Title: The Conserved Third Transmembrane Segment of YidC Contacts Nascent Escherichia coli Inner Membrane Proteins
Abstract: 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 α-helix is oriented toward the transmembrane domains of nascent inner membrane proteins that, in contrast, appear quite flexibly positioned at this stage in biogenesis. 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 α-helix is oriented toward the transmembrane domains of nascent inner membrane proteins that, in contrast, appear quite flexibly positioned at this stage in biogenesis. Approximately 20% of the proteins encoded by the Escherichia coli genome are destined for the inner membrane. It is generally assumed that the Sec-translocon is required for the insertion of most inner membrane proteins (IMPs), 2The abbreviations used are: IMP, inner membrane protein; BMOE, Bis-maleimidoethane; BMH, Bis-maleimidohexane; BPM, benzophenone-4-maleimide; CuPhe, Cu2+[phenanthroline]2; IMV, inverted inner membrane vesicle; TM, transmembrane segment; CBP, calmodulin-binding peptide. although a recent proteomic analysis showed that a defective Sec-translocon has surprisingly little effect on the steady state levels of most IMPs (1.Baars L. Wagner S. Wickström D. Klepsch M. Ytterberg A.J. van Wijk K.J. de Gier J.W. J. Bacteriol. 2008; 190: 3505-3525Crossref PubMed Scopus (38) Google Scholar). The core of the Sec-translocon consists of the integral IMPs SecY, SecE, and SecG. SecD, SecF, and YajC form an accessory complex that facilitates protein insertion and translocation but is not essential for survival (2.Luirink J. von Heijne G. Houben E. de Gier J.W. Annu. Rev. Microbiol. 2005; 59: 329-355Crossref PubMed Scopus (157) Google Scholar, 3.Xie K. Dalbey R.E. Nat. Rev. Microbiol. 2008; 6: 234-244Crossref PubMed Scopus (90) Google Scholar). In addition, the essential IMP YidC was co-purified with the Sec-translocon (4.Scotti P.A. Urbanus M.L. Brunner J. de Gier J.W. von Heijne G. van der Does C. Driessen A.J. Oudega B. Luirink J. EMBO J. 2000; 19: 542-549Crossref PubMed Scopus (305) Google Scholar). YidC has been characterized as an evolutionary conserved factor that is involved in the integration of many IMPs, including those that insert independent from the Sec-translocon (2.Luirink J. von Heijne G. Houben E. de Gier J.W. Annu. Rev. Microbiol. 2005; 59: 329-355Crossref PubMed Scopus (157) Google Scholar, 3.Xie K. Dalbey R.E. Nat. Rev. Microbiol. 2008; 6: 234-244Crossref PubMed Scopus (90) Google Scholar). The exact function of YidC is unknown. Cross-linking studies have indicated that YidC contacts the transmembrane segments (TMs) of substrate IMPs upon their lateral exit from the Sec-translocon to facilitate their integration into the lipid bilayer (5.Houben E.N. Ten Hagen-Jongman C.M. Brunner J. Oudega B. Luirink J. EMBO Rep. 2004; 5: 970-975Crossref PubMed Scopus (39) Google Scholar, 6.Urbanus M.L. Scotti P.A. Fröderberg L. Sääf A. de Gier J.W. Brunner J. Samuelson J.C. Dalbey R.E. Oudega B. Luirink J. EMBO Rep. 2001; 2: 524-529Crossref PubMed Scopus (154) Google Scholar, 7.Beck K. Eisner G. Trescher D. Dalbey R.E. Brunner J. Müller M. EMBO Rep. 2001; 2: 709-714Crossref PubMed Scopus (139) Google Scholar). Recent studies on the biogenesis of CyoA (subunit II of the cytochrome o oxidase) have shown that YidC can also function upstream of the Sec-translocon in the initial insertion of the N-terminal region of nascent CyoA followed by translocation of the (more complex) C-terminal domain by the Sec-translocon (8.Van Bloois E. Haan G.J. de Gier J.W. Oudega B. Luirink J. J. Biol. Chem. 2006; 281: 10002-10009Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 9.Du Plessis D.J. Nouwen N. Driessen A.J. J. Biol. Chem. 2006; 281: 12248-12252Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 10.Celebi N. Yi L. Facey S.J. Kuhn A. Dalbey R.E. J. Biol. Chem. 2006; 357: 1428-1436Google Scholar). Furthermore, YidC has been implicated in the folding and quality control of Sec-dependent IMPs (5.Houben E.N. Ten Hagen-Jongman