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1.
The YidC/Oxa1/Alb3 family proteins are involved in membrane protein biogenesis in bacteria, mitochondria, and chloroplasts. Recent studies show that YidC uses a channel-independent mechanism to insert a class of membrane proteins into the membrane. Bacillus subtilis has two YidC homologs, SpoIIIJ (YidC1) and YidC2 (YqjG); the former is expressed constitutively, while the latter is induced when the SpoIIIJ activity is compromised. MifM is a substrate of SpoIIIJ, and its failure in membrane insertion is accompanied by stable ribosome stalling on the mifM-yidC2 mRNA, which ultimately facilitates yidC2 translation. While mutational inactivation of SpoIIIJ has been known to induce yidC2 expression, here, we show that the level of this induction is lower than that observed when the membrane insertion signal of MifM is defective. Moreover, this partial induction of YidC2 translation is lowered further when YidC2 is overexpressed in trans. These results suggest that YidC2 is able to insert MifM into the membrane and to release its translation arrest. Thus, under SpoIIIJ-deficient conditions, YidC2 expression is subject to MifM-mediated autogenous feedback repression. Our results show that YidC2 uses a mechanism that is virtually identical to that used by SpoIIIJ; Arg75 of YidC2 in its intramembrane yet hydrophilic cavity is functionally indispensable and requires negatively charged residues of MifM as an insertion substrate. From these results, we conclude that MifM monitors the total activities of the SpoIIIJ and the YidC2 pathways to control the synthesis of YidC2 and to maintain the cellular capability of the YidC mode of membrane protein biogenesis. 相似文献
2.
《Journal of molecular biology》2023,435(15):168172
YidC is a member of the YidC/Oxa1/Alb3 protein family that is crucial for membrane protein biogenesis in the bacterial plasma membrane. While YidC facilitates the folding and complex assembly of membrane proteins along with the Sec translocon, it also functions as a Sec-independent membrane protein insertase in the YidC-only pathway. However, little is known about how membrane proteins are recognized and sorted by these pathways, especially in Gram-positive bacteria, for which only a small number of YidC substrates have been identified to date. In this study, we aimed to identify Bacillus subtilis membrane proteins whose membrane insertion depends on SpoIIIJ, the primary YidC homolog in B. subtilis. We took advantage of the translation arrest sequence of MifM, which can monitor YidC-dependent membrane insertion. Our systematic screening identified eight membrane proteins as candidate SpoIIIJ substrates. Results of our genetic study also suggest that the conserved arginine in the hydrophilic groove of SpoIIIJ is crucial for the membrane insertion of the substrates identified here. However, in contrast to MifM, a previously identified YidC substrate, the importance of the negatively charged residue on the substrates for membrane insertion varied depending on the substrate. These results suggest that B. subtilis YidC uses substrate-specific interactions to facilitate membrane insertion. 相似文献
3.
Yuanyuan Chen Raunak Soman Sri Karthika Shanmugam Andreas Kuhn Ross E. Dalbey 《The Journal of biological chemistry》2014,289(51):35656-35667
Recently, the structure of YidC2 from Bacillus halodurans revealed that the conserved positively charged residue within transmembrane segment one (at position 72) is located in a hydrophilic groove that is embedded in the inner leaflet of the lipid bilayer. The arginine residue was essential for the Bacillus subtilis SpoIIIJ (YidC1) to insert MifM and to complement a SpoIIIJ mutant strain. Here, we investigated the importance of the conserved positively charged residue for the function of the Escherichia coli YidC, Streptococcus mutans YidC2, and the chloroplast Arabidopsis thaliana Alb3. Like the Gram-positive B. subtilis SpoIIIJ, the conserved arginine was required for functioning of the Gram-positive S. mutans YidC2 and was necessary to complement the E. coli YidC depletion strain and to promote insertion of a YidC-dependent membrane protein synthesized with one but not two hydrophobic segments. In contrast, the conserved positively charged residue was not required for the E. coli YidC or the A. thaliana Alb3 to functionally complement the E. coli YidC depletion strain or to promote insertion of YidC-dependent membrane proteins. Our results also show that the C-terminal half of the helical hairpin structure in cytoplasmic loop C1 is important for the activity of YidC because various deletions in the region either eliminate or impair YidC function. The results here underscore the importance of the cytoplasmic hairpin region for YidC and show that the arginine is critical for the tested Gram-positive YidC homolog but is not essential for the tested Gram-negative and chloroplast YidC homologs. 相似文献
4.
