). https://doi.org/10.1091/mbc.e03-08-0625\u003c/li\u003e\n\u003cli\u003eTai, G.\u003cem\u003e et al.\u003c/em\u003e Participation of the syntaxin 5/Ykt6/GS28/GS15 SNARE complex in transport from the early/recycling endosome to the trans-Golgi network. \u003cem\u003eMol Biol Cell\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 4011-4022 (2004). https://doi.org/10.1091/mbc.e03-12-0876\u003c/li\u003e\n\u003cli\u003eGordon, D. E.\u003cem\u003e et al.\u003c/em\u003e VAMP3/Syb and YKT6 are required for the fusion of constitutive secretory carriers with the plasma membrane. \u003cem\u003ePLoS Genet\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, e1006698 (2017). https://doi.org/10.1371/journal.pgen.1006698\u003c/li\u003e\n\u003cli\u003eSun, C.\u003cem\u003e et al.\u003c/em\u003e LncRNA PVT1 promotes exosome secretion through YKT6, RAB7, and VAMP3 in pancreatic cancer. \u003cem\u003eAging (Albany NY)\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 10427-10440 (2020). https://doi.org/10.18632/aging.103268\u003c/li\u003e\n\u003cli\u003eGross, J. C., Chaudhary, V., Bartscherer, K. \u0026amp; Boutros, M. Active Wnt proteins are secreted on exosomes. \u003cem\u003eNat Cell Biol\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 1036-1045 (2012). https://doi.org/10.1038/ncb2574\u003c/li\u003e\n\u003cli\u003eCuddy, L. K.\u003cem\u003e et al.\u003c/em\u003e Stress-Induced Cellular Clearance Is Mediated by the SNARE Protein ykt6 and Disrupted by \u0026alpha;-Synuclein. \u003cem\u003eNeuron\u003c/em\u003e \u003cstrong\u003e104\u003c/strong\u003e, 869-884.e811 (2019). https://doi.org/10.1016/j.neuron.2019.09.001\u003c/li\u003e\n\u003cli\u003eLinnemannst\u0026ouml;ns, K.\u003cem\u003e et al.\u003c/em\u003e Ykt6-dependent endosomal recycling is required for Wnt secretion in the \u003cem\u003eDrosophila\u003c/em\u003e wing epithelium. \u003cem\u003eDevelopment\u003c/em\u003e \u003cstrong\u003e147\u003c/strong\u003e, dev185421 (2020). https://doi.org/10.1242/dev.185421\u003c/li\u003e\n\u003cli\u003eBas, L.\u003cem\u003e et al.\u003c/em\u003e Reconstitution reveals Ykt6 as the autophagosomal SNARE in autophagosome-vacuole fusion. \u003cem\u003eJ Cell Biol\u003c/em\u003e \u003cstrong\u003e217\u003c/strong\u003e, 3656-3669 (2018). https://doi.org/10.1083/jcb.201804028\u003c/li\u003e\n\u003cli\u003eGao, J., Reggiori, F. \u0026amp; Ungermann, C. A novel in vitro assay reveals SNARE topology and the role of Ykt6 in autophagosome fusion with vacuoles. \u003cem\u003eJ Cell Biol\u003c/em\u003e \u003cstrong\u003e217\u003c/strong\u003e, 3670-3682 (2018). https://doi.org/10.1083/jcb.201804039\u003c/li\u003e\n\u003cli\u003eDavidson, H. W., Rhodes, C. J. \u0026amp; Hutton, J. C. Intraorganellar calcium and pH control proinsulin cleavage in the pancreatic beta cell via two distinct site-specific endopeptidases. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e333\u003c/strong\u003e, 93-96 (1988). https://doi.org/10.1038/333093a0\u003c/li\u003e\n\u003cli\u003eBarg, S.\u003cem\u003e et al.\u003c/em\u003e Priming of insulin granules for exocytosis by granular Cl(-) uptake and acidification. \u003cem\u003eJ Cell Sci\u003c/em\u003e \u003cstrong\u003e114\u003c/strong\u003e, 2145-2154 (2001). https://doi.org/10.1242/jcs.114.11.2145\u003c/li\u003e\n\u003cli\u003eChaudhry, N.\u003cem\u003e et al.\u003c/em\u003e Lamp1 mediates lipid transport, but is dispensable for autophagy in \u003cem\u003eDrosophila\u003c/em\u003e. \u003cem\u003eAutophagy\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 2443-2458 (2022). https://doi.org/10.1080/15548627.2022.2038999\u003c/li\u003e\n\u003cli\u003eCook, N. R., Row, P. E. \u0026amp; Davidson, H. W. Lysosome associated membrane protein 1 (Lamp1) traffics directly from the TGN to early endosomes. \u003cem\u003eTraffic\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 685-699 (2004). https://doi.org/10.1111/j.1600-0854.2004.00212.x\u003c/li\u003e\n\u003cli\u003eBurgess, J.\u003cem\u003e et al.\u003c/em\u003e Type II phosphatidylinositol 4-kinase regulates trafficking of secretory granule proteins in Drosophila. \u003cem\u003eDevelopment (Cambridge)\u003c/em\u003e \u003cstrong\u003e139\u003c/strong\u003e, 3040-3050 (2012). https://doi.org/10.1242/dev.077644\u003c/li\u003e\n\u003cli\u003eMa, C. I. J.\u003cem\u003e et al.\u003c/em\u003e An early endosome-derived retrograde trafficking pathway promotes secretory granule maturation. \u003cem\u003eJournal of Cell Biology\u003c/em\u003e \u003cstrong\u003e219\u003c/strong\u003e, e201808017 (2020). https://doi.org/10.1083/jcb.201808017\u003c/li\u003e\n\u003cli\u003eNeuman, S. D., Terry, E. L., Selegue, J. E., Cavanagh, A. T. \u0026amp; Bashirullah, A. Mistargeting of secretory cargo in retromer-deficient cells. \u003cem\u003eDis Model Mech\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, dmm046417 (2021). https://doi.org/10.1242/dmm.046417\u003c/li\u003e\n\u003cli\u003eMa, C. J. \u0026amp; Brill, J. A. Endosomal Rab GTPases regulate secretory granule maturation in \u003cem\u003eDrosophila\u003c/em\u003e larval salivary glands. \u003cem\u003eCommun Integr Biol\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 15-20 (2021). https://doi.org/10.1080/19420889.2021.1874663\u003c/li\u003e\n\u003cli\u003ePols, M. S.