C.M. Brunner J. Oudega B. Luirink J. EMBO Rep. 2004; 5: 970-975Crossref PubMed Scopus (39) Google Scholar, 7.Beck K. Eisner G. Trescher D. Dalbey R.E. Brunner J. Müller M. EMBO Rep. 2001; 2: 709-714Crossref PubMed Scopus (139) Google Scholar, 11.Nagamori S. Smirnova I.N. Kaback H.R. J. Cell Biol. 2004; 165: 53-62Crossref PubMed Scopus (161) Google Scholar, 12.Van Bloois E. Dekker H.L. Fröderberg L. Houben E.N. Urbanus M.L. De Koster C.G. De Gier J.W. Luirink J. FEBS Lett. 2008; 582: 1419-1424Crossref PubMed Scopus (57) Google Scholar). Strikingly, YidC is also able to function as an insertase for small proteins such as F0c (the subunit c of F1F0-ATPase) and M13 and Pf3 phage coat proteins (2.Luirink J. von Heijne G. Houben E. de Gier J.W. Annu. Rev. Microbiol. 2005; 59: 329-355Crossref PubMed Scopus (157) Google Scholar, 3.Xie K. Dalbey R.E. Nat. Rev. Microbiol. 2008; 6: 234-244Crossref PubMed Scopus (90) Google Scholar). Little is known about the mechanism and timing of the recognition of substrate TMs by YidC both in its Sec-dependent and -independent operational mode. To address directly which region of YidC is involved in substrate recognition, we have used an in vitro site-specific cross-linking approach. We have found that during membrane integration, the TMs of several tested Sec-dependent and -independent IMPs are adjacent to a specific region in YidC TM3 that belongs to the most conserved part of YidC. The TM in the nascent substrate is flexibly oriented toward YidC, both lateral and vertical, whereas YidC TM3 displays a fixed orientation toward its substrate. We hypothesize that TM3 is (part of) a generic docking site for hydrophobic domains in growing nascent IMPs and provides a protected environment that facilitates their lipid partitioning and folding. Enzymes, Reagents, and Sera—Restriction enzymes were from Roche Applied Science. Megashortscript T7 transcription kit was from Ambion Inc. [35S]Methionine was from Amersham Biosciences. Bis-Maleimidoethane (BMOE) and Bis-maleimidohexane (BMH) were from Pierce. Benzophenone-4-maleimide (BPM) was from Molecular Probes. Phenanthroline, N-ethylmaleimide, and all other chemicals were supplied by Sigma. Antisera against YidC, PspA, and Lep have been described previously or were from our own collection (13.Van der Laan M. Urbanus M.L. Ten Hagen-Jongman C.M. Nouwen N. Oudega B. Harms N. Driessen A.J. Luirink J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5801-5806Crossref PubMed Scopus (127) Google Scholar). Strains and Plasmids—Strain MC4100 was used to obtain translation lysate (14.De Vrije T. Tommassen J. De Kruijff B. Biochim. Biophys. Acta. 1987; 900: 63-72Crossref PubMed Scopus (111) Google Scholar). Strain Top10F′ (Invitrogen) was used for routine cloning and maintenance of plasmid constructs. Strain JS7131, in which yidC is under the control of the araBAD operator/promoter, was used to express YidC cysteine mutants (15.Samuelson J.C. Chen M. Jiang F. Möller I. Wiedmann M. Kuhn A. Phillips G.J. Dalbey R.E. Nature. 2000; 406: 637-641Crossref PubMed Scopus (428) Google Scholar). pCL1921YidC-StrepII-CBP derivatives encoding the YidC cysteine mutants were constructed as follows. The coding region for the calmodulin-binding peptide (CBP) was PCR-amplified from pCalKC (Stratagene) with a forward primer containing a SacI restriction site and the Streptavidine tag II sequence and with a reverse primer containing the EcoRI restriction site (primer sequences available upon request). The PCR product was digested with SacI and EcoRI and introduced 3′ of the yidC gene into pEH1YidC (16.Urbanus M.L. Fröderberg L. Drew D. Bjork P. De Gier J.W. Brunner J. Oudega B. Luirink J. J. Biol. Chem. 2002; 277: 12718-12723Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), resulting in pEH1YidC-StrepII-CBP. This plasmid was digested with KpnI and EcoRI, and the restriction product containing the 3′ end of YidC gene with the StrepII-CBP tag was cloned into the low copy plasmid pCL1921YidC (17.Lerner C.G. Inouye M. Nucleic Acids Res. 1990; 18: 