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The Mechanosensitive Channel Protein MscL Is Targeted by the SRP to The Novel YidC Membrane Insertion Pathway of Escherichia coli
J. Mol. Biol. (2007) 365, 995–1004
doi:10.1016/j.jmb.2006.10.083
The Mechanosensitive Channel Protein MscL Is Targeted by the SRP to The Novel YidC Membrane Insertion Pathway of Escherichia coli Sandra J. Facey, Stella A. Neugebauer, Susanne Krauss and Andreas Kuhn⁎ Institute of Microbiology, University of Hohenheim, 70599 Stuttgart, Germany
The mechanosensitive channel MscL in the inner membrane of Escherichia coli is a homopentameric complex involved in homeostasis when cells are exposed to hypo-osmotic conditions. The E. coli MscL protein is synthesized as a polypeptide of 136 amino acid residues and uses the bacterial signal recognition particle (SRP) for membrane targeting. The protein is inserted into the membrane independently of the Sec translocon. Mutants affected in the Sec-components are competent for MscL assembly. Translocation of the periplasmic domain was detected using a membrane-impermeant, sulfhydryl-specific gel-shift reagent. The modification of a single cysteine residue at position 68 indicated its translocation across the inner membrane. From these in vivo experiments, it is concluded that the electrical chemical membrane potential is not necessary for membrane insertion of MscL. However, depletion of the membrane insertase YidC inhibits translocation of the protein across the membrane. We show here that YidC is essential for efficient membrane insertion of the MscL protein. YidC is a component of a recently identified membrane insertion pathway that is evolutionarily conserved in bacteria, mitochondria and chloroplasts. © 2006 Elsevier Ltd. All rights reserved.
*Corresponding author
Keywords: MscL; proton motive force; Sec translocase; SRP; YidC
Introduction YidC is a 61 kDa protein in the inner membrane of Escherichia coli. It was first identified as a homolog of the Oxa1 protein, which is involved in assembly of the cytochrome oxidase of Saccharomyces cerevisiae.1 YidC was found to be associated with nascent chains of bacterial membrane proteins, suggesting that it is involved in the membrane assembly as well.2 The proof that YidC is involved directly in the membrane insertion process came from a mutant E. coli strain in which YidC was depleted within 3 h of growth, accumulating the non-inserted membrane proteins in the cytoplasm, whereas exported proteins were not affected.3 Intriguingly, Sec-independent proteins, such as the major coat proteins of Abbreviations used: AMS, 4-acetamido-4′maleimidylstilbene-2, 2′-disulfonic acid disodium salt; CCCP, carbonyl cyanide p-chlorophenylhydrazone; SRP, signal recognition particle. E-mail address of the corresponding author: [email protected]
bacteriophage M13 and Pf3, were inhibited dramatically, whereas the Sec-dependent proteins were only retarded for membrane insertion.3,4 The Sec-dependent proteins contact YidC either shortly after they bind to SecYEG,2 or to both simultaneously.5 Most likely, YidC forms together with the Sec complex, a collection site for the transmembrane regions of multispanning proteins by shielding the protein chain from lipids and allowing the three-dimensional folding.6 In accordance with this, YidC has been found to be bound to the Sec translocase, specifically with the SecDFYajC complex.7 The SecDFYajC complex connects YidC to the SecYEG machinery. Since YidC is present in the E. coli membrane in molecular excess, only a fraction of YidC is bound to the Sec translocase and most of the YidC forms an independent entity.8 Biochemical purification and reconstitution into proteoliposomes showed that YidC functions per se as a membrane insertase and catalyses the insertion of Sec-independent proteins into the membrane.9,10 Therefore, YidC by itself resembles a novel membrane insertion pathway that can operate independent of the Sec translocon. It is
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996 not known how many membrane proteins use the YidC or the Sec pathway. The fact that YidC is essential for growth suggests that phage proteins are not the only substrates. Recently, subunit c from the F1F0 ATP synthase complex was shown to use the YidC-only pathway, which could explain the lethal effect of YidC depletion.10,11 The YidC substrates are limited to small periplasmic regions with only a few charged residues, since mutations that change these features switch to the Sec-dependent pathway.12,13 The MscL protein of the mechanosensitive channel with large conductance appears to play a primary role in protecting cells exposed to osmotic downshock.14 MscL is believed to open in response to high levels of tension within the membrane, allowing ions and other solutes to be released from the cell and thus preventing rupture of the cell membrane. The crystal structure of the MscL homolog from Mycobacterium tuberculosis revealed a homopentameric channel.15 Each subunit spans the inner membrane twice, and has a small periplasmic region of 29 residues that includes five charged residues. The structure of MscL shows that the N-terminal transmembrane helix forms an inner ring in the pentameric structure, whereas the Cterminal transmembrane helix is located at the periphery of the channel. The periplasmic regions of each subunit form the extracellular surface of the pore. We show here that the MscL protein is targeted to the membrane by the signal recognition particle (SRP) pathway, and inserted into the inner membrane of E. coli independently of SecA, SecE and SecDF, but uses the membrane insertase YidC. Previously, it was thought that the bacterial SRP targets proteins specifically to the Sec translocon.16 Since MscL uses SRP and YidC, this underlines that the membrane targeting, insertion and the translocation components operate as individual modules.
