F1F0 ATP synthase subunit c is a substrate of the novel YidC pathway for membrane protein biogenesis

The Escherichia coli YidC protein belongs to the Oxa1 family of membrane proteins that have been suggested to facilitate the insertion and assembly of membrane proteins either in cooperation with the Sec translocase or as a separate entity. Recently, we have shown that depletion of YidC causes a specific defect in the functional assembly of F1F0 ATP synthase and cytochrome o oxidase. We now demonstrate that the insertion of in vitro-synthesized F1F0 ATP synthase subunit c (F0c) into inner membrane vesicles requires YidC. Insertion is independent of the proton motive force, and proteoliposomes containing only YidC catalyze the membrane insertion of F0c in its native transmembrane topology whereupon it assembles into large oligomers. Co-reconstituted SecYEG has no significant effect on the insertion efficiency. Remarkably, signal recognition particle and its membrane-bound receptor FtsY are not required for the membrane insertion of F0c. In conclusion, a novel membrane protein insertion pathway in E. coli is described in which YidC plays an exclusive role.     

Subunit a of cytochrome o oxidase requires both YidC and SecYEG for membrane insertion

The Escherichia coli YidC protein belongs to the Oxa1 family of membrane proteins that facilitate the insertion of membrane proteins. Depletion of YidC in E. coli leads to a specific defect in the functional assembly of major energy transducing complexes such as the F1F0 ATPase and cytochrome bo3 oxidase. Here we report on the in vitro reconstitution of the membrane insertion of the CyoA subunit of cytochrome bo3 oxidase. Efficient insertion of in vitro synthesized pre-CyoA into proteoliposomes requires YidC, SecYEG, and SecA and occurs independently of the proton motive force. These data demonstrate that pre-CyoA is a substrate of a novel pathway that involves both SecYEG and YidC.     

YidC is involved in the biogenesis of anaerobic respiratory complexes in the inner membrane of Escherichia coli

YidC of Escherichia coli belongs to the evolutionarily conserved Oxa1/Alb3/YidC family. Members of this family have all been implicated in membrane protein biogenesis of aerobic respiratory and energy-transducing proteins. YidC is essential for the insertion of subunit c of the F(1)F(0)-ATP synthase and subunit a of cytochrome o oxidase. The aim of this study was to investigate whether YidC plays a role during anaerobic growth of Escherichia coli, specifically when either nitrate or fumarate are used as terminal electron acceptors or under fermentative conditions. The effect of YidC depletion on the growth, enzyme activities, and protein levels in the inner membrane was determined. YidC is essential for all anaerobic growth conditions tested, and this is not because of the decreased levels of F(1)F(0)-ATP synthase in the inner membrane only. The results suggest a role for YidC in the membrane biogenesis of integral membrane parts of the anaerobic respiratory chain.     

YidC is strictly required for membrane insertion of subunits a and c of the F(1)F(0)ATP synthase and SecE of the SecYEG translocase

YidC was previously discovered to play a critical role for the insertion of the Sec-independent M13 procoat and Pf3 coat phage proteins into the Escherichia coli inner membrane. To determine whether there is an absolute requirement of YidC for membrane protein insertion of any endogenous E. coli proteins, we investigated a few representative membrane proteins. We found that membrane subunits of the F(0) sector of the F(1)F(0)ATP synthase and the SecE protein of the SecYEG translocase are highly dependent on YidC for membrane insertion, based on protease mapping and immunoblot analysis. We found that the SecE dependency on YidC for membrane insertion does not contradict the observation that depletion of YidC does not block SecYEG-dependent protein export at 37 degrees C. YidC depletion does not decrease the SecE level low enough to block export at 37 degrees C. In contrast, we found that protein export of OmpA is severely blocked at 25 degrees C when YidC is depleted, which may be due to the decreased SecE level, as a 50% decrease in the SecE levels drastically affects protein export at the cold temperature [Schatz, P. J., Bieker, K. L., Ottemann, K. M., Silhavy, T. J., and Beckwith, J. (1991) EMBO J. 10, 1749-57]. These studies reported here establish that physiological substrates of YidC include subunits of the ATP synthase and the SecYEG translocase, demonstrating that YidC plays a vital role for insertion of endogenous membrane proteins in bacteria.     