4631Crossref PubMed Scopus (233) Google Scholar), replacing the 3′ end of YidC, resulting in pCL1921YidC-StrepII-CBP (we will refer to this plasmid as pCLYidC). The plasmid pCLYidC was used as a template for constructing single cysteine mutants in the YidC gene using the QuikChange site-directed mutagenesis kit (Stratagene). First, the codon for cysteine 423 was changed into an alanine codon resulting in pCLYidC C423A. This plasmid was subsequently used as a template to construct further single cysteine mutants in yidC gene. The nucleotide sequences of the mutant genes were confirmed by DNA sequencing. YidC wild type and cysteine derivatives (see Table 1) were expressed in the YidC depletion strain JS7131 carrying pEH3. pEH3 encodes the LacI repressor and is used to minimize leakage expression from the pCLYidC plasmid. Expression from pCLYidC was induced by 200 μm isopropyl-1-thio-β-d-galactopyranoside. Strains were used to make IMVs for insertion assays (14.De Vrije T. Tommassen J. De Kruijff B. Biochim. Biophys. Acta. 1987; 900: 63-72Crossref PubMed Scopus (111) Google Scholar). Single cysteine mutants of FtsQ, F0c, and Lep were constructed in pC4Meth by nested PCR as described previously (18.Valent Q.A. De Gier J.W. Von Heijne G. Kendall D.A. Ten Hagen-Jongman C.M. Oudega B. Luirink J. Mol. Microbiol. 1997; 25: 53-64Crossref PubMed Scopus (149) Google Scholar).TABLE 1Plasmids encoding YidC derivativesNamePlasmidTM2TM3pCLpCLYidCpCL.YidC-StrepII-CBPC423ApCL.YidC-StrepII-CBP C423AC432AF356CpCL.YidC-StrepII-CBP C423A/F356CF356CC432AS357CpCL.YidC-StrepII-CBP C423A/S357CS357CC432AI358CpCL.YidC-StrepII-CBP C423A/I358CI358CC432AF424CpCL.YidC-StrepII-CBP C423A/F424CC423A/F424CP425CpCL.YidC-StrepII-CBP C423A/P425CC423A/P425CL426CpCL.YidC-StrepII-CBP C423A/L426CC423A/L426CQ429CpCL.YidC-StrepII-CBP C423A/Q429CC423A/Q429CM430CpCL.YidC-StrepII-CBP C423A/M430CC423A/M430CP431CpCL.YidC-StrepII-CBP C423A/P431CC423A/P431C Open table in a new tab In Vitro Transcription, Translation, Integration, and Cross-linking—Truncated mRNA was prepared as described previously (4.Scotti P.A. Urbanus M.L. Brunner J. de Gier J.W. von Heijne G. van der Does C. Driessen A.J. Oudega B. Luirink J. EMBO J. 2000; 19: 542-549Crossref PubMed Scopus (305) Google Scholar) from HindIII-linearized pC4Meth derivatives. Translation and membrane integration of nascent chains were carried out as described previously (4.Scotti P.A. Urbanus M.L. Brunner J. de Gier J.W. von Heijne G. van der Does C. Driessen A.J. Oudega B. Luirink J. EMBO J. 2000; 19: 542-549Crossref PubMed Scopus (305) Google Scholar). Long range cysteine cross-linking was carried out with BMOE (1 mm) or BMH (0.5 mm) for 10 min at 25 °C and quenched with 5 mm β-mercaptoethanol for 10 min on ice. To separate integral membrane from soluble and peripheral cross-linked complexes, the samples were extracted with 0.18 m Na2CO3 (pH 11.5). The carbonate pellet fractions were analyzed by SDS-PAGE. Short range cysteine cross-linking was induced with 1 mm Cu2+[phenanthroline]2 (CuPhe) for 10 min at 25 °C and subsequently quenched with 10 mm N-ethylmaleimide for 10 min on ice. CuPhe was prepared as described previously (19.Kaufmann A. Manting E.H. Veenendaal A.K. Driessen A.J. Van der Does C. Biochemistry. 1999; 38: 9115-9125Crossref PubMed Scopus (108) Google Scholar), and the carbonate-resistant fraction was dissolved in SDS sample buffer without dithiothreitol. For photocross-linking, nascent chains (prepared as described previously (4.Scotti P.A. Urbanus M.L. Brunner J. de Gier J.W. von Heijne G. van der Does C. Driessen A.J. Oudega B. Luirink J. EMBO J. 2000; 19: 542-549Crossref PubMed Scopus (305) Google Scholar)) and BPM (1 mm) were incubated for 10 min at 25 °C and quenched with 10 mm N-ethylmaleimide on ice for 10 min. Subsequently, the nascent chains were purified through a high salt sucrose cushion (500 mm KOAc, 500 mm sucrose, 5 mm Mg(OAc)2, 50 mm Hepes-KOH, pH 7.9) and resuspended in RN buffer (100 mm KOAc, 5 mm Mg(OAc)2, 50 mm Hepes-KOH, pH 7.9). IMVs were added to allow