Regulatory Interplay Between FtsH, HflKC and YidC in Bacterial Membrane Protein Biogenesis and Quality Control

Regulatory Interplay Between FtsH, HflKC and YidC in Bacterial Membrane Protein Biogenesis and Quality Control | 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 Regulatory Interplay Between FtsH, HflKC and YidC in Bacterial Membrane Protein Biogenesis and Quality Control Mehmet Caliseki, Sarah Zorman, Christiane Schaffitzel, Burak Veli Kabasakal This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6895763/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The quality control of membrane proteins is essential for maintaining cellular homeostasis, as misfolded or damaged proteins can disrupt essential cellular functions. FtsH, a membrane-bound AAA + metalloprotease, is central to bacterial proteostasis, responsible for degrading misfolded or damaged membrane proteins. YidC, a membrane protein insertase, facilitates the folding, insertion, and assembly of membrane proteins into the lipid bilayer. This study investigates the physical interaction between FtsH, HflKC, and YidC, indicating a potential functional relationship between these proteins in maintaining membrane protein quality control in bacteria. Overexpression of YidC in Escherichia coli led to a disruption in the interaction between FtsH and its regulatory proteins HflK and HflC, possibly due to competition for binding sites. This was supported by the depletion of HflK and HflC in both Western blot and mass spectrometry analyses using detergent-solubilized membrane extracts. Additionally, the co-overexpression of FtsH and YidC induced cellular stress, as evidenced by the increased recruitment of stress-related proteins such as GroEL and DnaK. These findings suggest that FtsH and YidC collaborate in membrane protein biogenesis and participate in stress-responsive regulatory mechanisms that contribute to protein homeostasis. Figures Figure 1 Figure 2 2. Introduction The quality control of membrane proteins are fundamental processes that ensure bacterial cellular homeostasis and functionality [ 1 – 3 ]. Membrane proteins are essential for diverse functions, including signal transduction, molecular transport, and energy generation [ 4 – 7 ]. Their proper insertion, folding, and assembly in the lipid bilayer are crucial for maintaining membrane integrity [ 8 ]. Any defects in these processes, such as misfolding or improper insertion of membrane proteins, can disrupt cellular physiology, leading to proteotoxic stress and compromised viability [ 9 , 10 ]. Thus, bacteria have evolved sophisticated quality control mechanisms for membrane protein biogenesis and degradation. FtsH and YidC are two key players in bacterial membrane protein homeostasis [ 1 , 11 ]. FtsH is an ATP-dependent AAA + metalloprotease that selectively degrades misfolded, unassembled, or damaged membrane proteins, preventing their accumulation [ 2 , 12 , 13 ]. Structurally, FtsH forms a hexameric complex with its proteolytic and ATPase domains facing the cytoplasm, enabling the recognition and unfolding of both cytosolic and membrane-embedded substrates prior to their degradation [ 14 – 16 ]. FtsH function is modulated by interactions with HflK and HflC, which form a regulatory membrane-associated complex that influences substrate specificity and stability [ 1 , 14 – 20 ]. Structural studies have described the FtsH-HflK-HflC assembly as a bell- or nautilus-shaped complex that modulates FtsH activity by regulating substrate access across the inner membrane [ 16 ]. YidC is a highly conserved membrane protein insertase that facilitates the integration, folding, and assembly of membrane proteins [ 21 – 25 ]. It functions via both Sec-dependent and Sec-independent pathways, ensuring the correct localization and stability of membrane proteins [ 26 – 33 ]. In Sec-dependent pathways, YidC cooperates with the SecYEG translocon, while in Sec-independent pathways, it acts autonomously to insert proteins directly into the membrane [ 11 , 26 , 29 , 32 , 34 ]. Structurally, YidC consists of six transmembrane helices (TM1-TM6) and a hydrophilic groove that interacts with substrates, facilitating their integration into the lipid bilayer [ 35 – 37 ]. Beyond protein insertion, YidC has been proposed to contribute to membrane protein quality control by collaborating with FtsH [ 1 , 11 ]. Together, these proteins may help recognize and degrade misassembled membrane proteins, preventing their accumulation and ensuring membrane integrity [ 1 , 10 , 38 ]. Although biochemical and genetic studies support their potential interplay, the molecular basis of their interaction remains unresolved, necessitating further structural and biochemical studies. Here, we investigate the stability of the interactions between FtsH, YidC, and HflKC, aiming to determine whether they form a stable complex or exist as transiently interacting proteins. Previous studies have shown that FtsH, HflKC and YidC can be co-purified, suggesting possible physical interactions [ 1 ]. However, it remains unclear whether this interaction is stable under physiological conditions or transient and regulated by additional factors. By assessing their stability and potential regulatory mechanisms, this study provides insights into bacterial membrane protein quality control complexes and their involvement in membrane homeostasis. 3. Materials and Methods Expression Systems and Plasmid Design Two different expression systems were used for recombinant protein production. The first system, named FYHC Co-T expression system, employed a co-expression strategy using the pETDuet-1 (Novagen, 71146-3) and pRSFDuet-1 (Novagen, 71341) plasmids, both of which contain T7 promoters. The pETDuet-1 plasmid encoded YidC-10×His and FtsH-3×StrepTag II (Supp. Figure 1 A), while the pRSFDuet-1 plasmid carried untagged HflK and HflC (Supp. Table 1 ). These plasmids confer ampicillin and kanamycin resistance, respectively, allowing co-transformation into Escherichia coli BL21 (DE3) or C43 (DE3) cells for simultaneous expression of multiple proteins. This system was primarily used for pull-down experiments, Western blotting, and mass spectrometry analyses.mThe second system was based on the pFYHC expression construct, which was generated using the ACEMBL system [ 39 ]. It encodes FtsH-3×StrepTag, HflKC-10×His, and YidC-3×Flag, each under the control of different inducible promoters: arabinose, T7, and trc (Supp. Figure 1 B). The pFYHC plasmid was transformed into E. coli BL21 (DE3) or C43 (DE3) cells, enabling regulated, stoichiometrically balanced expression of the target protein complex. Selection was maintained using ampicillin, kanamycin, and spectinomycin. This expression system was used in preparative purifications and structural studies. Membrane Protein Expression, Isolation and Purification E. coli BL21 (DE3) or C43 (DE3) cells harboring either the FYHC Co-T expression system or the pFYHC construct were cultured overnight in LB medium at 37°C with appropriate antibiotics to establish starter cultures. A 5 mL aliquot was inoculated into 500 mL of Terrific Broth (TB; Sigma-Aldrich, T0918) in 2 L baffled flasks, yielding a total culture volume of 6 L. Cultures were grown at 37°C until OD₆₀₀ reached ~ 0.8. In the FYHC Co-T system, protein expression was induced with 0.4 mM IPTG and cultures were incubated either at 18°C for 18 h or at 37°C for 4 h. For the pFYHC system, expression was induced by sequential addition of 1 mM rhamnose, 0.4 mM IPTG, and 2% (w/v) arabinose, under the same temperature conditions. Cells were harvested by centrifugation at 5,000 × g for 30 min at 4°C, and pellets were resuspended in lysis buffer containing 20 mM HEPES (pH 7.5), 150 mM NaCl, 8% glycerol, 2 mM MgCl₂, 1 mM PMSF (Roche 11359061001), one EDTA-free protease inhibitor tablet (Pierce, A32965), and 1 µg/mL benzonase (Millipore, E1014). The suspension was incubated at 4°C for 1 h with gentle agitation, followed by sonication. Lysates were clarified by centrifugation at 10,000 × g for 1.5 h at 4°C. Membrane fractions were isolated by ultracentrifugation at 100,000 × g for 1 h at 4°C and resuspended in solubilization buffer (same as lysis buffer plus 1% DDM). After 1 h solubilization with gentle agitation at 4°C, insoluble debris was removed by another ultracentrifugation step and supernatants were filtered through a 0.45 µm filter. Solubilized membrane proteins were purified by affinity chromatography using either Ni-NTA or Strep-Tactin resin, depending on the tag. For Ni-NTA purification, His-tagged proteins were captured on Ni-NTA resin (Thermo Scientific, 25215) and eluted with 300 mM imidazole following standard wash steps with 10 mM and 50 mM imidazole. For Strep-tagged proteins, solubilized lysates were incubated with Strep-Tactin Sepharose (IBA, 2-1201-025) and eluted with 5 mM desthiobiotin in detergent-containing buffer. Final purification was performed using size-exclusion chromatography (SEC) on a Superose 6 Increase 3.2/300 column (Cytiva, 29091598) pre-equilibrated with SEC buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 2 mM MgCl₂, 0.05% DDM). Target proteins eluted as a single, symmetric peak (Supp. Figure 2 A) and were collected for downstream biochemical and structural proteomics analyses. Protein Crosslinking Crosslinking experiments were conducted using Dithiobis(succinimidyl