\u003cem\u003e et al.\u003c/em\u003e hVps41 and VAMP7 function in direct TGN to late endosome transport of lysosomal membrane proteins. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 1361 (2013). https://doi.org/10.1038/ncomms2360\u003c/li\u003e\n\u003cli\u003eZhou, L.\u003cem\u003e et al.\u003c/em\u003e Convergence of secretory, endosomal, and autophagic routes in trans-Golgi-associated lysosomes. \u003cem\u003eJ Cell Biol\u003c/em\u003e \u003cstrong\u003e222\u003c/strong\u003e, e202203045 (2023). https://doi.org/10.1083/jcb.202203045\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"5a59f365-aa57-49c5-8994-ed1d6ed8d578","identifier":"10.13039/501100011019","name":"Nemzeti Kutatási Fejlesztési és Innovációs Hivatal","awardNumber":"OTKA FK_142508","order_by":0},{"identity":"837a9a27-4f60-49f5-9dc4-ad816c3ece03","identifier":"10.13039/501100011019","name":"Nemzeti Kutatási Fejlesztési és Innovációs Hivatal","awardNumber":"Elvonal KKP129797","order_by":1},{"identity":"de2eee40-461a-4642-a5e3-557c33c9c033","identifier":"10.13039/501100011019","name":"Nemzeti Kutatási Fejlesztési és Innovációs Hivatal","awardNumber":"HunProtExc 2018-1.2.1-NKP-2018–00005","order_by":2},{"identity":"66d652d2-bc9d-489b-907f-4e5071c8edd5","identifier":"10.13039/501100003825","name":"Magyar Tudományos Akadémia","awardNumber":"BO/00400/23","order_by":3},{"identity":"4878c1bd-020e-475e-bcdd-fc7670088a96","identifier":"10.13039/501100009934","name":"Eötvös Loránd Tudományegyetem","awardNumber":"EKA_2022/045-P302-1","order_by":4},{"identity":"fc809a80-7001-4f38-a1ff-cba7d494a5df","identifier":"10.13039/501100005881","name":"Emberi Eroforrások Minisztériuma","awardNumber":"ÚNKP-23-5-ELTE-1257","order_by":5}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"Eötvös Loránd University","isAcceptedByJournal":true,"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":"lysosome, secretory granule, crinophagy, SNARE, Ykt6","lastPublishedDoi":"10.21203/rs.3.rs-3917956/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3917956/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn the Drosophila larval salivary gland, developmentally programmed fusions between lysosomes and secretory granules (SGs) and their subsequent acidification promote the maturation of SGs that are secreted shortly before puparium formation. Subsequently, ongoing fusions between non-secreted SGs and lysosomes give rise to degradative crinosomes, where the superfluous secretory material is degraded. Lysosomal fusions control both the quality and quantity of SGs, however, its molecular mechanism is incompletely characterized. Here we identify the R-SNARE Ykt6 as a novel regulator of crinosome formation, but not the acidification of maturing SGs. We show that Ykt6 localizes to Lamp1\u0026thinsp;+\u0026thinsp;carrier vesicles, and forms a SNARE complex with Syntaxin 13 and Snap29 to mediate fusion with SGs. These Lamp1 carriers represent a distinct vesicle population that are functionally different from canonical Arl8+, Cathepsin L\u0026thinsp;+\u0026thinsp;lysosomes, which also fuse with maturing SGs but are controlled by another SNARE complex composed of Syntaxin 13, Snap29 and Vamp7. Ykt6- and Vamp7-mediated vesicle fusions also determine the fate of SGs, as loss of either of these SNAREs prevents crinosomes from acquiring endosomal PI3P. Our results highlight that fusion events between SGs and different lysosome-related vesicle populations are critical for fine regulation of the maturation and crinophagic degradation of SGs.\u003c/p\u003e","manuscriptTitle":"The Ykt6-Snap29-Syx13 SNARE complex promotes crinophagy via secretory granule fusion with Lamp1 carrier vesicles","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-02 08:38:42","doi":"10.21203/rs.3.rs-3917956/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":"afc36b8b-1c38-4fb8-a697-c7db727da5ee","owner":[],"postedDate":"February 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":28516091,"name":"General Cell Biology \u0026 Physiology"}],"tags":[],"updatedAt":"2024-02-14T14:22:02+00:00","versionOfRecord":{"articleIdentity":"rs-3917956","link":"https://doi.org/10.1038/s41598-024-53607-x","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-02-08 00:00:00","publishedOnDateReadable":"February 8th, 2024"},"versionCreatedAt":"2024-02-02 08:38:42","video":"","vorDoi":"10.1038/s41598-024-53607-x","vorDoiUrl":"https://doi.org/10.1038/s41598-024-53607-x","workflowStages":[]},"version":"v1","identity":"rs-3917956","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3917956","identity":"rs-3917956","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}