targeting and integration into the membrane, and the samples were UV-irradiated to induce cross-linking to membrane components as described previously (4.Scotti P.A. Urbanus M.L. Brunner J. de Gier J.W. von Heijne G. van der Does C. Driessen A.J. Oudega B. Luirink J. EMBO J. 2000; 19: 542-549Crossref PubMed Scopus (305) Google Scholar). The carbonate pellet fractions were analyzed by SDS-PAGE. Radiolabeled proteins were visualized by phosporimaging using a Molecular Dynamics PhosphorImager 473. Experimental Strategy—To investigate the interaction of YidC with nascent IMPs during their insertion into the E. coli inner membrane, we have cross-linked integration intermediates in an in vitro approach (4.Scotti P.A. Urbanus M.L. Brunner J. de Gier J.W. von Heijne G. van der Does C. Driessen A.J. Oudega B. Luirink J. EMBO J. 2000; 19: 542-549Crossref PubMed Scopus (305) Google Scholar). In this technique, translation of a truncated mRNA (a mRNA that lacks a stop codon) is translated in a cell-free E. coli extract to produce nascent, ribosome-bound polypeptides of predefined length. Translation is carried out in the presence of [35S]methionine to label the nascent chains, and IMVs are added to allow co-translational insertion of the nascent IMP into the Sec/YidC-translocon or YidC insertase. Upon insertion, interactions of the nascent chain and the translocon/insertase are probed by cross-linking using homo-bifunctional cysteine specific reagents. Introduction of single cysteines in both the nascent chain and the interacting partner protein makes the procedure site-specific. Thus, this approach yields spatial information regarding the proximity of the integration intermediate and its partner at the residue level. The integration intermediates used in this study were derived from the model IMPs FtsQ, leader peptidase (Lep), and the F0c subunit of the F1F0-ATPase. FtsQ is a single spanning, type II IMP involved in cell division (see Fig. 3A) (20.Goehring N.W. Beckwith J. Curr. Biol. 2005; 15: R514-R526Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar). Lep, the major signal peptidase, spans the membrane twice with translocated N and C termini (see Fig. 6A). F0c has the same topology as Lep but comprises a much shorter translocated C terminus (Fig. 7A). FtsQ and Lep have been shown to use the Sec/YidC-translocon for membrane integration (4.Scotti P.A. Urbanus M.L. Brunner J. de Gier J.W. von Heijne G. van der Does C. Driessen A.J. Oudega B. Luirink J. EMBO J. 2000; 19: 542-549Crossref PubMed Scopus (305) Google Scholar, 5.Houben E.N. Ten Hagen-Jongman C.M. Brunner J. Oudega B. Luirink J. EMBO Rep. 2004; 5: 970-975Crossref PubMed Scopus (39) Google Scholar, 15.Samuelson J.C. Chen M. Jiang F. Möller I. Wiedmann M. Kuhn A. Phillips G.J. Dalbey R.E. Nature. 2000; 406: 637-641Crossref PubMed Scopus (428) Google Scholar, 21.Houben E.N. Scotti P.A. Valent Q.A. Brunner J. de Gier J.L. Oudega B. Luirink J. FEBS Lett. 2000; 476: 229-233Crossref PubMed Scopus (73) Google Scholar). In contrast, F0c only requires the YidC insertase (Fig. 7A) (22.Yi L. Celebi N. Chen M. Dalbey R.E. J. Biol. Chem. 2004; 279: 39260-39267Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 23.Van der Laan M. Bechtluft P. Kol S. Nouwen N. Driessen A.J. J. Cell Biol. 2004; 165: 213-222Crossref PubMed Scopus (178) Google Scholar, 24.Van Bloois E. Haan G.J. De Gier J.W. Oudega B. Luirink J. FEBS Lett. 2004; 576: 97-100Crossref PubMed Scopus (75) Google Scholar).FIGURE 6Lep cross-linking to YidC TM3 cysteine mutants. A, topology of Lep in the inner membrane. B, nascent Lep derivatives used in this study. The white circle represents the position of the unique cysteine at position 10 in nascent Lep. The black rectangle represents the TM. C, in vitro translation of nascent 60LepCys10 was carried out in the presence of IMVs containing YidC TM3 cysteine mutants and cross-linked using BMOE. Carbonate-resistant pellet fractions are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 7F0c cross-linking to YidC TM3 cysteine mutants. A, topology