propionate) (DSP) and Disuccinimidyl dibutyric urea (DSBU) to stabilize protein–protein interactions at different stages of the purification workflow. DSP (Pierce, A35393) was applied during membrane protein solubilization, immediately after membrane isolation. The membrane resuspension was incubated with 0.25 mM or 1 mM DSP (dissolved in DMSO) at room temperature for 1 hour. The reaction was quenched with 20 mM Tris-HCl (pH 7.5), followed by a 15-minute incubation. The DSP-treated membranes were subsequently solubilized with DDM and used for downstream analysis including affinity purification and crosslinking mass spectrometry (XL-MS). DSBU (Thermo Scientific) was used post-purification, following Ni-NTA or Strep-Tactin chromatography, to capture stable oligomeric interactions in detergent-solubilized samples. Purified protein complexes were incubated with 3 mM DSBU (in DMSO) for 1 hour at room temperature. The reaction was quenched with 20 mM Tris-HCl (pH 7.5), achieved by addition of 1 M Tris-HCl stock, and incubated for 30 minutes at 4°C. Crosslinked samples were analyzed by SDS-PAGE, used for XL-MS, or subjected to SEC for further isolation of crosslinked oligomeric species. SDS-PAGE and Western Blot Analysis Protein samples were analyzed by SDS-PAGE using 4–12% Bolt™ Bis-Tris Plus Mini Gels (Invitrogen) in MES running buffer at 180 V. Following electrophoresis, gels were stained with Coomassie Brilliant Blue to evaluate protein purity and expression efficiency. For Western blotting, proteins were transferred from unstained gels onto nitrocellulose membranes (0.2 µm pore size, Bio-Rad Trans-Blot Turbo system) under constant voltage (25 V, 7 minutes). Membranes were blocked with 3% (w/v) BSA in TBS-T for 1 hour at room temperature and subsequently incubated with HRP-conjugated antibodies against the relevant affinity tags: anti-His (Qiagen, 1:5000), anti-StrepTagII (Sigma-Aldrich, 1:5000), or anti-Flag (Rockland, 1:10000), all diluted in TBS-T. After incubation for 1 hour at room temperature, membranes were washed thoroughly with TBS-T to remove unbound antibodies. Protein bands were visualized using the Pierce™ ECL Western Blotting Substrate and detected by chemiluminescence. Western blot analysis was used to monitor the expression of affinity-tagged constructs and confirm co-purification or crosslinked interactions in selected samples. XL-MS Analysis Following membrane protein purification, samples were analyzed by mass spectrometry (MS) either in liquid form or excised from SDS-PAGE gels at University of Bristol Biomedical Sciences Proteomics Facility (Bristol, UK) and Scientific and Technological Research Council of Türkiye (TÜBİTAK) National Metrology Institute (UME) Chemistry Group-Bioanalysis Laboratory (İstanbul, Türkiye). DSBU-crosslinked membrane proteins were purified and enzymatically digested using the FASP Protein Digestion Kit (Abcam) according to the manufacturer’s protocol. The protein sample was reduced with dithiothreitol (DTT), alkylated with iodoacetamide (IAA), and subsequently digested with trypsin in 50 mM ammonium bicarbonate at 37°C overnight. Following digestion, peptides were eluted, concentrated using a Speed-Vac, and resuspended in 5% v/v acetonitrile (ACN) and 0.1% v/v formic acid (FA) for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The analysis was conducted using a Thermo Scientific™ UltiMate™ 3000 RSLC Ultra Nano system coupled to a Thermo Scientific Q Exactive™ HF mass spectrometer. Peptide separation was performed via reverse-phase chromatography on a C18 Easy-Spray column, with solution-derived peptides analyzed over 90 minutes and gel-derived peptides over 55 minutes. Data processing was conducted using Thermo Scientific Proteome Discoverer 2.5 software [ 40 ], while crosslinking MS (XL-MS) data analysis was performed using MSAnnika [ 41 ]. Growth Curve Analysis To evaluate the impact of heterologous expression of membrane protein complexes on bacterial growth, growth curve experiments were performed using E. coli BL21 (DE3) cells transformed with different plasmid combinations. Constructs included individual expression of FtsH (pETDuet-FtsH, Amp^R), YidC (pETDuet-YidC, Amp^R), HflKC (pRSFDuet-HflK-HflC, Kan^R), and co-expression of all three proteins using pETDuet-FtsH-YidC (Amp^R) and pRSFDuet-HflK-HflC (Kan^R). Freshly transformed colonies were grown overnight in 10 mL 2xYT medium supplemented with the appropriate antibiotics. The following day, 2 mL of each starter culture was inoculated into 200 mL of 2xYT in 2 L baffled flasks. Cultures were incubated at 37°C with shaking until OD₆₀₀ reached ~ 0.6, at which point protein expression was induced using 0.4 mM IPTG. Bacterial growth was subsequently monitored by measuring OD₆₀₀ at 30-minute intervals for up to 6 hours. 4. Results 4.1. Co-purification of FtsH, YidC, and HflKC Under Detergent-Solubilized Conditions Membrane proteins were expressed using the FYHC Co-T expression system, which enables the co-expression of Strep-tagged FtsH, His-tagged YidC, and untagged HflK and HflC in E. coli. After membrane isolation and detergent solubilization, purification was performed via the His-tag on YidC using Ni²⁺-NTA affinity chromatography. FtsH was detected in the eluates by SDS-PAGE (Fig. 1A). The FtsH signal decreased in later fractions of the gradient. To improve retention, 0.25 mM DSP was added during solubilization. Under these conditions, FtsH was observed in later elution fractions as well (Fig. 1B). In a separate purification experiment targeting the Strep-tag on FtsH, YidC was detected in the eluates without crosslinker (Fig. 1C). HflK and HflC were not detected in SDS-PAGE analyses in the FYHC Co-T system. Since these proteins lacked affinity tags, Western blot analysis was not applicable. In the pFYHC system, in which HflK contains an N-terminal His-tag, HflK was detected by Western blot following Ni²⁺-NTA purification (Fig. 1D). FtsH was also observed in the eluates, while FLAG-tagged YidC was not detected under the same conditions. 4.2 MS-Based Identification of Proteins Co-purified with FtsH and YidC Protein complexes expressed under different conditions were purified using Ni-NTA affinity, Strep-Tactin affinity, and size-exclusion chromatography. The resulting samples were subsequently analyzed by SDS-PAGE and mass spectrometry (MS) to identify proteins co-purifying with FtsH and YidC. Mass spectrometry results revealed several proteins associated with the purified complexes, including GroEL [42–44], DnaK [44–46], ClpA [47], SlyD [48,49], and SecA [50,51] (Table 1). These proteins are involved in various aspects of proteostasis, including protein folding, membrane insertion, and degradation. GroEL and DnaK were consistently identified in samples where FtsH and YidC were co-expressed, particularly under overexpression conditions. The presence of ClpA, SlyD, and SecA varied depending on the expression system and purification approach. In samples expressing FtsH, HflKC, and YidC together, phage shock protein A (PspA) [52,53], a well-known stress response protein, was detected by MS. PspA was not observed in samples where only FtsH and YidC were expressed. HflK and HflC were detected at low levels in the MS datasets (Table 1), but were not detected by Western blot analysis (Figure 1). 4.3 XL-MS Analysis of FtsH–YidC Interaction and Associated Protein Network XL-MS analysis identified a broad network of protein associations involving FtsH and YidC (Table 2). Two crosslinks were detected between these proteins: residue 1 of YidC, located in the cytoplasm, interacted with residues 33 and 34 of FtsH, both positioned in the periplasmic region. These observations suggest that the FtsH–YidC interaction is not stable under detergent-solubilized conditions and likely occurs transiently. Beyond this interaction, FtsH was found to associate with several other proteins. In the cytoplasm, it interacted with SodA [54] at residue 138 of FtsH and 119 of SodA, with MdtP [55] at residue 60 of FtsH and 407 of MdtP, and with RpoA [56] at residue 391 of FtsH and 309 of RpoA. HflK [14–16] was crosslinked to FtsH at residues 338 and 486. In the periplasm, FtsH interacted with RuvA [57] at residue 332 of FtsH and 2 of RuvA, and with FdoG [58] at residue 332 of FtsH and 330 of FdoG. YidC was associated with CsiE [59] through residue 1 of YidC and 373 of CsiE, and with PutP [60] through residue 377 of YidC and 482 of PutP. Additionally, a crosslink was identified between YidC residue 401 and RpoH residue 173, a stress-response factor previously reported to interact with FtsH [61]. Although a stable complex between FtsH and YidC was not detected, the interaction data support the view that both proteins are components of broader cellular pathways, contributing to protein quality control, membrane protein biogenesis, and stress response through multiple independent associations. 