Manfred J. Saller Fabrizia Fusetti Arnold J. M. Driessen 《Journal of bacteriology》2009,191(21):6749-6757
In all domains of life Oxa1p-like proteins are involved in membrane protein biogenesis. Bacillus subtilis, a model organism for gram-positive bacteria, contains two Oxa1p homologs: SpoIIIJ and YqjG. These molecules appear to be mutually exchangeable, although SpoIIIJ is specifically required for spore formation. SpoIIIJ and YqjG have been implicated in a posttranslocational stage of protein secretion. Here we show that the expression of either spoIIIJ or yqjG functionally compensates for the defects in membrane insertion due to YidC depletion in Escherichia coli. Both SpoIIIJ and YqjG complement the function of YidC in SecYEG-dependent and -independent membrane insertion of subunits of the cytochrome o oxidase and F1Fo ATP synthase complexes. Furthermore, SpoIIIJ and YqjG facilitate membrane insertion of F1Fo ATP synthase subunit c from both E. coli and B. subtilis into inner membrane vesicles of E. coli. When isolated from B. subtilis cells, SpoIIIJ and YqjG were found to be associated with the entire F1Fo ATP synthase complex, suggesting that they have a role late in the membrane assembly process. These data demonstrate that the Bacillus Oxa1p homologs have a role in membrane protein biogenesis rather than in protein secretion.The YidC/OxaI/Alb3 protein family plays a crucial role in membrane protein biogenesis by facilitating the insertion of a specific subset of membrane proteins (for reviews, see references 20 and 24). In mitochondria, the OxaI protein is essential for insertion of both nucleus- and mitochondrion-encoded proteins into the inner membrane (39). The OxaI homolog of Escherichia coli, designated YidC, is known to play a role in two different membrane protein insertion pathways. Some proteins, such as subunit c of the rotary domain of the F1Fo ATP synthase (Foc) (47), MscL (10), M13 (34), and Pf3 (5), insert via the YidC-only pathway. YidC also functions in concert with the protein-conducting channel SecYEG in membrane insertion of subunit a of cytochrome o oxidase (CyoA) (8, 44) and subunit a of the F1Fo ATP synthase (23, 53, 54). In addition, YidC has been implicated in the folding of a membrane-inserted lactose permease (30) and the binding protein-dependent maltose ABC transporter (50).Members of the YidC/OxaI/Alb3 protein family are found in all three domains of life, and the number of paralogs per cell or organelle ranges from one (most gram-negative bacteria) to six (Arabidopsis thaliana). The length of Oxa1p-like proteins varies considerably, from just over 200 amino acids (in most gram-positive bacteria) to 795 amino acids (Chlamydophila pneumoniae) (52). However, in all Oxa1p proteins, a conserved region consisting of about 200 amino acids can be recognized, which comprises five putative transmembrane segments, as experimentally demonstrated for E. coli YidC (33). Overall, the amino acid sequence conservation among Oxa1p homologs is low (17). Bacillus subtilis contains two membrane proteins, SpoIIIJ and YqjG, with significant similarity to proteins belonging to the YidC/OxaI/Alb3 family. Previous gene inactivation analysis showed that a single paralog is sufficient for cell viability during vegetative growth of B. subtilis, while a double knockout led to a lethal phenotype (29, 41). SpoIIIJ is essential for activation of a prespore-specific sigma factor (9, 36), and cells with spoIIIJ deleted are incapable of spore formation. Sporulation is blocked at stage III, directly after completion of prespore engulfment (9). YqjG cannot complement SpoIIIJ in this process, but the exact reason for the specific requirement for SpoIIIJ is unknown. Previous studies indicated that the stability of various secretory proteins (e.g., LipA and PhoA) was strongly affected under YqjG- and SpoIIIJ-limiting conditions, while the insertion or stability of a number of membrane proteins tested appeared to be unaffected (41). These data suggested that YqjG and SpoIIIJ, unlike the other Oxa1p-like proteins, play a role in protein secretion. Here we show that both YidC homologs in B. subtilis complement the E. coli growth defect due to a YidC depletion and functionally replace YidC in Sec-dependent and -independent membrane protein insertion. In vitro insertion assays demonstrated that membrane insertion of Foc of both E. coli and B. subtilis is mediated by SpoIIIJ and YqjG. In addition, the entire F1Fo ATP synthase of B. subtilis was found to copurify with both SpoIIIJ and YqjG, suggesting that these proteins have a role in a late stage of the assembly of this membrane protein complex. 相似文献
5.
