Article |
Address correspondence to Arnold J.M. Driessen, Dept. of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, Netherlands. Tel.: 31-50-3632164. Fax: 31-50-3632154. email: a.j.m.driessen{at}biol.rug.nl
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Abstract |
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Key Words: YidC; F1F0 ATP synthase; membrane insertion; membrane targeting; complex assembly
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Introduction |
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E. coli F1F0 ATP synthase consists of a membrane-integral F0 part (subunit composition a1b2c10) and a peripherally bound, catalytic F1 subcomplex (3ß3
; Capaldi and Aggeler, 2002). During the catalytic cycle, the reversible protonation of F0c at residue Asp61 induces a rotation of
,
, and the c ring relative to the a3b3 hexagon. Subunits F0b and F1
form a so-called "stator" that ensures that F0a and the
3ß3 hexagon do not rotate together with
cring. This causes a rotor torque that is believed to be translated into conformational changes of the catalytic residues by elastic power transmission finally leading the synthesis of ATP from ADP and phosphate (Weber and Senior, 2003). Although the mechanisms of energy transduction have been studied in great detail, remarkably little is known about the assembly of large energy-transducing membrane protein complexes like F1F0 ATP synthases or cytochrome oxidases. In yeast, proteins have been identified that are required for their biogenesis, but their precise function is not understood (Ackerman and Tzagoloff, 1990; Ackerman, 2002; Carr and Winge, 2003). The E. coli F1F0 ATP synthase represents the simplest form of this enzyme, containing only the core subunits described above. The observed YidC requirement for the assembly of functional F1F0 ATP synthase in vivo, and particularly of the membrane insertion of the F0c rotor ring subunit, indicates that the biogenesis of this key enzyme is more complex than anticipated so far (Arechaga et al., 2002). To understand the role of YidC in the membrane insertion and oligomeric assembly of F0c, we have used an in vitro translation/insertion assay that has been successfully used to determine the minimal requirements for the Sec-dependent membrane insertion of FtsQ (van der Laan et al., 2004). Here, we demonstrate that in vitro membrane insertion of F0c is blocked by YidC depletion and can be reconstituted with proteoliposomes containing only YidC, whereas SecYEG, the PMF, and the SRP pathway are not required.
Recently, Serek et al. (2004) reported that YidC alone reconstituted into proteoliposomes stimulates the in vitro membrane insertion of Pf3 coat, indicating that YidC can function as a separate membrane protein insertase. Our data demonstrate an essential role of the YidC insertase in the assembly of a Sec-independent, endogenous E. coli membrane protein. F0c is the first described natural substrate of this novel membrane protein biogenesis pathway, which appears to be used by bacteriophages to assemble their coat proteins in the host cell membrane.
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Results |
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YidC proteoliposomes catalyze the SecYEG-independent membrane insertion of F0c
The observation that YidC is required for the insertion of F0c into IMVs did not exclude that other membrane proteins, like the Sec translocase or other unknown components, are involved as well. Only the reconstitution of F0c insertion into proteoliposomes could clearly define the minimal requirements for this process. Therefore, we prepared (proteo-)liposomes containing either YidC together with SecYEG, YidC, or SecYEG alone, or containing no protein, and titrated them into the translation mixture. Upon addition of protein-free liposomes, small amounts of protease-protected F0c were detected (Fig. 3 A), indicating a low level of spontaneous membrane insertion. However, proteoliposomes containing YidC supported highly efficient membrane insertion of F0c (Fig. 3, A and B). Insertion efficiency achieved in the presence of saturating amounts of proteoliposomes was 40%. Co-reconstitution of SecYEG did not significantly increase the amount of membrane-inserted F0c (Fig. 3, A and B). Moreover, the presence of SecYEG alone did not stimulate F0c insertion compared with protein-free liposomes (Fig. 3, A and B). In contrast, proteoliposomes containing SecYEG mediated the translocation of proOmpA independently of YidC, showing that the reconstituted Sec translocase is functional (Fig. 3 A). These data indicate that YidC alone is able to catalyze membrane integration of F0c independently of the Sec translocase. To further examine a possible role of SecYEG in YidC-mediated insertion of F0c, experiments were performed using limiting YidC concentrations. YidC was reconstituted at different protein-to-lipid ratios in the presence or absence of a fixed amount of SecYEG. However, in no case was a significant stimulation of F0c insertion by SecYEG observed (Fig. 4, A and B).