4.4. Assessment of Bacterial Growth Under Co-Expression of FtsH, YidC, and HflKC During recombinant protein production, it was observed that E. coli cells expressing FtsH and YidC exhibited normal growth profiles, whereas cells co-expressing FtsH, YidC, and HflKC showed delayed growth. To investigate this observation systematically, bacterial growth was monitored under five different expression conditions using the FYHC Co-T expression system: (i) FtsH (pETDuet-FtsH), (ii) YidC (pETDuet-YidC), (iii) FtsH and YidC (pETDuet-FtsH + pETDuet-YidC), (iv) HflKC (pRSFDuet-HflK-HflC), and (v) FtsH, YidC, and HflKC (pETDuet-YidC-FtsH + pRSFDuet-HflK-HflC). Bacterial cultures were grown in 2×YT medium at 37 °C with continuous shaking at 100, 150, or 200 rpm. Cell growth was monitored throughout the entire cultivation, including both pre-induction and post-induction phases. Protein expression was induced with 0.4 mM IPTG when cultures reached OD600 = 0.6. As shown in Figure 2A–C, expression of FtsH, YidC, or their combination did not adversely affect cell growth under the tested conditions. In contrast, expression of HflKC alone (Figure 2D) or in combination with FtsH and YidC (Figure 2E) led to a significant reduction in growth, particularly at higher agitation speeds. These results confirm that the delayed growth observed during co-expression of FtsH, YidC, and HflKC is linked to HflKC overexpression. 5. Discussion This study provides observations on the interactions between the membrane proteins YidC, FtsH, and HflKC, which are known to play roles in protein quality control, membrane protein biogenesis, and cellular homeostasis. The results indicate that the interaction between FtsH and YidC is likely transient, and may depend on cellular context or the presence of specific substrates. The identification of several co-purified proteins suggests that both FtsH and YidC are part of a broader protein interaction network associated with membrane protein folding and degradation. Additionally, under YidC overexpression conditions, HflKC was only minimally detected in purified samples, whereas FtsH and YidC co-purified. This observation may reflect a competitive or mutually exclusive association with FtsH, although further investigation is required to clarify the underlying mechanism. The bacterial growth assays showed a delayed growth phenotype upon co-expression of FtsH, YidC, and HflKC. While the exact cause of this delay is unclear, the data may point to a role for HflKC in cellular responses to oxygen availability, suggesting a possible link between HflKC expression and oxygen metabolism under overexpression conditions. 5.1 Interaction Dynamics Between YidC and FtsH In the FYHC Co-T expression system, membrane proteins were co-expressed using pETDuet-YidC-FtsH and pRSFDuet-HflK-HflC plasmids. Under detergent-solubilized conditions, Ni²⁺-NTA affinity purification targeting His₁₀-tagged YidC resulted in co-purification of Strep-tagged FtsH even in the absence of a chemical crosslinker (Fig. 1 A). This finding contrasts with an earlier study, where co-purification of FtsH and YidC required chemical crosslinking [ 1 ]. The gradual loss of FtsH signal across elution fractions suggests that the interaction may be transient or sensitive to purification conditions. Upon addition of 0.25 mM DSP as a crosslinker, FtsH was retained more efficiently (Fig. 1 B), indicating stabilization of the interaction. Consistently, when the purification was performed using Strep-Tactin affinity chromatography targeting Strep-tagged FtsH, His-tagged YidC was co-purified even without a crosslinker (Fig. 1 C). Together, these results support that FtsH and YidC interact under detergent-solubilized conditions, though the interaction may be condition-dependent. HflK and HflC were not detected in these experiments using the FYHC Co-T expression system. As this setup did not include affinity tags for either HflK or HflC, their detection by Western blot was not feasible (Supp. Figure 3). Affinity tagging for HflK was incorporated in the pFYHC expression system, where HflK carries an N-terminal His₁₀ tag. Under these conditions, HflK could be successfully detected following Ni²⁺-NTA purification (Fig. 1 D). FtsH was also detected via anti-Strep-tag Western blot, although at lower levels than those observed with the FYHC Co-T system. Notably, YidC-FLAG was not detected under these conditions. 5.2 Potential Competition Between HflKC and YidC for FtsH Binding HflK and HflC were not detected in SDS-PAGE analysis when co-expressed with FtsH and YidC using the FYHC Co-T expression system (Supp. Figure 3A). Conversely, YidC was not observed in Western blot analysis when expressed with FtsH, HflK, and HflC from the pFYHC single-plasmid expression system (Supp. Figure 3D). These findings suggest a competitive relationship between YidC and HflKC for association with FtsH under detergent-solubilized conditions. The low detection levels of HflK and HflC in the mass spectrometry analysis of Ni-NTA eluates in the FYHC Co-T system, where YidC was used for pull-down, further support this interpretation (Table 1 ). Previous studies reported that YidC depletion leads to increased levels of FtsH, HflK, and HflC, which was proposed to reflect a compensatory response to membrane stress [ 9 , 10 , 62 ]. In contrast, the current findings indicate that YidC overexpression may reduce HflKC recruitment, potentially altering the interaction network involving FtsH. Since HflK and HflC are known to regulate FtsH proteolytic activity [ 14 – 18 ], changes in their association could influence the efficiency or substrate preference of membrane protein degradation. Further investigation is required to understand how the presence or relative abundance of YidC modulates the interaction between FtsH and its regulatory subunits HflK and HflC under different cellular or experimental conditions. 5.3 Protein Interactions Identified by XL-MS Suggest Broader Functional Associations Our XL-MS analysis identified multiple proteins that interact with FtsH and YidC, suggesting their involvement in a broader proteostasis and membrane biogenesis network (Table 2 ). For FtsH, crosslinked partners included proteins with roles in oxidative stress response (e.g., SodA) [ 54 , 63 ], drug resistance (e.g., MdtP) [ 55 ], and RNA transcription (e.g., RpoA) [ 56 ]. The interaction between FtsH and SodA may be linked to the regulation of oxidative damage, in line with previous studies reporting FtsH’s role in stress adaptation. Similarly, the connection with MdtP supports a role in maintaining membrane integrity under toxic compound exposure, while the link to RpoA might indicate transcriptional regulation under proteotoxic conditions. In the periplasm, FtsH interacted with proteins such as RuvA [ 57 ], involved in DNA recombination and repair, and FdoG [ 58 ], a component of formate dehydrogenase complex, pointing to additional functions in DNA maintenance and metabolism. These findings are consistent with earlier reports that FtsH’s activity extends beyond membrane protein degradation, contributing to cellular homeostasis under stress conditions. YidC was found to interact with proteins related to membrane protein insertion and folding such as PutP [ 60 ], reflecting its central role in co-translational insertion and stabilization of membrane proteins. The observed interaction between YidC and RpoH, a sigma factor that activates stress response genes, aligns with its role in adaptive proteostasis. Interestingly, while the interaction between FtsH and RpoH is well-documented [ 61 ], the link between YidC and RpoH may reflect a coordinated regulation of membrane stress responses. Collectively, these interactions suggest that FtsH and YidC are not isolated actors but part of a dynamic and interconnected regulatory network, which ensures proper membrane protein folding, quality control, and stress response. 5.4 Implications of HflKC Expression in Bacterial Adaptation to Oxygen Availability Bacterial growth analyses suggest that HflKC overexpression affects cell proliferation under different oxygenation conditions (Fig. 2 D, E). In particular, delayed growth was observed at higher agitation rates when HflKC was co-expressed, indicating a potential link between HflKC expression and oxygen-dependent metabolic adaptation. These findings are consistent with a recent study reporting that deletion of hflK or hflC alters ubiquinone biosynthesis, thereby affecting aerobic respiration [ 64 ]. In this context, HflKC overexpression may influence components of the respiratory chain or regulatory networks that respond to oxygen availability. The observed growth pattern at lower agitation rates, which are typically associated with reduced oxygen transfer, suggests that elevated levels of HflKC might alter the balance of cellular pathways sensitive to oxidative conditions. Given the known interaction of HflKC with the FtsH protease [ 17 , 18 ], it is possible that HflKC modulates the turnover of proteins involved in respiration or redox homeostasis. However, further analysis is needed to determine whether HflKC directly impacts components such as cytochrome oxidase complexes or ubiquinone levels. Increased oxygen levels can elevate reactive oxygen species (ROS) production, leading to oxidative stress [ 65 – 67 ]. Some SPFH domain-containing proteins, including eukaryotic prohibitins, have been implicated in managing oxidative stress and mitochondrial homeostasis [ 68 ]. Whether bacterial HflKC plays a comparable role remains to be determined. Notably, FtsH is involved in degrading stress response regulators, such as RpoS, which coordinate bacterial responses to oxidative damage [ 69 ]. It is therefore conceivable that HflKC may influence FtsH substrate specificity or protease dynamics under oxidative stress conditions. Interestingly, YidC overexpression was