Yu Z Lavèn M Klepsch M de Gier JW Bitter W van Ulsen P Luirink J 《Journal of bacteriology》2011,193(19):5242-5251
YidC has an essential but poorly defined function in membrane protein insertion and folding in bacteria. The yidC gene is located in a gene cluster that is highly conserved in Gram-negative bacteria, the gene order being rpmH, rnpA, yidD, yidC, and trmE. Here, we show that Escherichia coli yidD, which overlaps with rnpA and is only 2 bp upstream of yidC, is expressed and localizes to the inner membrane, probably through an amphipathic helix. Inactivation of yidD had no discernible effect on cell growth and viability. However, compared to control cells, ΔyidD cells were affected in the insertion and processing of three YidC-dependent inner membrane proteins. Furthermore, in vitro cross-linking showed that YidD is in proximity of a nascent inner membrane protein during its localization in the Sec-YidC translocon, suggesting that YidD might be involved in the insertion process. 相似文献
6.
Yanping Geng Jeanine de Keyzer Dirk-Jan Scheffers Arnold J. M. Driessen 《Journal of bacteriology》2014,196(7):1318-1324
Proteins of the YidC/OxaI/Alb3 family play a crucial role in the insertion, folding, and/or assembly of membrane proteins in prokaryotes and eukaryotes. Bacillus subtilis has two YidC-like proteins, denoted SpoIIIJ and YqjG. SpoIIIJ and YqjG are largely exchangeable in function, but SpoIIIJ has a unique role in sporulation, while YqjG stimulates competence development. To obtain more insight into the regions important for the sporulation specificity of SpoIIIJ, a series of SpoIIIJ/YqjG chimeras was constructed. These chimeras were tested for functionality during vegetative growth and for their ability to complement the sporulation defect of a spoIIIJ deletion strain. The data suggest an important role for the domain comprising transmembrane segment 2 (TMS2) and its flanking loops in sporulation specificity, with lesser contributions to specificity by TMS1 and TMS3. 相似文献
7.
Dong Y Palmer SR Hasona A Nagamori S Kaback HR Dalbey RE Brady LJ 《Journal of bacteriology》2008,190(7):2458-2469
Oxa/YidC/Alb family proteins are chaperones involved in membrane protein insertion and assembly. Streptococcus mutans has two YidC paralogs. Elimination of yidC2, but not yidC1, results in stress sensitivity with decreased membrane-associated F(1)F(o) ATPase activity and an inability to initiate growth at low pH or high salt concentrations (A. Hasona, P. J. Crowley, C. M. Levesque, R. W. Mair, D. G. Cvitkovitch, A. S. Bleiweis, and L. J. Brady, Proc. Natl. Acad. Sci. USA 102:17466-17471, 2005). We now show that Escherichia coli YidC complements for acid tolerance, and partially for salt tolerance, in S. mutans lacking yidC2 and that S. mutans YidC1 or YidC2 complements growth in liquid medium, restores the proton motive force, and functions to assemble the F(1)F(o) ATPase in a previously engineered E. coli YidC depletion strain (J. C. Samuelson, M. Chen, F. Jiang, I. Moller, M. Wiedmann, A. Kuhn, G. J. Phillips, and R. E. Dalbey, Nature 406:637-641, 2000). Both YidC1 and YidC2 also promote membrane insertion of known YidC substrates in E. coli; however, complete membrane integrity is not fully replicated, as evidenced by induction of phage shock protein A. While both function to rescue E. coli growth in broth, a different result is observed on agar plates: growth of the YidC depletion strain is largely restored by 247YidC2, a hybrid S. mutans YidC2 fused to the YidC targeting region, but not by a similar chimera, 247YidC1, nor by YidC1 or YidC2. Simultaneous expression of YidC1 and YidC2 improves complementation on plates. This study demonstrates functional redundancy between YidC orthologs in gram-negative and gram-positive organisms but also highlights differences in their activity depending on growth conditions and species background, suggesting that the complete functional spectrum of each is optimized for the specific bacteria and environment in which they reside. 相似文献
8.