Different regions of the nonconserved large periplasmic domain of Escherichia coli YidC are involved in the SecF interaction and membrane insertase activity

The YidC protein of Escherichia coli is required for inserting Sec-independent membrane proteins and has a supportive role for the insertion of Sec-dependent proteins into the membrane bilayer. Because a portion of YidC copurifies with the Sec translocase, this interaction might be necessary to assist in the membrane insertion of Sec-dependent proteins. This study describes a deletion analysis that investigates which parts of YidC are required for its interaction with the SecDF complex of the Sec translocase and for the function of YidC as an insertase for the Sec-dependent membrane proteins. The results suggest that the first periplasmic region, which includes residues 24-346, is required for the interaction of YidC with the Sec translocase, in particular with the SecF protein. Further studies showed that residues 215-265 of YidC are sufficient for SecF binding. Surprisingly, the interaction of YidC with SecF is not critical for cell viability as YidC, lacking residues 24-264, was fully functional to support the growth of E. coli. It was also observed that this YidC mutant was fully functional to insert the Sec-dependent subunit A of the F(1)F(o) ATP synthase and an M13 procoat derivative, as well as the Sec-independent M13 procoat protein and subunit C of the ATP synthase. Only when additional residues of the periplasmic region were deleted (265-346) was the membrane insertase function of YidC inhibited.     

Saccharomyces cerevisiae Cox18 complements the essential Sec-independent function of Escherichia coli YidC

Members of the YidC/Oxa1/Alb3 protein family function in the biogenesis of membrane proteins in bacteria, mitochondria and chloroplasts. In Escherichia coli, YidC plays a key role in the integration and assembly of many inner membrane proteins. Interestingly, YidC functions both in concert with the Sec-translocon and as a separate insertase independent of the translocon. Mitochondria of higher eukaryotes contain two distant homologues of YidC: Oxa1 and Cox18/Oxa2. Oxa1 is required for the insertion of membrane proteins into the mitochondrial inner membrane. Cox18/Oxa2 plays a poorly defined role in the biogenesis of the cytochrome c oxidase complex. Employing a genetic complementation approach by expressing the conserved region of yeast Cox18 in E. coli, we show here that Cox18 is able to complement the essential Sec-independent function of YidC. This identifies Cox18 as a bona fide member of the YidC/Oxa1/Alb3 family.     

YidC-mediated membrane insertion of assembly mutants of subunit c of the F1F0 ATPase

YidC is a member of the OxaI family of membrane proteins that has been implicated in the membrane insertion of inner membrane proteins in Escherichia coli. We have recently demonstrated that proteoliposomes containing only YidC support both the stable membrane insertion and the oligomerization of the c subunit of the F(1)F(0) ATP synthase (F(0)c). Here we have shown that two mutants of F(0)c unable to form a functional F(1)F(0) ATPase interact with YidC, require YidC for membrane insertion, but fail to oligomerize. These data show that oligomerization is not essential for the stable YidC-dependent membrane insertion of F(0)c consistent with a function of YidC as a membrane protein insertase.     

Oxal/Alb3/YidC system for insertion of membrane proteins in mitochondria,chloroplasts and bacteria (Review)

Recent studies have shown that there is a pathway that is evolutionarily conserved for the insertion of proteins into the membrane in mitochondria, chloroplasts, and bacteria. In this pathway, the Oxa1/Alb3/YidC proteins are believed to function as membrane insertases that play an important role in the membrane protein biogenesis of respiratory and energy transduction proteins. Additional roles of the Oxa1/Alb3/YidC members may be in the lateral integration of proteins into the lipid bilayer, and in the folding and assembly of proteins into membrane protein complexes.     

Oxa1/Alb3/YidC system for insertion of membrane proteins in mitochondria, chloroplasts and bacteria (review)

Recent studies have shown that there is a pathway that is evolutionarily conserved for the insertion of proteins into the membrane in mitochondria, chloroplasts, and bacteria. In this pathway, the Oxa1/Alb3/YidC proteins are believed to function as membrane insertases that play an important role in the membrane protein biogenesis of respiratory and energy transduction proteins. Additional roles of the Oxa1/Alb3/YidC members may be in the lateral integration of proteins into the lipid bilayer, and in the folding and assembly of proteins into membrane protein complexes.     