associated with a reduced detection of HflK and HflC according to MS-based interaction analyses [ 9 , 10 , 62 ]. This observation raises the possibility that elevated YidC levels may indirectly affect the ability of HflKC to interact with FtsH. Since FtsH function has been linked to the degradation of several stress regulators, including those involved in oxidative response, competition between YidC and HflKC for FtsH binding may represent an additional regulatory mechanism modulating bacterial adaptation to oxidative conditions. Overall, these observations suggest that HflKC contributes to cellular adaptation mechanisms that extend beyond protein quality control. Its expression appears to affect bacterial growth in response to oxygen availability, possibly through modulation of FtsH activity. Furthermore, competition with YidC for FtsH binding may influence the composition and function of membrane proteostasis machinery under stress conditions. Further biochemical and structural studies are needed to clarify the molecular basis of these interactions and their physiological consequences. Conclusion This study presents observations on the interactions between FtsH, YidC, and HflKC, with a focus on their potential roles in membrane protein quality control. FtsH and YidC were found to co-purify under detergent-solubilized conditions without the use of crosslinkers, suggesting a possible interaction that may be transient and context-dependent. The lack of a stable association is consistent with the detection of multiple additional proteins co-purifying with FtsH and YidC, several of which are involved in proteostasis-related pathways. Growth-based analyses indicate that co-expression of FtsH, YidC, and HflKC can influence bacterial proliferation, particularly under varying oxygenation conditions, pointing to the importance of regulated expression levels. The low recovery of HflKC in the presence of YidC may reflect a potential competition for FtsH binding, although further investigation is required to clarify this relationship. Overall, these findings provide preliminary insights into the network of interactions surrounding FtsH, YidC, and HflKC, and highlight the need for additional studies to better define the molecular mechanisms underlying their coordination in bacterial cells. Declarations Author Contribution M.Ç., C.S., and B.V.K. conceived the study. S.Z. generated the pFYHC plasmid. M.Ç. performed all other experiments, and wrote the manuscript with input from C.S. and B.V.K. All authors have read and agreed to the final manuscript. Acknowledgement M.Ç., and B.V.K. were funded by the Scientific and Technological Council of Türkiye (TÜBİTAK) BİDEB 2232 International Outstanding Researchers Program (Project No: 118C225). M.Ç. was also funded by TÜBİTAK 2211-A National Graduate Fellowship Program. C.S. acknowledges funding by a BBSRC Responsive Mode Grant (BB/P000940/1) and a Wellcome Trust Investigator Grant (210701/Z/18/Z). M.Ç. was also supported by a TÜBİTAK 2214-A - International Research Fellowship Program for carrying out a part of his PhD thesis studies at the University of Bristol, School of Biochemistry. We acknowledge Dr. Süreyya Özcan and Hatice Akkulak from METU for their help in XL-MS analyses. Data Availability Data is provided within the manuscript or supplementary information files. References E. Van Bloois, H.L. Dekker, L. Fröderberg, E.N.G. Houben, M.L. Urbanus, C.G. De Koster, J.-W. De Gier, J. Luirink, Detection of cross‐links between FtsH, YidC, HflK/C suggests a linked role for these proteins in quality control upon insertion of bacterial inner membrane proteins, FEBS Lett. 582 (2008) 1419–1424. https://doi.org/10.1016/j.febslet.2008.02.082. Y. Akiyama, Quality Control of Cytoplasmic Membrane Proteins in Escherichia coli, J. Biochem. (Tokyo) 146 (2009) 449–454. https://doi.org/10.1093/jb/mvp071. D.W. Watkins, S.L. Williams, I. 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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-6895763","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":472350596,"identity":"e853066e-3ab8-4c58-a7d3-153c17004014","order_by":0,"name":"Mehmet Caliseki","email":"","orcid":"","institution":"Turkish Accelerator and Radiation Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Mehmet","middleName":"","lastName":"Caliseki","suffix":""},{"id":472350597,"identity":"aa819766-a7b6-475b-8029-2e5546f2f11b","order_by":1,"name":"Sarah Zorman","email":"","orcid":"","institution":"European Molecular Biology Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Sarah","middleName":"","lastName":"Zorman","suffix":""},{"id":472350598,"identity":"828cc223-03bb-4506-a0ae-ad8bbb2e472c","order_by":2,"name":"Christiane Schaffitzel","email":"","orcid":"","institution":"University of Bristol","correspondingAuthor":false,"prefix":"","firstName":"Christiane","middleName":"","lastName":"Schaffitzel","suffix":""},{"id":472350599,"identity":"de0d426d-d640-4da6-9835-ccac9c18e8d3","order_by":3,"name":"Burak Veli Kabasakal","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFElEQVRIie2Rz0rDQBCHZxlYLyu9rkS6r7DBgwQr+igbCrka8FKwhEhge+s5pdB38A02BNKL4lUQNH0CA0IpCMXk7kp6E9nvMnP5+M0fAIfjDyKBAnYNB7yv23oKQGqo+ykkkwaAAaAE1VPRvJdyzqPwM54m4mRW6Ltm+sYGKZJawehGXP+sBHlUenlV+ksW6hdT3TJuEKWCKHgwlsEe16nHqCEr6BSqGLx/VFxBKf3UqmRfbJ9crQYbHZu9YsLg0e5XZa0r71hjuOShhkIrJg1S6BRhWT+Y6ehiMS/Hi3yT8ae5Yn67C1cyktJ2MaRnr/E2ucyfx0Uz2arh0CBpmslICstgNtqI7rEHcmiKw+Fw/Fu+AfbbW7xSriNLAAAAAElFTkSuQmCC","orcid":"","institution":"University of Bristol","correspondingAuthor":true,"prefix":"","firstName":"Burak","middleName":"Veli","lastName":"Kabasakal","suffix":""}],"badges":[],"createdAt":"2025-06-14 22:08:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6895763/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6895763/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84898394,"identity":"ad891893-990c-4a9e-9a01-f7dcaaf9019f","added_by":"auto","created_at":"2025-06-18 14:25:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":330036,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWestern blot analysis of FtsH co-purification with YidC and HflKC.\u003c/strong\u003e Overexpression of FtsH, YidC, and HflKC was performed using two different expression systems: FYHC Co-T (Panels A–C) and pFYHC (Panel D). Membrane proteins were purified by affinity chromatography, either using Ni-NTA resin (Panels A, B, D) or Strep-Tactin resin (Panel C). For the crosslinked condition (Panel B), membranes were treated with 0.25 mM DSP prior to Ni-NTA purification. For the non-crosslinked conditions, no crosslinker was applied. Western blotting was performed using HRP-conjugated antibodies against specific affinity tags: anti-His, anti-Strep, or anti-Flag.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6895763/v1/1985b6f7079588aa456ff7a0.png"},{"id":84897617,"identity":"c4d441dd-37d3-4986-a236-4ab9d6101e9f","added_by":"auto","created_at":"2025-06-18 14:17:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":160464,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGrowth curve analysis of FtsH, YidC, HflKC expressed in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e Bl21 (DE3) cells. \u003c/strong\u003eOD 600 nm was monitored during the expression of proteins from plasmids coding for FtsH (pETDuet-FtsH, Amp+) \u003cstrong\u003e(A)\u003c/strong\u003e, YidC (pETDuet-YidC Amp+) \u003cstrong\u003e(B)\u003c/strong\u003e, FtsH and YidC \u003cstrong\u003e(C)\u003c/strong\u003e, HflKC (pRSFDuet-HflKC, Kan+) \u003cstrong\u003e(D)\u003c/strong\u003e, and FtsH, YidC, HflKC (pETDuet-FtsH-YidC Amp+ and pRSFDuet-HflK-HflC Kan+) \u003cstrong\u003e(E)\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6895763/v1/e33cb8d6478170b29556b85b.png"},{"id":85561784,"identity":"423021dc-c17e-4947-940f-19262ccbaf52","added_by":"auto","created_at":"2025-06-27 13:08:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1241186,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6895763/v1/b5099087-18c3-4223-8579-cae7c8b0bde4.pdf"},{"id":84897626,"identity":"9e358c84-358e-412b-bc21-710ee23319f4","added_by":"auto","created_at":"2025-06-18 14:17:19","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":8639397,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFtsHYidCrev.docx","url":"https://assets-eu.researchsquare.com/files/rs-6895763/v1/bb1cac197234615d3a6d29d7.docx"},{"id":84897615,"identity":"cdaddd5d-76e0-42ab-b953-127593df6a92","added_by":"auto","created_at":"2025-06-18 14:17:18","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":27172,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-6895763/v1/21e500a77230d3b2d0e6e0e5.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eRegulatory Interplay Between FtsH, HflKC and YidC in Bacterial Membrane Protein Biogenesis and Quality Control\u003c/p\u003e","fulltext":[{"header":"2. Introduction","content":"\u003cp\u003eThe quality control of membrane proteins are fundamental processes that ensure bacterial cellular homeostasis and functionality [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Membrane proteins are essential for diverse functions, including signal transduction, molecular transport, and energy generation [\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Their proper insertion, folding, and assembly in the lipid bilayer are crucial for maintaining membrane integrity [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Any defects in these processes, such as misfolding or improper insertion of membrane proteins, can disrupt cellular physiology, leading to proteotoxic stress and compromised viability [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Thus, bacteria have evolved sophisticated quality control mechanisms for membrane protein biogenesis and degradation.