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YidC-mediated membrane insertion of F0c does not require the SRP pathway
Co-translational targeting to the Sec translocase occurs via the SRP pathway (Herskovits et al., 2000). SRP binds to particularly hydrophobic signal sequences or transmembrane segments as they emerge from the ribosome. Upon interaction of SRP with its membrane-bound receptor FtsY, GTP hydrolysis drives the release of SRP from the nascent chain and the transfer of the translating ribosome to the Sec translocase. It is not clear whether the SRP pathway also delivers proteins directly to YidC. The YidC-dependent phage proteins M13 procoat (De Gier et al., 1998) and Pf3 coat (Chen et al., 2002) do not require SRP. However, Fröderberg et al. (2003) have constructed a fusion protein that does not depend on SecYEG, but requires YidC as well as the SRP pathway in vivo. In addition, the chloroplast YidC homologue Alb3 forms a complex with cpSRP and cpFtsY that can be stabilized by the addition of the nonhydrolysable GTP analogue GMP-PNP (Moore et al., 2003). As F0c represents the first "native" substrate of a novel membrane protein insertion pathway in which YidC seems to play a key role, we analyzed the involvement of the SRP pathway as well as the SecA motor protein. SecA is strictly required for the SecYEG-dependent translocation of the large periplasmic domain of FtsQ (van der Laan et al., 2004). As expected, immunodepletion of SecA from the translation lysate had no effect on the insertion of F0c into YidC proteoliposomes (Fig. 5 A, lane 2), whereas under identical conditions it completely blocks FtsQ insertion (van der Laan et al., 2004). Remarkably, immunodepletion of the SRP receptor FtsY also did not significantly affect YidC-mediated F0c insertion (Fig. 5 A, lane 3). Efficient FtsY depletion was demonstrated by Western blotting (Fig. 5 B) and by the inhibitory effect on membrane insertion of FtsQ (Fig. 5 C) as described before (van der Laan et al., 2004). To confirm this observation, we applied a second, independent experimental approach using a translation lysate that had been depleted from SRP in vivo. E. coli SRP consists of a 4.5S RNA and a 48-kD protein called Ffh. Strain HDB51 (Lee and Bernstein, 2001) carries the ffh gene under control of the arabinose promoter. Therefore, cells can be depleted from Ffh by growing them in the presence of glucose. Translation lysates were prepared from cells grown under Ffh depletion conditions as well as from Ffh-containing control cells grown in the presence of arabinose. Efficiency of depletion was monitored by Western blotting (Fig. 5 D). In agreement with the results obtained with FtsY-depleted lysate, we did not observe any major effect of Ffh depletion on the insertion of F0c into IMVs, YidC proteoliposomes, or YidC/SecYEG proteoliposomes (Fig. 5 E). In contrast, the insertion of FtsQ into either IMVs or SecYEG proteoliposomes was strongly inhibited upon depletion of Ffh (Fig. 5 F). Together, these data demonstrate that membrane targeting and YidC-mediated membrane insertion of F0c does not require the SRP pathway.