YidC family members are involved in the membrane insertion, lateral integration, folding, and assembly of membrane proteins

Members of the YidC family exist in all three domains of life, where they control the assembly of a large variety of membrane protein complexes that function as transporters, energy devices, or sensor proteins. Recent studies in bacteria have shown that YidC functions on its own as a membrane protein insertase independent of the Sec protein-conducting channel. YidC can also assist in the lateral integration and folding of membrane proteins that insert into the membrane via the Sec pathway.     

Bacillus subtilis YqjG is required for genetic competence development

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.     

The Sec-independent function of Escherichia coli YidC is evolutionary-conserved and essential

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.     

Crystal structure of the major periplasmic domain of the bacterial membrane protein assembly facilitator YidC

The essential bacterial membrane protein YidC facilitates insertion and assembly of proteins destined for integration into the inner membrane. It has homologues in both mitochondria and chloroplasts. Here we report the crystal structure of the Escherichia coli YidC major periplasmic domain (YidCECP1) at 2.5A resolution. This domain is present in YidC from Gram-negative bacteria and is more than half the size of the full-length protein. The structure reveals that YidCECP1 is made up of a large twisted beta-sandwich protein fold with a C-terminal alpha-helix that packs against one face of the beta-sandwich. Our structure and sequence analysis reveals that the C-terminal alpha-helix and the beta-sheet that it lays against are the most conserved regions of the domain. The region corresponding to the C-terminal alpha-helix was previously shown to be important for the protein insertase function of YidC and is conserved in other YidC-like proteins. The structure reveals that a region of YidC that was previously shown to be involved in binding to SecF maps to one edge of the beta-sandwich. Electrostatic analysis of the molecular surface for this region of YidC reveals a predominantly charged surface and suggests that the SecF-YidC interaction may be electrostatic in nature. Interestingly, YidCECP1 has significant structural similarity to galactose mutarotase from Lactococcus lactis, suggesting that this domain may have another function besides its role in membrane protein assembly.     

The charge distribution in the cytoplasmic loop of subunit C of the F1F0 ATPase is a determinant for YidC targeting

YidC is a member of the Oxa1 family of proteins that facilitates the membrane insertion of a subset of inner membrane proteins in Escherichia coli. YidC acts as an insertase for membrane insertion of subunit c of the F(1)F(0) ATP synthase (F(0)c), but the requirements for substrate recognition have remained unclear. Here, we have analyzed the role of the charged aminoacyl residues in F(0)c in YidC targeting and membrane insertion. Binding experiments demonstrate that F(0)c is targeted directly to YidC without the presence of a stable lipid surface-bound intermediate. Positive charges in the cytoplasmic loop of F(0)c are important determinants for YidC binding and subsequent membrane insertion. These data support a model in which F(0)c binds directly to YidC prior to its membrane insertion.     

Conditional lethal mutations separate the M13 procoat and Pf3 coat functions of YidC: different YIDC structural requirements for membrane protein insertion

Conditional lethal YidC mutants have been isolated to decipher the role of YidC in the assembly of Sec-dependent and Sec-independent membrane proteins. We now show that the membrane insertion of the Sec-independent M13 procoat-lep protein is inhibited in a short time in a temperature-sensitive mutant when shifted to the nonpermissive temperature. This provides an additional line of evidence that YidC plays a direct role in the insertion of the Sec-independent M13 procoat protein. However, in the temperature-sensitive mutant, the insertion of the Sec-independent Pf3 phage coat protein and the Sec-dependent leader peptidase were not strongly inhibited at the restricted temperatures. Conversely, using a cold-sensitive YidC strain, we find that the membrane insertion of the Sec-independent Pf3 coat protein is blocked, and the Sec-dependent leader peptidase is inhibited at the nonpermissive temperature, whereas the insertion of the M13 procoat protein is nearly normal. These data show that the YidC function for procoat and its function for Pf3 coat and possibly leader peptidase are genetically separable and suggest that the YidC structural requirements are different for the Sec-independent M13 procoat and Pf3 coat phage proteins that insert by different mechanisms.     

YidC and SecY mediate membrane insertion of a Type I transmembrane domain

YidC has been identified recently as an evolutionary conserved factor that is involved in the integration of inner membrane proteins (IMPs) in Escherichia coli. The discovery of YidC has inspired the reevaluation of membrane protein assembly pathways in E. coli. In this study, we have analyzed the role of YidC in membrane integration of a widely used model IMP, leader peptidase (Lep). Site-directed photocross-linking experiments demonstrate that both YidC and SecY contact nascent Lep very early during biogenesis, at only 50-amino acid nascent chain length. At this length the first transmembrane domain (TM), which acquires a type I topology, is not even fully exposed outside the ribosome. The pattern of interactions appears dependent on the position of the cross-linking probe in the nascent chain. Upon elongation, nascent Lep remains close to YidC and comes into contact with lipids as well. Our results suggest a role for YidC in both the reception and lipid partitioning of type I TMs.     