\u003c/p\u003e \u003cp\u003eFtsH and YidC are two key players in bacterial membrane protein homeostasis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. FtsH is an ATP-dependent AAA\u0026thinsp;+\u0026thinsp;metalloprotease that selectively degrades misfolded, unassembled, or damaged membrane proteins, preventing their accumulation [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Structurally, FtsH forms a hexameric complex with its proteolytic and ATPase domains facing the cytoplasm, enabling the recognition and unfolding of both cytosolic and membrane-embedded substrates prior to their degradation [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. FtsH function is modulated by interactions with HflK and HflC, which form a regulatory membrane-associated complex that influences substrate specificity and stability [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan additionalcitationids=\"CR15 CR16 CR17 CR18 CR19\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Structural studies have described the FtsH-HflK-HflC assembly as a bell- or nautilus-shaped complex that modulates FtsH activity by regulating substrate access across the inner membrane [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eYidC is a highly conserved membrane protein insertase that facilitates the integration, folding, and assembly of membrane proteins [\u003cspan additionalcitationids=\"CR22 CR23 CR24\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. It functions via both Sec-dependent and Sec-independent pathways, ensuring the correct localization and stability of membrane proteins [\u003cspan additionalcitationids=\"CR27 CR28 CR29 CR30 CR31 CR32\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In Sec-dependent pathways, YidC cooperates with the SecYEG translocon, while in Sec-independent pathways, it acts autonomously to insert proteins directly into the membrane [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Structurally, YidC consists of six transmembrane helices (TM1-TM6) and a hydrophilic groove that interacts with substrates, facilitating their integration into the lipid bilayer [\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBeyond protein insertion, YidC has been proposed to contribute to membrane protein quality control by collaborating with FtsH [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Together, these proteins may help recognize and degrade misassembled membrane proteins, preventing their accumulation and ensuring membrane integrity [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Although biochemical and genetic studies support their potential interplay, the molecular basis of their interaction remains unresolved, necessitating further structural and biochemical studies.\u003c/p\u003e \u003cp\u003eHere, we investigate the stability of the interactions between FtsH, YidC, and HflKC, aiming to determine whether they form a stable complex or exist as transiently interacting proteins. Previous studies have shown that FtsH, HflKC and YidC can be co-purified, suggesting possible physical interactions [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. However, it remains unclear whether this interaction is stable under physiological conditions or transient and regulated by additional factors. By assessing their stability and potential regulatory mechanisms, this study provides insights into bacterial membrane protein quality control complexes and their involvement in membrane homeostasis.\u003c/p\u003e"},{"header":"3. Materials and Methods","content":"\u003cp\u003e \u003cb\u003eExpression Systems and Plasmid Design\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTwo different expression systems were used for recombinant protein production. The first system, named FYHC Co-T expression system, employed a co-expression strategy using the pETDuet-1 (Novagen, 71146-3) and pRSFDuet-1 (Novagen, 71341) plasmids, both of which contain T7 promoters. The pETDuet-1 plasmid encoded YidC-10\u0026times;His and FtsH-3\u0026times;StrepTag II (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), while the pRSFDuet-1 plasmid carried untagged HflK and HflC (Supp. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These plasmids confer ampicillin and kanamycin resistance, respectively, allowing co-transformation into \u003cem\u003eEscherichia coli\u003c/em\u003e BL21 (DE3) or C43 (DE3) cells for simultaneous expression of multiple proteins. This system was primarily used for pull-down experiments, Western blotting, and mass spectrometry analyses.mThe second system was based on the pFYHC expression construct, which was generated using the ACEMBL system [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. It encodes FtsH-3\u0026times;StrepTag, HflKC-10\u0026times;His, and YidC-3\u0026times;Flag, each under the control of different inducible promoters: arabinose, T7, and trc (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The pFYHC plasmid was transformed into \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3) or C43 (DE3) cells, enabling regulated, stoichiometrically balanced expression of the target protein complex. Selection was maintained using ampicillin, kanamycin, and spectinomycin. This expression system was used in preparative purifications and structural studies.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMembrane Protein Expression, Isolation and Purification\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3) or C43 (DE3) cells harboring either the FYHC Co-T expression system or the pFYHC construct were cultured overnight in LB medium at 37\u0026deg;C with appropriate antibiotics to establish starter cultures. A 5 mL aliquot was inoculated into 500 mL of Terrific Broth (TB; Sigma-Aldrich, T0918) in 2 L baffled flasks, yielding a total culture volume of 6 L. Cultures were grown at 37\u0026deg;C until OD₆₀₀ reached\u0026thinsp;~\u0026thinsp;0.8. In the FYHC Co-T system, protein expression was induced with 0.4 mM IPTG and cultures were incubated either at 18\u0026deg;C for 18 h or at 37\u0026deg;C for 4 h. For the pFYHC system, expression was induced by sequential addition of 1 mM rhamnose, 0.4 mM IPTG, and 2% (w/v) arabinose, under the same temperature conditions.\u003c/p\u003e \u003cp\u003eCells were harvested by centrifugation at 5,000 \u0026times; g for 30 min at 4\u0026deg;C, and pellets were resuspended in lysis buffer containing 20 mM HEPES (pH 7.5), 150 mM NaCl, 8% glycerol, 2 mM MgCl₂, 1 mM PMSF (Roche 11359061001), one EDTA-free protease inhibitor tablet (Pierce, A32965), and 1 \u0026micro;g/mL benzonase (Millipore, E1014). The suspension was incubated at 4\u0026deg;C for 1 h with gentle agitation, followed by sonication. Lysates were clarified by centrifugation at 10,000 \u0026times; g for 1.5 h at 4\u0026deg;C. Membrane fractions were isolated by ultracentrifugation at 100,000 \u0026times; g for 1 h at 4\u0026deg;C and resuspended in solubilization buffer (same as lysis buffer plus 1% DDM). After 1 h solubilization with gentle agitation at 4\u0026deg;C, insoluble debris was removed by another ultracentrifugation step and supernatants were filtered through a 0.45 \u0026micro;m filter.\u003c/p\u003e \u003cp\u003eSolubilized membrane proteins were purified by affinity chromatography using either Ni-NTA or Strep-Tactin resin, depending on the tag. For Ni-NTA purification, His-tagged proteins were captured on Ni-NTA resin (Thermo Scientific, 25215) and eluted with 300 mM imidazole following standard wash steps with 10 mM and 50 mM imidazole. For Strep-tagged proteins, solubilized lysates were incubated with Strep-Tactin Sepharose (IBA, 2-1201-025) and eluted with 5 mM desthiobiotin in detergent-containing buffer.\u003c/p\u003e \u003cp\u003eFinal purification was performed using size-exclusion chromatography (SEC) on a Superose 6 Increase 3.2/300 column (Cytiva, 29091598) pre-equilibrated with SEC buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 2 mM MgCl₂, 0.05% DDM). Target proteins eluted as a single, symmetric peak (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and were collected for downstream biochemical and structural proteomics analyses.\u003c/p\u003e \u003cp\u003e \u003cb\u003eProtein Crosslinking\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCrosslinking experiments were conducted using Dithiobis(succinimidyl propionate) (DSP) and Disuccinimidyl dibutyric urea (DSBU) to stabilize protein\u0026ndash;protein interactions at different stages of the purification workflow. DSP (Pierce, A35393) was applied during membrane protein solubilization, immediately after membrane isolation. The membrane resuspension was incubated with 0.25 mM or 1 mM DSP (dissolved in DMSO) at room temperature for 1 hour. The reaction was quenched with 20 mM Tris-HCl (pH 7.5), followed by a 15-minute incubation. The DSP-treated membranes were subsequently solubilized with DDM and used for downstream analysis including affinity purification and crosslinking mass spectrometry (XL-MS).