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Discussion |
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The first step toward the discovery of the native substrates of YidC was the observation that YidC-depleted IMVs contain strongly reduced amounts of cytochrome o oxidase and F1F0 ATP synthase (van der Laan et al., 2003), two major energy-transducing membrane protein complexes. In the case of F1F0 ATP synthase, the amount of the small ring-forming F0c protein was especially affected. Here, we show by means of in vitro insertion experiments that YidC alone mediates the membrane integration of F0c. Proteoliposomes containing only YidC catalyze efficient F0c insertion. Although F0c shows some spontaneous insertion into liposomes, reconstituted YidC dramatically stimulates F0c integration into proteoliposomes. At all YidC concentrations tested, co-reconstitution of the SecYEG complex has no effect on the insertion efficiency, indicating that the Sec translocase is not required for membrane integration of F0c.
It has been shown that membrane partitioning of M13 and Pf3 coat occurs in the absence of a PMF, but that the proteins do not acquire their native topology under these conditions (Date et al., 1980; Chen et al., 2002). Insertion of F0c does not depend on the PMF. We demonstrate by cysteine accessibility experiments that the COOH terminus of the protein, which is located in the periplasm in vivo, is translocated into YidC proteoliposomes, as it is protected from labeling with a membrane-impermeable probe. In contrast, the cytoplasmic loop connecting the two transmembrane helices remains accessible on the outer surface of the proteoliposomes. Thus, the final topology of F0c differs from M13 procoat and Pf3 coat. Although with M13 procoat the hydrophilic loop must be translocated across the membrane, the corresponding domain of F0c remains cytosolic. The first step in the membrane insertion of both M13 procoat and F0c might be the binding to the membrane surface mediated by electrostatic interactions with charged phospholipid headgroups. Presumably, the three positive charges in the cytosolic loop of F0c then prevent membrane translocation, and partitioning of the hydrophobic transmembrane domains into the lipid bilayer is assisted by YidC. The short NH2-terminal, periplasmic tail of F0c contains two negative charges. However, a possible electrophoretic contribution to their translocation is obviously not required. Interestingly, stepwise readdition of charged amino acids into an uncharged Pf3 variant (Pf3-4N) demonstrated that negatively charged residues in the periplasmic NH2-terminal domain show a clear electrophoretic response only when the hydrophobicity of the transmembrane segment is limiting (Kiefer and Kuhn, 1999). However, F0c is a very hydrophobic protein. This could explain why YidC-mediated hydrophobic interactions seem to be the sole driving force for F0c membrane insertion.
It has been established that the YidC-dependent proteins M13 procoat and Pf3 do not require the SRP-targeting pathway to become inserted into the membrane (De Gier et al., 1998; Chen et al., 2002). Consistently, we now demonstrate by two independent approaches, i.e., the in vitro depletion of FtsY and in vivo depletion of Ffh, that inactivation of the SRP pathway has no effect on the insertion of the physiological YidC substrate F0c into both IMVs and YidC proteoliposomes. Although an SRP requirement was not observed, proteoliposomes have to be present cotranslationally. Recently, a direct interaction between mitochondrial ribosomes and the YidC homologue Oxa1p has been reported (Jia et al., 2003; Szyrach et al., 2003). YidC lacks the COOH-terminal cytosolic domain that is required for ribosome binding to Oxa1p (Jia et al., 2003). So far, there is no evidence for a direct coupling between translation and membrane insertion of F0c. However, it seems likely that a very hydrophobic protein like F0c quickly aggregates in the absence of membranes, and this may complicate the post-translational insertion reaction, as the time window between translation and membrane insertion of such small membrane proteins is probably very short in vivo. In this respect, F0c does not seem to differ much from M13 procoat, a protein of similar length that also does not require SRP for membrane targeting.