of F0c in the IM. B, nascent F0c derivatives used in this study. The white circle represents the position of the unique cysteine at position 15 in nascent F0c. The black rectangle represents the TM. C, in vitro translation of nascent 79F0cCys15 was carried out in the presence of IMVs containing YidC TM3 cysteine mutants and cross-linked using BMOE. Carbonate-resistant pellet fractions are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Construction and Characterization of Single Cysteine YidC Mutants—Using site-specific photo cross-linking, it has been shown previously that nascent FtsQ is targeted to the Sec-translocon by the SRP (18.Valent Q.A. De Gier J.W. Von Heijne G. Kendall D.A. Ten Hagen-Jongman C.M. Oudega B. Luirink J. Mol. Microbiol. 1997; 25: 53-64Crossref PubMed Scopus (149) Google Scholar). The FtsQ TM inserts initially at SecY moving to a combined YidC/lipid environment upon elongation as suggested by sequential contacts during insertion of nascent FtsQ (6.Urbanus M.L. Scotti P.A. Fröderberg L. Sääf A. de Gier J.W. Brunner J. Samuelson J.C. Dalbey R.E. Oudega B. Luirink J. EMBO Rep. 2001; 2: 524-529Crossref PubMed Scopus (154) Google Scholar). For instance, a relatively long nascent FtsQ of 108 amino acids harboring a photo-probe at position 40 in the TM was shown to primarily cross-link YidC. Furthermore, 108FtsQ with a single cysteine engineered at position 40 was shown to cross-link YidC reconstituted in proteoliposomes together with SecYEG using the homo-bifunctional cysteine specific cross-linker BMOE (8 Å) (25.Van der Laan M. Houben E.N. Nouwen N. Luirink J. Driessen A.J. EMBO Rep. 2001; 2: 519-523Crossref PubMed Scopus (96) Google Scholar). Because YidC only contains one cysteine at position 423 in the third TM, this observation implied that TM3 of YidC is close to the TM of nascent FtsQ at this stage in the integration process. Interestingly, TM3 belongs (together with TM2) to the most conserved regions in the Oxa1/Alb3/YidC family members (26.Kiefer D. Kuhn A. Int. Rev. Cytol. 2007; 259: 113-138Crossref PubMed Scopus (45) Google Scholar). To examine the proximity between YidC TM3/TM2 and nascent IMPs in more detail, we have used cysteine scanning cross-linking. First, the single endogenous cysteine in YidC was replaced by alanine to generate cysteine-free YidC. This derivative served as a negative control in our cross-link studies and as a starting point to introduce single cysteine residues at selected positions in TM3 and TM2 (Table 1), covering more than two turns of the presumably α-helical TM3 structure and one turn in TM2 (Fig. 1, B and C). All of the yidC constructs were cloned in the low copy expression vector pCL1921 under lac promoter control. To distinguish the plasmid-encoded YidC derivatives from endogenous YidC, a StrepII-CBP tag was added at the C terminus of all constructs, increasing the size of YidC by ∼5 kDa. Tagged YidC that contains the cysteine at position 423 will be further referred to as wild type. Functionality of the YidC derivatives was assessed by investigating their ability to complement the in vivo growth defect that accompanies depletion of YidC. To this end, the constructs were introduced in the YidC depletion strain JS7131 in which expression of the chromosomal yidC is under control of the araBAD promoter (15.Samuelson J.C. Chen M. Jiang F. Möller I. Wiedmann M. Kuhn A. Phillips G.J. Dalbey R.E. Nature. 2000; 406: 637-641Crossref PubMed Scopus (428) Google Scholar). Complementation was assayed on solid medium in the absence of l-arabinose to repress the chromosomal yidC but in the presence of isopropyl-1-thio-β-d-galactopyranoside to induce expression of the plasmid-encoded yidC derivative (Fig. 2A). As a control, JS7131 transformed with the cloning vector only grew on the plate containing l-arabinose as expected. pCL1921 encoding wild type YidC complemented growth in the absence of l-arabinose, indicating that YidC with a C-terminal StrepII-CBP tag is functional, consistent with the permissive nature of the C-terminus