\u003c/p\u003e \u003cp\u003eDSBU (Thermo Scientific) was used post-purification, following Ni-NTA or Strep-Tactin chromatography, to capture stable oligomeric interactions in detergent-solubilized samples. Purified protein complexes were incubated with 3 mM DSBU (in DMSO) for 1 hour at room temperature. The reaction was quenched with 20 mM Tris-HCl (pH 7.5), achieved by addition of 1 M Tris-HCl stock, and incubated for 30 minutes at 4\u0026deg;C. Crosslinked samples were analyzed by SDS-PAGE, used for XL-MS, or subjected to SEC for further isolation of crosslinked oligomeric species.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSDS-PAGE and Western Blot Analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eProtein samples were analyzed by SDS-PAGE using 4\u0026ndash;12% Bolt\u0026trade; Bis-Tris Plus Mini Gels (Invitrogen) in MES running buffer at 180 V. Following electrophoresis, gels were stained with Coomassie Brilliant Blue to evaluate protein purity and expression efficiency. For Western blotting, proteins were transferred from unstained gels onto nitrocellulose membranes (0.2 \u0026micro;m pore size, Bio-Rad Trans-Blot Turbo system) under constant voltage (25 V, 7 minutes). Membranes were blocked with 3% (w/v) BSA in TBS-T for 1 hour at room temperature and subsequently incubated with HRP-conjugated antibodies against the relevant affinity tags: anti-His (Qiagen, 1:5000), anti-StrepTagII (Sigma-Aldrich, 1:5000), or anti-Flag (Rockland, 1:10000), all diluted in TBS-T. After incubation for 1 hour at room temperature, membranes were washed thoroughly with TBS-T to remove unbound antibodies. Protein bands were visualized using the Pierce\u0026trade; ECL Western Blotting Substrate and detected by chemiluminescence. Western blot analysis was used to monitor the expression of affinity-tagged constructs and confirm co-purification or crosslinked interactions in selected samples.\u003c/p\u003e \u003cp\u003e \u003cb\u003eXL-MS Analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFollowing membrane protein purification, samples were analyzed by mass spectrometry (MS) either in liquid form or excised from SDS-PAGE gels at University of Bristol Biomedical Sciences Proteomics Facility (Bristol, UK) and Scientific and Technological Research Council of T\u0026uuml;rkiye (T\u0026Uuml;BİTAK) National Metrology Institute (UME) Chemistry Group-Bioanalysis Laboratory (İstanbul, T\u0026uuml;rkiye). DSBU-crosslinked membrane proteins were purified and enzymatically digested using the FASP Protein Digestion Kit (Abcam) according to the manufacturer\u0026rsquo;s protocol. The protein sample was reduced with dithiothreitol (DTT), alkylated with iodoacetamide (IAA), and subsequently digested with trypsin in 50 mM ammonium bicarbonate at 37\u0026deg;C overnight. Following digestion, peptides were eluted, concentrated using a Speed-Vac, and resuspended in 5% v/v acetonitrile (ACN) and 0.1% v/v formic acid (FA) for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The analysis was conducted using a Thermo Scientific\u0026trade; UltiMate\u0026trade; 3000 RSLC Ultra Nano system coupled to a Thermo Scientific Q Exactive\u0026trade; HF mass spectrometer. Peptide separation was performed via reverse-phase chromatography on a C18 Easy-Spray column, with solution-derived peptides analyzed over 90 minutes and gel-derived peptides over 55 minutes. Data processing was conducted using Thermo Scientific Proteome Discoverer 2.5 software [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], while crosslinking MS (XL-MS) data analysis was performed using MSAnnika [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eGrowth Curve Analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the impact of heterologous expression of membrane protein complexes on bacterial growth, growth curve experiments were performed using \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3) cells transformed with different plasmid combinations. Constructs included individual expression of FtsH (pETDuet-FtsH, Amp^R), YidC (pETDuet-YidC, Amp^R), HflKC (pRSFDuet-HflK-HflC, Kan^R), and co-expression of all three proteins using pETDuet-FtsH-YidC (Amp^R) and pRSFDuet-HflK-HflC (Kan^R).\u003c/p\u003e \u003cp\u003eFreshly transformed colonies were grown overnight in 10 mL 2xYT medium supplemented with the appropriate antibiotics. The following day, 2 mL of each starter culture was inoculated into 200 mL of 2xYT in 2 L baffled flasks. Cultures were incubated at 37\u0026deg;C with shaking until OD₆₀₀ reached\u0026thinsp;~\u0026thinsp;0.6, at which point protein expression was induced using 0.4 mM IPTG. Bacterial growth was subsequently monitored by measuring OD₆₀₀ at 30-minute intervals for up to 6 hours.\u003c/p\u003e"},{"header":"4. Results","content":"\u003cp\u003e\u003cstrong\u003e4.1. Co-purification of FtsH, YidC, and HflKC Under Detergent-Solubilized Conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMembrane proteins were expressed using the FYHC Co-T expression system, which enables the co-expression of Strep-tagged FtsH, His-tagged YidC, and untagged HflK and HflC in E. coli. After membrane isolation and detergent solubilization, purification was performed via the His-tag on YidC using Ni\u0026sup2;⁺-NTA affinity chromatography. FtsH was detected in the eluates by SDS-PAGE (Fig. 1A). The FtsH signal decreased in later fractions of the gradient.\u003c/p\u003e\n\u003cp\u003eTo improve retention, 0.25 mM DSP was added during solubilization. Under these conditions, FtsH was observed in later elution fractions as well (Fig. 1B). In a separate purification experiment targeting the Strep-tag on FtsH, YidC was detected in the eluates without crosslinker (Fig. 1C).\u003c/p\u003e\n\u003cp\u003eHflK and HflC were not detected in SDS-PAGE analyses in the FYHC Co-T system. Since these proteins lacked affinity tags, Western blot analysis was not applicable. In the pFYHC system, in which HflK contains an N-terminal His-tag, HflK was detected by Western blot following Ni\u0026sup2;⁺-NTA purification (Fig. 1D). FtsH was also observed in the eluates, while FLAG-tagged YidC was not detected under the same conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2 MS-Based Identification of Proteins Co-purified with FtsH and YidC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtein complexes expressed under different conditions were purified using Ni-NTA affinity, Strep-Tactin affinity, and size-exclusion chromatography. The resulting samples were subsequently analyzed by SDS-PAGE and mass spectrometry (MS) to identify proteins co-purifying with FtsH and YidC.\u003c/p\u003e\n\u003cp\u003eMass spectrometry results revealed several proteins associated with the purified complexes, including GroEL [42\u0026ndash;44], DnaK [44\u0026ndash;46], ClpA [47], SlyD [48,49], and SecA [50,51] (Table 1). These proteins are involved in various aspects of proteostasis, including protein folding, membrane insertion, and degradation. GroEL and DnaK were consistently identified in samples where FtsH and YidC were co-expressed, particularly under overexpression conditions. The presence of ClpA, SlyD, and SecA varied depending on the expression system and purification approach.\u003c/p\u003e\n\u003cp\u003eIn samples expressing FtsH, HflKC, and YidC together, phage shock protein A (PspA) [52,53], a well-known stress response protein, was detected by MS. PspA was not observed in samples where only FtsH and YidC were expressed. HflK and HflC were detected at low levels in the MS datasets (Table 1), but were not detected by Western blot analysis (Figure 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.3 XL-MS Analysis of FtsH\u0026ndash;YidC Interaction and Associated Protein Network\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXL-MS analysis identified a broad network of protein associations involving FtsH and YidC (Table 2). Two crosslinks were detected between these proteins: residue 1 of YidC, located in the cytoplasm, interacted with residues 33 and 34 of FtsH, both positioned in the periplasmic region. These observations suggest that the FtsH\u0026ndash;YidC interaction is not stable under detergent-solubilized conditions and likely occurs transiently.\u003c/p\u003e\n\u003cp\u003eBeyond this interaction, FtsH was found to associate with several other proteins. In the cytoplasm, it interacted with SodA [54] at residue 138 of FtsH and 119 of SodA, with MdtP [55] at residue 60 of FtsH and 407 of MdtP, and with RpoA [56] at residue 391 of FtsH and 309 of RpoA. HflK [14\u0026ndash;16] was crosslinked to FtsH at residues 338 and 486. In the periplasm, FtsH interacted with RuvA [57] at residue 332 of FtsH and 2 of RuvA, and with FdoG [58] at residue 332 of FtsH and 330 of FdoG.\u003c/p\u003e\n\u003cp\u003eYidC was associated with CsiE [59] through residue 1 of YidC and 373 of CsiE, and with PutP [60] through residue 377 of YidC and 482 of PutP. Additionally, a crosslink was identified between YidC residue 401 and RpoH residue 173, a stress-response factor previously reported to interact with FtsH [61].