Our data represent the first direct demonstration of a physiologically important catalytic activity of YidC, which casts a light on the essential role of YidC in E. coli. We have functionally reconstituted a novel membrane protein insertion pathway, and we have shown that the F0c protein is a substrate of it. Viruses and phages generally make use of key biogenesis pathways of their host cells in order to assure their own propagation. For the assembly of filamentous phages like M13 and Pf3, the accumulation of coat proteins in the cytoplasmic membrane of the host is an essential step. It now appears that M13 and Pf3 make use of a cellular machinery that plays a central role the biogenesis of major energy-transducing membrane protein complexes like F1F0 ATP synthase. The mechanism of YidC-mediated membrane protein integration remains to be elucidated. Classical protein translocation and insertion machineries like Sec or Tat translocases use ATP and/or the PMF as energy sources to actively transport proteins or domains of proteins across the membrane. In contrast, no PMF requirement was found for the YidC-mediated insertion of F0c, and YidC contains no obvious conserved nucleotide-binding domain.
It has been suggested that the function of YidC might be analogous to a chaperone that stabilizes folding and assembly intermediates of membrane proteins or membrane protein complexes. Remarkably, all three substrates of the YidC pathway are small and very hydrophobic proteins that assemble into large oligomeric structures like the phage coat or the rotor ring of the F1F0 ATP synthase. The mechanisms of assembly may be vastly different. However, phage coat assembly takes place at the outer surface of the inner membrane, and the final oligomer is not a membrane-inserted complex. Also, substrates of Oxa1, like Cox2p (He and Fox, 1997), and Alb3, like light-harvesting complex protein (Moore et al., 2000), are part of energy-transducing membrane protein complexes. YidC could play an important role as a chaperone in the assembly of these complexes. It might stabilize the transmembrane topology of a single F0c or act as a platform on which subunits can accumulate and organize into a ring structure. This ring is a very rigid complex (Arechaga et al., 2002) However, some intermediates in the process of complex formation might have to be stabilized by YidC. The role of YidC as well as the Sec translocase in the membrane insertion of other F0 subunits is unclear. Yi et al. (2003) have reported that the polytopic subunit F0a also requires YidC to become integrated into the membrane. However, F0a is known to be unstable in the absence of F0c, so that the F0a insertion defect might be at least partly indirect (Hermolin and Fillingame, 1995).
The reconstituted system described here will be a valuable tool in the detailed analysis of YidC-mediated membrane protein integration. Important questions are where the initial interaction of YidC with its substrates takes place, and how binding to and release from YidC are regulated. To understand the role of YidC in the formation of membrane protein complexes, the reconstituted assay described in this paper should be expanded to all subunits of the F0 complex, e.g., by combination with the reconstitution of Sec-dependent membrane protein insertion on which we have recently reported (van der Laan et al., 2004). This will be a challenge for future analyses.
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Materials and methods |
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Depletion of YidC and Ffh
Depletion of YidC and Ffh was performed essentially as described by Nouwen et al. (2001) for SecDFyajC. For YidC depletion, an overnight culture E. coli JS7131 grown in Luria-Bertoni (LB) medium supplemented with 0.2% (wt/vol) arabinose and 25 µg/ml spectinomycin was diluted 1:100 into the same medium without the antibiotic and grown until an OD660 of 0.8. Wild-type IMVs were prepared from these cells. A part of the same culture was washed once with warm LB and resuspended in LB containing 0.2% (wt/vol) glucose at an OD660 of 0.4. After every generation, the culture was diluted with 1 vol of the same medium until the cells stopped growing. YidC-depleted IMVs were prepared from this culture. E. coli HDB51 was grown overnight in LB with 25 µg/ml kanamycin, 10 µg/ml tetracycline, 100 µg/ml ampicillin, and 0.2% (wt/vol) arabinose. Cells were diluted 1:200 into the same medium without antibiotic and grown until an OD660 of 0.2. HDB51 wild-type lysate was prepared from these cells. Depletion of Ffh was induced in the same way as described for YidC, with the exception that cells were initially resuspended at an OD660 of 0.1.