of YidC with respect to alterations (16.Urbanus M.L. Fröderberg L. Drew D. Bjork P. De Gier J.W. Brunner J. Oudega B. Luirink J. J. Biol. Chem. 2002; 277: 12718-12723Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 27.Jiang F. Chen M. Yi L. De Gier J.W. Kuhn A. Dalbey R.E. J. Biol. Chem. 2003; 278: 48965-48972Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Importantly, all plasmid-encoded single cysteine YidC mutants were able to complement growth of JS7131 in the absence of l-arabinose, indicating that they are functional. For in vitro cross-link experiments, IMVs were prepared of all JS7131 derivatives grown in liquid LB in the absence of l-arabinose and the presence of isopropyl-1-thio-β-d-galactopyranoside to specifically express the plasmid-encoded YidC. YidC was detected in the IMVs of all mutants, albeit at different levels for reasons that are unknown. As expected, the YidC mutants ran slower in SDS-PAGE compared with untagged YidC (faintly visible in the empty pCL vector control) (Fig. 2B). We have shown previously that depletion of YidC leads to up-regulation of PspA, which is in part associated with the inner membrane (13.Van der Laan M. Urbanus M.L. Ten Hagen-Jongman C.M. Nouwen N. Oudega B. Harms N. Driessen A.J. Luirink J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5801-5806Crossref PubMed Scopus (127) Google Scholar). PspA is a stress protein that responds to dissipation of the proton motive force (28.Darwin A.J. Mol. Microbiol. 2005; 57: 621-628Crossref PubMed Scopus (228) Google Scholar). YidC depletion affects the proton motive force by defects in the functional assembly of the F1F0-ATPase and the cytochrome o oxidase (13.Van der Laan M. Urbanus M.L. Ten Hagen-Jongman C.M. Nouwen N. Oudega B. Harms N. Driessen A.J. Luirink J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5801-5806Crossref PubMed Scopus (127) Google Scholar, 29.Van der Laan M. Nouwen N.P. Driessen A.J. Curr. Opin. Microbiol. 2005; 8: 182-187Crossref PubMed Scopus (53) Google Scholar). As shown in Fig. 2B, the induction of PspA seen in the empty pCL vector control is completely prevented by expression of all YidC derivatives. This indicates that all of the mutants are able to sustain the proton motive force and are functional in the assembly of respiratory chain complexes. Together, the data suggest that the YidC derivatives, although for unknown reasons expressed at different levels compared with the Lep loading control (Fig. 2B), are functional. The TM of 108FtsQ Inserts in Proximity of YidC TM3—To probe the environment of the TM in the 108FtsQ integration intermediate near YidC TM3, 108FtsQCys40 (Fig. 3B) was inserted in IMVs that contain YidC derivatives with single cysteine substitutions in TM3 (Fig. 1C). Upon integration, the samples were cross-linked using the homo-bifunctional cysteine-specific reagent BMOE. Strong and specific cross-linking was observed to YidC (verified by immunoprecipitation, not shown) harboring a cysteine at positions 425, 426, 429, or, to a lesser extent, 424 (Fig. 3C). No adducts were observed in the absence of BMOE (not shown) or when using IMVs that contain the cysteine-less YidC mutant (Fig. 3C), confirming the specificity of the procedure. Notably, the positions that strongly cross-link to 108FtsQCys40 seem to cluster at one side of the putative α-helical YidC TM3, although more positions should be analyzed to confirm this notion (Fig. 1C). To rule out that, despite their functionality, some YidC mutants are structurally altered in such a way that they are no longer in proximity of nascent 108FtsQCys40, cross-linking was performed with the hetero-bifunctional reagent BPM. BPM reacts with the sulfhydryl groups of cysteine residues via a maleimide moiety. Upon ultraviolet irradiation, a reactive species is generated that can form covalent bonds with nearby groups. The linker arm connecting the two reactive groups is ∼10 Å. 108FtsQCys40 nascent chains were produced, purified through a high salt sucrose cushion, and incubated with BPM to allow