Luísa C?rte Filipa Valente Mónica Serrano Cláudio M. Gomes Charles P. Moran Jr Adriano O. Henriques 《PloS one》2014,9(8)
During sporulation in Bacillus subtilis, the onset of activity of the late forespore-specific sigma factor σG coincides with completion of forespore engulfment by the mother cell. At this stage, the forespore becomes a free protoplast, surrounded by the mother cell cytoplasm and separated from it by two membranes that derive from the asymmetric division septum. Continued gene expression in the forespore, isolated from the surrounding medium, relies on the SpoIIIA-SpoIIQ secretion system assembled from proteins synthesised both in the mother cell and in the forespore. The membrane protein insertase SpoIIIJ, of the YidC/Oxa1/Alb3 family, is involved in the assembly of the SpoIIIA-SpoIIQ complex. Here we show that SpoIIIJ exists as a mixture of monomers and dimers stabilised by a disulphide bond. We show that residue Cys134 within transmembrane segment 2 (TM2) of SpoIIIJ is important to stabilise the protein in the dimeric form. Labelling of Cys134 with a Cys-reactive reagent could only be achieved under stringent conditions, suggesting a tight association at least in part through TM2, between monomers in the membrane. Substitution of Cys134 by an Ala results in accumulation of the monomer, and reduces SpoIIIJ function in vivo. Therefore, SpoIIIJ activity in vivo appears to require dimer formation. 相似文献
9.
Reconstitution of Sec-dependent membrane protein insertion: nascent FtsQ interacts with YidC in a SecYEG-dependent manner 总被引:3,自引:0,他引:3
Martin van der Laan Edith N.G. Houben Nico Nouwen Joen Luirink Arnold J.M. Driessen 《EMBO reports》2001,2(6):519-523
The inner membrane protein YidC is associated with the preprotein translocase of Escherichia coli and contacts transmembrane segments of nascent inner membrane proteins during membrane insertion. YidC was purified to homogeneity and co-reconstituted with the SecYEG complex. YidC had no effect on the SecA/SecYEG-mediated translocation of the secretory protein proOmpA; however, using a crosslinking approach, the transmembrane segment of nascent FtsQ was found to gain access to YidC via SecY. These data indicate the functional reconstitution of the initial stages of YidC-dependent membrane protein insertion via the SecYEG complex. 相似文献
10.
Members of the evolutionary conserved Oxa1/Alb3/YidC family have been shown to play an important role in membrane protein insertion, folding and/or assembly. Bacillus subtilis contains two YidC-like proteins, denoted as SpoIIIJ and YqjG. SpoIIIJ and YqjG are largely exchangeable, but SpoIIIJ is essential for spore formation and YqjG cannot complement this activity. To elucidate the role of YqjG, we determined the membrane proteome and functional aspects of B. subtilis cells devoid of SpoIIIJ, YqjG or both. The data show that SpoIIIJ and YqjG have complementary functions in membrane protein insertion and assembly. The reduced levels of F(1)F(O) ATP synthase in cells devoid of both SpoIIIJ and YqjG are due to a defective assembly of the F(1)-domain onto the F(0)-domain. Importantly, for the first time, a specific function is demonstrated for YqjG in genetic competence development. 相似文献
11.
Yanping Geng Alexej Kedrov Joseph J. Caumanns Alvaro H. Crevenna Don C. Lamb Roland Beckmann Arnold J. M. Driessen 《The Journal of biological chemistry》2015,290(28):17250-17261
Members of the YidC/Oxa1/Alb3 protein family mediate membrane protein insertion, and this process is initiated by the assembly of YidC·ribosome nascent chain complexes at the inner leaflet of the lipid bilayer. The positively charged C terminus of Escherichia coli YidC plays a significant role in ribosome binding but is not the sole determinant because deletion does not completely abrogate ribosome binding. The positively charged cytosolic loops C1 and C2 of YidC may provide additional docking sites. We performed systematic sequential deletions within these cytosolic domains and studied their effect on the YidC insertase activity and interaction with translation-stalled (programmed) ribosome. Deletions within loop C1 strongly affected the activity of YidC in vivo but did not influence ribosome binding or substrate insertion, whereas loop C2 appeared to be involved in ribosome binding. Combining the latter deletion with the removal of the C terminus of YidC abolished YidC-mediated insertion. We propose that these two regions play an crucial role in the formation and stabilization of an active YidC·ribosome nascent chain complex, allowing for co-translational membrane insertion, whereas loop C1 may be involved in the downstream chaperone activity of YidC or in other protein-protein interactions. 相似文献
12.