Proteinase K pretreatment of IMVs
To inactivate membrane proteins, IMVs were treated with 0.2 mg/ml proteinase K for 20 min on ice. Proteinase K activity was subsequently blocked by the addition of 0.5 mM PMSF, and IMVs were collected by centrifugation through a cushion consisting of 50 mM Hepes-KOH, pH 7.5, 0.5 mM PMSF, and 20% (wt/vol) sucrose. IMVs were washed with 50 mM Hepes-KOH, pH 7.5, and resuspended in 50 mM Hepes-KOH, pH 7.5, and 20% glycerol. Control IMVs were subjected to the same treatment, with the exception that proteinase K was left out.
In vitro transcription, translation, and insertion reaction
The RiboMAXTM in vitro transcription kit (Promega) was used for the synthesis of mRNA. Plasmids pBSKftsQ (FtsQ), pET20atpE (wild-type F0c), pET20atpE-A40C (F0c A40C, containing a unique cysteine residue at position 40), or pET20atpE-A79C (F0c A79C, containing a unique cysteine at position 79), respectively, served as DNA templates. In vitro translationinsertion reactions were performed for 20 min at 37°C in the presence of the indicated amounts of IMVs or (proteo-)liposomes as described previously (van der Laan et al., 2004).
Labeling of F0c A40C and F0c A79C
To determine the transmembrane topology of in vitrosynthesized and membrane-inserted F0c, translation reactions using F0c A40C or F0c A79C mRNAs and YidC proteoliposomes were performed in the absence of any radioactively labeled amino acid. Subsequently, 0.4 mg/ml protease K was added to degrade nonincorporated F0c. After 30 min on ice, 0.5 mM PMSF was added to block proteinase K activity. Proteoliposomes were isolated from the reaction mixture by centrifugation through a cushion consisting of 50 mM Hepes-KOH, pH 7.0, 50 mM KCl, and 20% (wt/vol) glycerol. Membranes were resuspended in 50 mM Hepes-KOH, pH 7.0, and 50 mM KCl, and were incubated for 10 min on ice in the presence or absence of 0.5 mM AMdiS (Molecular Probes, Inc.) as indicated. Subsequently, 1 mM fluorescein maleimide (Molecular Probes, Inc.) was added, and incubation was continued for another 10 min. Labeling reactions were quenched with 5 mM DTT and samples were subjected to precipitation with TCA. Samples were analyzed by 17.5% SDS-PAGE, and fluorescently labeled proteins were visualized using a Lumi-Imager F1 workstation (Roche).
Oligomerization of in vitrosynthesized F0c
F0c was synthesized as radioactively labeled protein in the presence of proteoliposomes containing either YidC, SecYEG together with YidC, or with no proteins. 0.4 mg/ml proteinase K was added and samples were incubated for 30 min on ice to degrade noninserted F0c. 0.5 mM PMSF was used to inactivate proteinase K, and F0c-loaded proteoliposomes were isolated by centrifugation through a cushion consisting of 50 mM Hepes-KOH, pH 8.0, 50 mM KCl, and 20% (vol/vol) sucrose. Pellets were resuspended in 50 µl solubilization buffer (50 mM Hepes, pH 8.0, 50 mM KCl, 20% glycerol, and 0.05% dodecyl maltoside) and were incubated for 15 min on ice. Subsequently, samples were mixed with gel-loading buffer. Blue Native PAGE analysis was performed on 818% gradient gels as described previously (Schägger and von Jagow, 1991).
Other methods
SecYEG (Manting et al., 2000) and YidC (van der Laan et al., 2001) were purified as described. Reconstitutions were performed as described previously (van der Laan et al., 2001). Where indicated, SecA or FtsY was removed from the lysate by immunodepletion (van der Laan et al., 2004); depletion was verified by immunoblotting. ProOmpA translocation experiments were performed for 20 min at 37°C using fluorescently labeled precursor protein (De Keyzer et al., 2002).
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Acknowledgments |
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This work was supported by the Earth and Life Sciences Foundation, which is subsidized by the Netherlands Organization for Scientific Research (program grant 809.65.012).
Submitted: 18 February 2004
Accepted: 17 March 2004
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References |
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