Distinct requirements for translocation of the N-tail and C-tail of the Escherichia coli inner membrane protein CyoA

Inner membrane proteins (IMPs) of Escherichia coli use different pathways for membrane targeting and integration. YidC plays an essential but poorly defined role in the integration and folding of IMPs both in conjunction with the Sec translocon and as a Sec-independent insertase. Depletion of YidC only marginally affects the insertion of Sec-dependent IMPs, whereas it blocks the insertion of a subset of Sec-independent IMPs. Substrates of this latter "YidC-only" pathway include the relatively small IMPs M13 procoat, Pf3 coat protein, and subunit c of the F(1)F(0) ATPase. Recently, it has been shown that the steady state level of the larger and more complex CyoA subunit of the cytochrome o oxidase is also severely affected upon depletion of YidC. In the present study we have analyzed the biogenesis of the integral lipoprotein CyoA. Collectively, our data suggest that the first transmembrane segment of CyoA rather than the signal sequence recruits the signal recognition particle for membrane targeting. Membrane integration and assembly appear to occur in two distinct sequential steps. YidC is sufficient to catalyze insertion of the N-terminal domain consisting of the signal sequence, transmembrane segment 1, and the small periplasmic domain in between. Translocation of the large C-terminal periplasmic domain requires the Sec translocon and SecA, suggesting that for this particular IMP the Sec translocon might operate downstream of YidC.     

Dynamic disulfide scanning of the membrane-inserting Pf3 coat protein reveals multiple YidC substrate contacts

The membrane insertase YidC inserts newly synthesized proteins into the plasma membrane. While defects in YidC homologs in animals and plants cause diseases, YidC in bacteria is essential for life. Membrane insertion and assembly of ATP synthase and respiratory complexes is catalyzed by YidC. To investigate how YidC interacts with membrane-inserting proteins, we generated single cysteine mutants in YidC and in the model substrate Pf3 coat protein. The single cysteine mutants were expressed and analyzed for disulfide formation during 30 s of synthesis. The results show that the substrate contacts different YidC residues in four of the six transmembrane regions. The residues are located either in the region of the inner leaflet, in the center, as well as in the periplasmic leaflet, consistent with the hypothesis that YidC presents a hydrophobic platform for inserting membrane proteins. In a YidC mutant where most of the contacting residues were mutated to serines, YidC function was severely disturbed and no longer active in a complementation test, suggesting that the residues are important for function. In addition, a Pf3 mutant with a defect in membrane insertion was deficient to contact the periplasmic residues of YidC.     

Identification of Bacillus subtilis YidC Substrates Using a MifM-instructed Translation Arrest-based Reporter

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.     

Sec/SRP requirements and energetics of membrane insertion of subunits a, b, and c of the Escherichia coli F1F0 ATP synthase

Previously, the role of YidC in the membrane protein biogenesis of the F(0) sector of the Escherichia coli F(1)F(0) ATP synthase was investigated. Whereas subunits a and c of the F(1)F(0) ATP synthase were strictly dependent on YidC for membrane insertion, subunit b required YidC for efficient insertion (Yi, L., Jiang, F., Chen, M., Cain, B., Bolhuis, A., and Dalbey, R. E. (2003) Biochemistry 42, 10537-10544). In this paper, we investigated other protein components and energetics that are required in the membrane protein assembly of the F(0) sector subunits. We show here that the Sec translocase and the signal recognition particle (SRP) pathway are required for membrane insertion of subunits a and b. In contrast, subunit c required neither the Sec machinery nor the SRP pathway for insertion. While the proton motive force was not required for insertion of subunits b and c, it was required for translocation of the negatively charged periplasmic NH(2)-terminal tail of subunit a, whereas periplasmic loop 2 of subunit a could insert in a proton motive force-independent manner. Taken together, the in vivo data suggest that subunits a and b are inserted by the Sec/SRP pathway with the help of YidC, and subunit c is integrated into the membrane by the novel YidC pathway.