Zht Cheng Wu Jeanine de Keyzer Greetje A. Berrelkamp-Lahpor Arnold J. M. Driessen 《Journal of bacteriology》2013,195(19):4545-4551
The YidC/OxaI/Alb3 family of membrane proteins is involved in the biogenesis of integral membrane proteins in bacteria, mitochondria, and chloroplasts. Gram-positive bacteria often contain multiple YidC paralogs that can be subdivided into two major classes, namely, YidC1 and YidC2. The Streptococcus mutans YidC1 and YidC2 proteins possess C-terminal tails that differ in charges (+9 and + 14) and lengths (33 and 61 amino acids). The longer YidC2 C terminus bears a resemblance to the C-terminal ribosome-binding domain of the mitochondrial OxaI protein and, in contrast to the shorter YidC1 C terminus, can mediate the interaction with mitochondrial ribosomes. These observations have led to the suggestion that YidC1 and YidC2 differ in their abilities to interact with ribosomes. However, the interaction with bacterial translating ribosomes has never been addressed. Here we demonstrate that Escherichia coli ribosomes are able to interact with both YidC1 and YidC2. The interaction is stimulated by the presence of a nascent membrane protein substrate and abolished upon deletion of the C-terminal tail, which also abrogates the YidC-dependent membrane insertion of subunit c of the F1F0-ATPase into the membrane. It is concluded that both YidC1 and YidC2 interact with ribosomes, suggesting that the modes of membrane insertion by these membrane insertases are similar. 相似文献
13.
YidC, an assembly site for polytopic Escherichia coli membrane proteins located in immediate proximity to the SecYE translocon and lipids 总被引:2,自引:0,他引:2
Konstanze Beck Gottfried Eisner Dorothea Trescher Ross E. Dalbey Josef Brunner Matthias Müller 《EMBO reports》2001,2(8):709-714
Like its mitochondrial homolog Oxa1p, the inner membrane protein YidC of Escherichia coli is involved in the integration of membrane proteins. We have analyzed individual insertion steps of the polytopic E. coli membrane protein MtlA targeted as ribosome-nascent chain complexes to inner membrane vesicles. YidC can accommodate at least the first two transmembrane segments of MtlA at the protein lipid interface and retain them even though the length of the nascent chain would amply allow insertion into membrane lipids. An even longer insertion intermediate of MtlA is described that still has the first transmembrane helix bound to YidC while the third contacts SecE and YidC during integration. Our findings suggest that YidC forms a contiguous integration unit with the SecYE translocon and functions as an assembly site for polytopic membrane proteins mediating the formation of helix bundles prior to their release into the membrane lipids. 相似文献
14.
Preuss M Ott M Funes S Luirink J Herrmann JM 《The Journal of biological chemistry》2005,280(13):13004-13011
Members of the Oxa1/YidC family are involved in the biogenesis of membrane proteins. In bacteria, YidC catalyzes the insertion and assembly of proteins of the inner membrane. Mitochondria of animals, fungi, and plants harbor two distant homologues of YidC, Oxa1 and Cox18/Oxa2. Oxa1 plays a pivotal role in the integration of mitochondrial translation products into the inner membrane of mitochondria. It contains a C-terminal ribosome-binding domain that physically interacts with mitochondrial ribosomes to facilitate the co-translational insertion of nascent membrane proteins. The molecular function of Cox18/Oxa2 is not well understood. Employing a functional complementation approach with mitochondria-targeted versions of YidC we show that YidC is able to functionally replace both Oxa1 and Cox18/Oxa2. However, to integrate mitochondrial translation products into the inner membrane of mitochondria, the ribosome-binding domain of Oxa1 has to be appended onto YidC. On the contrary, the fusion of the ribosome-binding domain onto YidC prevents its ability to complement COX18 mutants suggesting an indispensable post-translational activity of Cox18/Oxa2. Our observations suggest that during evolution of mitochondria from their bacterial ancestors the two descendents of YidC functionally segregated to perform two distinct activities, one co-translational and one post-translational. 相似文献
15.
16.
Wagner S Pop OI Pop O Haan GJ Baars L Koningstein G Klepsch MM Genevaux P Luirink J de Gier JW 《The Journal of biological chemistry》2008,283(26):17881-17890
The polytopic inner membrane protein MalF is a constituent of the MalFGK(2) maltose transport complex in Escherichia coli. We have studied the biogenesis of MalF using a combination of in vivo and in vitro approaches. MalF is targeted via the SRP pathway to the Sec/YidC insertion site. Despite close proximity of nascent MalF to YidC during insertion, YidC is not required for the insertion of MalF into the membrane. However, YidC is required for the stability of MalF and the formation of the MalFGK(2) maltose transport complex. Our data indicate that YidC supports the folding of MalF into a stable conformation before it is incorporated into the maltose transport complex. 相似文献
17.