cross-linking to the Cys40 residue. Subsequently, the samples were incubated with β-mercaptoethanol to quench cysteine cross-linking. Then IMVs were added for membrane integration of 108FtsQCys40-BPM, and the samples were UV-irradiated to induce cross-linking to membrane components independent of closely spaced cysteine residues. By this procedure, all of the YidC derivatives were cross-linked to the FtsQ integration intermediate (Fig. 3D). In this and the following experiments, no YidC cross-linking adducts were observed in the absence of cross-linkers (data not shown). This shows that the IMVs with the YidC derivatives are still functional in accommodating nascent 108FtsQCys40 in accordance with the in vivo complementation data described above. The TM of 108FtsQ Is Dynamically Oriented Relative to YidC TM3—To obtain more spatial and structural information regarding the proximity of the TM in 108FtsQ and YidC TM3, four additional mutants were constructed that have single cysteines in the TM of 108FtsQ at positions 36, 39, 41, and 42 (Fig. 3B). These mutant nascent chains were integrated in the IMVs that contain YidC derivatives with single cysteine substitutions in TM3 (Fig. 1C) and cross-linked with BMOE. Interestingly, the cross-link patterns are very similar to that of 108FtsQCys40, i.e. cross-linking to YidC positions 425, 426, 429, and, to a lesser extent, 424 (Fig. 4A). Cross-linking is most efficient at position 425 except for 108FtsQCys36 where cross-linking peaks at position 426. The similarity in cross-linking profiles for all of the tested positions in the TM of 108FtsQ indicates that the FtsQ TM does not occupy a fixed position relative to YidC TM3. Rather, the FtsQ TM seems to orient itself flexibly along the more rigid YidC TM3 perpendicular to the plane of the membrane. Importantly, the analyzed region of the FtsQ TM did not show a clear helical asymmetry in cross-linking to YidC TM3. This could mean that the TM in the FtsQ integration intermediate has not yet adopted an α-helical conformation but rather constitutes a randomly coiled stretch of residues that is able to reposition up and down relative to YidC TM3. Alternatively, the FtsQ TM is α-helical at this stage, but the helix has sufficient flexibility and space to rotate relative to YidC TM3. Finally, it cannot be excluded that the YidC TM3 docking site may have some mobility toward its substrate. To verify that in this approach cross-linking to YidC TM3 is specific for the TM of 108FtsQ, single cysteines were introduced at positions 15 and 60 located in hydrophilic regions that flank the FtsQ TM. As expected, BMOE cross-linking yielded no YidC adducts (supplemental Fig. S1), consistent with previous photo cross-link data in which probes engineered at positions 10 and 59 of 108FtsQ did not show significant cross-linking to any region of YidC (4.Scotti P.A. Urbanus M.L. Brunner J. de Gier J.W. von Heijne G. van der Does C. Driessen A.J. Oudega B. Luirink J. EMBO J. 2000; 19: 542-549Crossref PubMed Scopus (305) Google Scholar). Together, the data indicate that the TM of membrane-integrated 108FtsQ contacts YidC TM3 with a high degree of conformational freedom. The TM of 108FtsQ Is Held at YidC TM3 during Membrane Integration—Next, we examined the effect of nascent chain length on the proximity between the FtsQ TM and YidC TM3. Previously, we have shown by photo cross-linking that the FtsQ TM in nascent chains as short as 97 residues contacts YidC (6.Urbanus M.L. Scotti P.A. Fröderberg L. Sääf A. de Gier J.W. Brunner J. Samuelson J.C. Dalbey R.E. Oudega B. Luirink J. EMBO Rep. 2001; 2: 524-529Crossref PubMed Scopus (154) Google Scholar). With our set of YidC cysteine mutants, we were now able to specifically examine the effect of nascent chain length on the interaction of the FtsQ TM with YidC TM3. Remarkably, 97FtsQCys40 integrated in the single cysteine YidC IMVs showed a BMOE cross-linking profile (Fig. 4B) almost identical to 108FtsQCys40 (Fig. 3C). These results suggest that the FtsQ TM, although flexibly oriented t