\u003c/p\u003e\n\u003cp\u003eAlthough a stable complex between FtsH and YidC was not detected, the interaction data support the view that both proteins are components of broader cellular pathways, contributing to protein quality control, membrane protein biogenesis, and stress response through multiple independent associations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.4. Assessment of Bacterial Growth Under Co-Expression of FtsH, YidC, and HflKC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring recombinant protein production, it was observed that \u003cem\u003eE. coli\u003c/em\u003e cells expressing FtsH and YidC exhibited normal growth profiles, whereas cells co-expressing FtsH, YidC, and HflKC showed delayed growth. To investigate this observation systematically, bacterial growth was monitored under five different expression conditions using the FYHC Co-T expression system: (i) FtsH (pETDuet-FtsH), (ii) YidC (pETDuet-YidC), (iii) FtsH and YidC (pETDuet-FtsH + pETDuet-YidC), (iv) HflKC (pRSFDuet-HflK-HflC), and (v) FtsH, YidC, and HflKC (pETDuet-YidC-FtsH + pRSFDuet-HflK-HflC).\u003c/p\u003e\n\u003cp\u003eBacterial cultures were grown in 2\u0026times;YT medium at 37 \u0026deg;C with continuous shaking at 100, 150, or 200 rpm. Cell growth was monitored throughout the entire cultivation, including both pre-induction and post-induction phases. Protein expression was induced with 0.4 mM IPTG when cultures reached OD600 = 0.6. As shown in Figure 2A\u0026ndash;C, expression of FtsH, YidC, or their combination did not adversely affect cell growth under the tested conditions. In contrast, expression of HflKC alone (Figure 2D) or in combination with FtsH and YidC (Figure 2E) led to a significant reduction in growth, particularly at higher agitation speeds.\u003c/p\u003e\n\u003cp\u003eThese results confirm that the delayed growth observed during co-expression of FtsH, YidC, and HflKC is linked to HflKC overexpression.\u003c/p\u003e"},{"header":"5. Discussion","content":"\u003cp\u003eThis study provides observations on the interactions between the membrane proteins YidC, FtsH, and HflKC, which are known to play roles in protein quality control, membrane protein biogenesis, and cellular homeostasis. The results indicate that the interaction between FtsH and YidC is likely transient, and may depend on cellular context or the presence of specific substrates. The identification of several co-purified proteins suggests that both FtsH and YidC are part of a broader protein interaction network associated with membrane protein folding and degradation.\u003c/p\u003e \u003cp\u003eAdditionally, under YidC overexpression conditions, HflKC was only minimally detected in purified samples, whereas FtsH and YidC co-purified. This observation may reflect a competitive or mutually exclusive association with FtsH, although further investigation is required to clarify the underlying mechanism. The bacterial growth assays showed a delayed growth phenotype upon co-expression of FtsH, YidC, and HflKC. While the exact cause of this delay is unclear, the data may point to a role for HflKC in cellular responses to oxygen availability, suggesting a possible link between HflKC expression and oxygen metabolism under overexpression conditions.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e5.1 Interaction Dynamics Between YidC and FtsH\u003c/h2\u003e \u003cp\u003eIn the FYHC Co-T expression system, membrane proteins were co-expressed using pETDuet-YidC-FtsH and pRSFDuet-HflK-HflC plasmids. Under detergent-solubilized conditions, Ni²⁺-NTA affinity purification targeting His₁₀-tagged YidC resulted in co-purification of Strep-tagged FtsH even in the absence of a chemical crosslinker (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). This finding contrasts with an earlier study, where co-purification of FtsH and YidC required chemical crosslinking [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The gradual loss of FtsH signal across elution fractions suggests that the interaction may be transient or sensitive to purification conditions. Upon addition of 0.25 mM DSP as a crosslinker, FtsH was retained more efficiently (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), indicating stabilization of the interaction. Consistently, when the purification was performed using Strep-Tactin affinity chromatography targeting Strep-tagged FtsH, His-tagged YidC was co-purified even without a crosslinker (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Together, these results support that FtsH and YidC interact under detergent-solubilized conditions, though the interaction may be condition-dependent.\u003c/p\u003e \u003cp\u003eHflK and HflC were not detected in these experiments using the FYHC Co-T expression system. As this setup did not include affinity tags for either HflK or HflC, their detection by Western blot was not feasible (Supp. Figure\u0026nbsp;3). Affinity tagging for HflK was incorporated in the pFYHC expression system, where HflK carries an N-terminal His₁₀ tag. Under these conditions, HflK could be successfully detected following Ni²⁺-NTA purification (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). FtsH was also detected via anti-Strep-tag Western blot, although at lower levels than those observed with the FYHC Co-T system. Notably, YidC-FLAG was not detected under these conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e5.2 Potential Competition Between HflKC and YidC for FtsH Binding\u003c/h2\u003e \u003cp\u003eHflK and HflC were not detected in SDS-PAGE analysis when co-expressed with FtsH and YidC using the FYHC Co-T expression system (Supp. Figure\u0026nbsp;3A). Conversely, YidC was not observed in Western blot analysis when expressed with FtsH, HflK, and HflC from the pFYHC single-plasmid expression system (Supp. Figure\u0026nbsp;3D). These findings suggest a competitive relationship between YidC and HflKC for association with FtsH under detergent-solubilized conditions. The low detection levels of HflK and HflC in the mass spectrometry analysis of Ni-NTA eluates in the FYHC Co-T system, where YidC was used for pull-down, further support this interpretation (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePrevious studies reported that YidC depletion leads to increased levels of FtsH, HflK, and HflC, which was proposed to reflect a compensatory response to membrane stress [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. In contrast, the current findings indicate that YidC overexpression may reduce HflKC recruitment, potentially altering the interaction network involving FtsH. Since HflK and HflC are known to regulate FtsH proteolytic activity [\u003cspan additionalcitationids=\"CR15 CR16 CR17\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e–\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], changes in their association could influence the efficiency or substrate preference of membrane protein degradation. Further investigation is required to understand how the presence or relative abundance of YidC modulates the interaction between FtsH and its regulatory subunits HflK and HflC under different cellular or experimental conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e5.3 Protein Interactions Identified by XL-MS Suggest Broader Functional Associations\u003c/h2\u003e \u003cp\u003eOur XL-MS analysis identified multiple proteins that interact with FtsH and YidC, suggesting their involvement in a broader proteostasis and membrane biogenesis network (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). For FtsH, crosslinked partners included proteins with roles in oxidative stress response (e.g., SodA) [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e], drug resistance (e.g., MdtP) [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], and RNA transcription (e.g., RpoA) [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. The interaction between FtsH and SodA may be linked to the regulation of oxidative damage, in line with previous studies reporting FtsH’s role in stress adaptation. Similarly, the connection with MdtP supports a role in maintaining membrane integrity under toxic compound exposure, while the link to RpoA might indicate transcriptional regulation under proteotoxic conditions.\u003c/p\u003e \u003cp\u003eIn the periplasm, FtsH interacted with proteins such as RuvA [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], involved in DNA recombination and repair, and FdoG [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], a component of formate dehydrogenase complex, pointing to additional functions in DNA maintenance and metabolism. These findings are consistent with earlier reports that FtsH’s activity extends beyond membrane protein degradation, contributing to cellular homeostasis under stress conditions.\u003c/p\u003e \u003cp\u003eYidC was found to interact with proteins related to membrane protein insertion and folding such as PutP [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e], reflecting its central role in co-translational insertion and stabilization of membrane proteins. The observed interaction between YidC and RpoH, a sigma factor that activates stress response genes, aligns with its role in adaptive proteostasis. Interestingly, while the interaction between FtsH and RpoH is well-documented [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e], the link between YidC and RpoH may reflect a coordinated regulation of membrane stress responses.\u003c/p\u003e \u003cp\u003eCollectively, these interactions suggest that FtsH and YidC are not isolated actors but part of a dynamic and interconnected regulatory network, which ensures proper membrane protein folding, quality control, and stress response.