Ines Seitl Stephan Wickles Roland Beckmann Andreas Kuhn Dorothee Kiefer 《Molecular microbiology》2014,91(2):408-421
The marine Gram‐negative bacteria Rhodopirellula baltica and Oceanicaulis alexandrii have, in contrast to Escherichia coli, membrane insertases with extended positively charged C‐terminal regions similar to the YidC homologues in mitochondria and Gram‐positive bacteria. We have found that chimeric forms of E. coli YidC fused to the C‐terminal YidC regions from the marine bacteria mediate binding of YidC to ribosomes and therefore may have a functional role for targeting a nascent protein to the membrane. Here, we show in E. coli that an extended C‐terminal region of YidC can compensate for a loss of SRP‐receptor function in vivo. Furthermore, the enhanced affinity of the ribosome to the chimeric YidC allows the isolation of a ribosome nascent chain complex together with the C‐terminally elongated YidC chimera. This complex was visualized at 8.6 Å by cryo‐electron microscopy and shows a close contact of the ribosome and a YidC monomer. 相似文献
18.
Escherichia coli YidC is a polytopic inner membrane protein that plays an essential and versatile role in the biogenesis of inner membrane proteins. YidC functions in Sec-dependent membrane insertion but acts also independently as a separate insertase for certain small membrane proteins. We have used a site-specific cross-linking approach to show that the conserved third transmembrane segment of YidC contacts the transmembrane domains of both nascent Sec-dependent and -independent substrates, indicating a generic recognition of insertion intermediates by YidC. Our data suggest that specific residues of the third YidC transmembrane segment alpha-helix is oriented toward the transmembrane domains of nascent inner membrane proteins that, in contrast, appear quite flexibly positioned at this stage in biogenesis. 相似文献
19.
van Bloois E Nagamori S Koningstein G Ullers RS Preuss M Oudega B Harms N Kaback HR Herrmann JM Luirink J 《The Journal of biological chemistry》2005,280(13):12996-13003
YidC plays a role in the integration and assembly of many (if not all) Escherichia coli inner membrane proteins. Strikingly, YidC operates in two distinct pathways: one associated with the Sec translocon that also mediates protein translocation across the inner membrane and one independent from the Sec translocon. YidC is homologous to Alb3 and Oxa1 that function in the integration of proteins into the thylakoid membrane of chloroplasts and inner membrane of mitochondria, respectively. Here, we have expressed the conserved region of yeast Oxa1 in a conditional E. coli yidC mutant. We find that Oxa1 restores growth upon depletion of YidC. Data obtained from in vivo protease protection assays and in vitro cross-linking and folding assays suggest that Oxa1 complements the insertion of Sec-independent proteins but is unable to take over the Sec-associated function of YidC. Together, our data indicate that the Sec-independent function of YidC is conserved and essential for cell growth. 相似文献
20.
Philip J. Robinson Cheryl A. Woolhead 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》2013,1833(12):2781-2788
Membrane protein insertion is controlled by proteinaceous factors embedded in the lipid bilayer. Bacterial inner membrane proteins utilise the Sec translocon as the major facilitator of insertion; however some proteins are Sec independent and instead require only YidC. A common feature of YidC substrates is the exposure of a signal anchor sequence when translation is close to completion; this allows minimal time for targeting and favours a post-translational insertion mechanism. Despite this there is little evidence of YidC's post-translational activity. Here we develop an experimental system that uncouples translation and insertion of the endogenous YidC substrate F0c (subunit c of the F0F1 ATP synthase). In this process we (i) develop a novel one step purification method for YidC, including an on column membrane reconstitution, (ii) isolate a soluble form of F0c and (iii) show that incubation of F0c with YidC proteoliposomes results in a high level of membrane integration. Conformational analyses of inserted F0c through Blue Native PAGE and fluorescence quenching reveal a native, oligomerised structure. These data show that YidC can act as a post-translational insertase, a finding which could explain the absence of a ribosome binding domain on YidC. This correlates with the post-translational activity of other YidC family members lacking the ribosome binding domain. 相似文献