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e5.4 Implications of HflKC Expression in Bacterial Adaptation to Oxygen Availability\u003c/h2\u003e \u003cp\u003eBacterial growth analyses suggest that HflKC overexpression affects cell proliferation under different oxygenation conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, E). In particular, delayed growth was observed at higher agitation rates when HflKC was co-expressed, indicating a potential link between HflKC expression and oxygen-dependent metabolic adaptation. These findings are consistent with a recent study reporting that deletion of hflK or hflC alters ubiquinone biosynthesis, thereby affecting aerobic respiration [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. In this context, HflKC overexpression may influence components of the respiratory chain or regulatory networks that respond to oxygen availability.\u003c/p\u003e \u003cp\u003eThe observed growth pattern at lower agitation rates, which are typically associated with reduced oxygen transfer, suggests that elevated levels of HflKC might alter the balance of cellular pathways sensitive to oxidative conditions. Given the known interaction of HflKC with the FtsH protease [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], it is possible that HflKC modulates the turnover of proteins involved in respiration or redox homeostasis. However, further analysis is needed to determine whether HflKC directly impacts components such as cytochrome oxidase complexes or ubiquinone levels.\u003c/p\u003e \u003cp\u003eIncreased oxygen levels can elevate reactive oxygen species (ROS) production, leading to oxidative stress [\u003cspan additionalcitationids=\"CR66\" citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e–\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Some SPFH domain-containing proteins, including eukaryotic prohibitins, have been implicated in managing oxidative stress and mitochondrial homeostasis [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Whether bacterial HflKC plays a comparable role remains to be determined. Notably, FtsH is involved in degrading stress response regulators, such as RpoS, which coordinate bacterial responses to oxidative damage [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. It is therefore conceivable that HflKC may influence FtsH substrate specificity or protease dynamics under oxidative stress conditions.\u003c/p\u003e \u003cp\u003eInterestingly, YidC overexpression was associated with a reduced detection of HflK and HflC according to MS-based interaction analyses [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. This observation raises the possibility that elevated YidC levels may indirectly affect the ability of HflKC to interact with FtsH. Since FtsH function has been linked to the degradation of several stress regulators, including those involved in oxidative response, competition between YidC and HflKC for FtsH binding may represent an additional regulatory mechanism modulating bacterial adaptation to oxidative conditions.\u003c/p\u003e \u003cp\u003eOverall, these observations suggest that HflKC contributes to cellular adaptation mechanisms that extend beyond protein quality control. Its expression appears to affect bacterial growth in response to oxygen availability, possibly through modulation of FtsH activity. Furthermore, competition with YidC for FtsH binding may influence the composition and function of membrane proteostasis machinery under stress conditions. Further biochemical and structural studies are needed to clarify the molecular basis of these interactions and their physiological consequences.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study presents observations on the interactions between FtsH, YidC, and HflKC, with a focus on their potential roles in membrane protein quality control. FtsH and YidC were found to co-purify under detergent-solubilized conditions without the use of crosslinkers, suggesting a possible interaction that may be transient and context-dependent. The lack of a stable association is consistent with the detection of multiple additional proteins co-purifying with FtsH and YidC, several of which are involved in proteostasis-related pathways. Growth-based analyses indicate that co-expression of FtsH, YidC, and HflKC can influence bacterial proliferation, particularly under varying oxygenation conditions, pointing to the importance of regulated expression levels. The low recovery of HflKC in the presence of YidC may reflect a potential competition for FtsH binding, although further investigation is required to clarify this relationship.\u003c/p\u003e\u003cp\u003eOverall, these findings provide preliminary insights into the network of interactions surrounding FtsH, YidC, and HflKC, and highlight the need for additional studies to better define the molecular mechanisms underlying their coordination in bacterial cells.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eM.\u0026Ccedil;., C.S., and B.V.K. conceived the study. S.Z. generated the pFYHC plasmid. M.\u0026Ccedil;. performed all other experiments, and wrote the manuscript with input from C.S. and B.V.K. All authors have read and agreed to the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eM.\u0026Ccedil;., and B.V.K. were funded by the Scientific and Technological Council of T\u0026uuml;rkiye (T\u0026Uuml;BİTAK) BİDEB 2232 International Outstanding Researchers Program (Project No: 118C225). M.\u0026Ccedil;. was also funded by T\u0026Uuml;BİTAK 2211-A National Graduate Fellowship Program. C.S. acknowledges funding by a BBSRC Responsive Mode Grant (BB/P000940/1) and a Wellcome Trust Investigator Grant (210701/Z/18/Z). M.\u0026Ccedil;. was also supported by a T\u0026Uuml;BİTAK 2214-A - International Research Fellowship Program for carrying out a part of his PhD thesis studies at the University of Bristol, School of Biochemistry. We acknowledge Dr. S\u0026uuml;reyya \u0026Ouml;zcan and Hatice Akkulak from METU for their help in XL-MS analyses.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData is provided within the manuscript or supplementary information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eE. Van Bloois, H.L. Dekker, L. Fröderberg, E.N.G. Houben, M.L. Urbanus, C.G. De Koster, J.-W. De Gier, J. Luirink, Detection of cross‐links between FtsH, YidC, HflK/C suggests a linked role for these proteins in quality control upon insertion of bacterial inner membrane proteins, FEBS Lett. 582 (2008) 1419–1424. https://doi.org/10.1016/j.febslet.2008.02.082.\u003c/li\u003e\n\u003cli\u003eY. Akiyama, Quality Control of Cytoplasmic Membrane Proteins in Escherichia coli, J. Biochem. (Tokyo) 146 (2009) 449–454. https://doi.org/10.1093/jb/mvp071.\u003c/li\u003e\n\u003cli\u003eD.W. Watkins, S.L. Williams, I. 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S.K, A comprehensive review on latent role of stress proteins in antibiotic resistance, The Microbe 4 (2024) 100151. https://doi.org/10.1016/j.microb.2024.100151.\u003c/li\u003e\n \u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6895763/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6895763/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe quality control of membrane proteins is essential for maintaining cellular homeostasis, as misfolded or damaged proteins can disrupt essential cellular functions. FtsH, a membrane-bound AAA\u0026thinsp;+\u0026thinsp;metalloprotease, is central to bacterial proteostasis, responsible for degrading misfolded or damaged membrane proteins. YidC, a membrane protein insertase, facilitates the folding, insertion, and assembly of membrane proteins into the lipid bilayer. This study investigates the physical interaction between FtsH, HflKC, and YidC, indicating a potential functional relationship between these proteins in maintaining membrane protein quality control in bacteria.\u003c/p\u003e \u003cp\u003eOverexpression of YidC in \u003cem\u003eEscherichia coli\u003c/em\u003e led to a disruption in the interaction between FtsH and its regulatory proteins HflK and HflC, possibly due to competition for binding sites. This was supported by the depletion of HflK and HflC in both Western blot and mass spectrometry analyses using detergent-solubilized membrane extracts. Additionally, the co-overexpression of FtsH and YidC induced cellular stress, as evidenced by the increased recruitment of stress-related proteins such as GroEL and DnaK. These findings suggest that FtsH and YidC collaborate in membrane protein biogenesis and participate in stress-responsive regulatory mechanisms that contribute to protein homeostasis.\u003c/p\u003e","manuscriptTitle":"Regulatory Interplay Between FtsH, HflKC and YidC in Bacterial Membrane Protein Biogenesis and Quality Control","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-18 14:17:14","doi":"10.21203/rs.3.rs-6895763/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":"18e8a15f-0f9c-48f1-b8b8-e771c348141b","owner":[],"postedDate":"June 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-27T13:08:31+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-18 14:17:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6895763","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6895763","identity":"rs-6895763","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}