MscL Insertion
immunoprecipitated. Derivatized and underivatized proteins were then separated by SDS-PAGE and examined by phosphorimaging. We chose the AMS derivatization method to analyse the topology of the MscL protein instead of the conventional protease mapping assay, because the periplasmic loop of MscL was mostly resistant to digestion by proteinase K. To apply the assay, several single-cysteine mutants of MscL were prepared at positions 59, 64 and 68 in the periplasmic loop, because it was not known which proteins would be expressed or would exhibit a gel mobility-shift with AMS. In all cases, a shift was observed for the cysteine mutants (data not shown), suggesting that all of the cysteine residues in the mutant proteins are derivatizable to AMS. The cysteine mutant I68C was selected for further analysis (Figure 1). In this mutant, cysteine is substituted for isoleucine at position 68 in the periplasmic loop. As expected, the cysteine mutant I68C, which is located in the periplasmic loop, is labelled by AMS in whole and disrupted cells (Figure 1(b), middle panel). In contrast, wild-type MscL, which contains no endogenous cysteine residue, shows no reaction with AMS (Figure 1(b), left panel). In the S136C mutant, serine was substituted for cysteine at position 136 in the C terminus of MscL, which is located in the cytoplasm.
Results Membrane topology analysed by AMS derivatization Insertion of MscL into the cytoplasmic membrane was monitored by rapid derivatization of a unique cysteine residue in the periplasmic loop of the protein with AMS, a membrane-impermeable, sulfhydryl reagent that shifts protein mobility on SDS/ polyacrylamide gels. 17 Derivatization of free cysteine residues retards the mobility of the protein on a SDS/polyacrylamide gel by an amount corresponding roughly to the added mass of AMS (∼ 0.5 kDa). Thus, the derivatized form of MscL will display a higher molecular mass than the underivatized form. Cells expressing the membrane protein were incubated in AMS and then pulselabelled and chased with non-radioactive Met. After radiolabelling, proteins were acid-precipitated and
Figure 1. Topological model of the E. coli MscL protein and location of cysteine substitutions and AMS derivatization of MscL mutants. (a) MscL is a 136 amino acid residue membrane protein that consists of two transmembrane α-helices, which are connected by a periplasmic loop of 29 residues. Arrows indicate the positions of the cysteine mutations introduced for AMS derivatization. (b) Cells expressing the mutant proteins were pulse-labelled with [35S]Met for 30 s, then chased with non-radioactive Met for 2 min. After pulse-chase labelling, the cells were incubated in the presence (+) or absence (−) of AMS or disrupted by sonification and then treated with AMS for 10 min. The AMS-treated aliquots were combined with an equal volume of 20 mM DTT in medium salts to quench the AMS reaction. The proteins were then acid-precipitated and immunoprecipitated with anti-His antiserum, and subjected to SDS-PAGE (17% (w/v) polyacrylamide). d and u denote the AMS-derivatized and underivatized protein, respectively.
MscL Insertion
Mutant S136C is our negative control, and exhibits no gel mobility-shift in the presence of AMS, which is membrane-impermeable (Figure 1(b), right panel). However, disruption of the cells by sonification should permit labelling of the available SH group upon addition of AMS. Figure 1(b) shows this was indeed the case. Mutant S136C shows a shift with AMS when the membrane is disrupted, indicating that AMS is membrane-impermeable under the assay conditions, and that derivatization of the periplasmic-localized cysteine residue is evidence for translocation. Depletion of the Sec translocase components does not affect the membrane insertion of MscL We investigated whether the SecYEG/DF translocase mediates the membrane insertion of MscL. Depletion strains that control the expression of SecE and SecDF were used to investigate the translocation of MscL. The role of SecE was examined in the depletion strain E. coli CM124.18 Expression of SecE is induced with arabinose and repressed tightly in the presence of glucose. Cells carrying the cysteine mutants were either grown in medium with arabinose (Figure 2(a), lanes 3, 4, 7 and 8) or with glucose for 8 h (Figure 2(a), lanes 1, 2, 5 and 6). To examine translocation, CM124 cells expressing the MscL mutant I68C were pulse-labelled with [35S] Met for 30 s and chased with non-radioactive Met for 2 min in the presence or in the absence of AMS. The results show that two major radiolabelled bands were observed when the cells carrying the cysteine mutant I68C were depleted of SecE (Figure 2(a), lane 6). Samples radiolabelled in the absence of AMS with or without SecE (lanes 5 and 7) yielded a single band that co-migrated with the lower band of lanes 6 and 8, indicating that the band corresponds to underivatized protein. The upper band in lanes 6 and 8 corresponds to derivatized protein. This demonstrates that MscL has inserted into the membrane without SecE (lane 6). In contrast, translocation and signal peptide cleavage of the Sec-dependent outer membrane protein (OmpA) was blocked completely by SecE depletion (Figure 2(b)). To ensure that only the translocated cysteine residues were derivatized, cells expressing a MscL mutant containing a cytoplasmic-localized cysteine residue (S136C) were radiolabelled in the presence of AMS (Figure 2(a), lanes 2 and 4). As expected, S136C exhibited no gel mobility-shift in the presence of AMS. Previously, it was shown that SecDF facilitates the insertion of Sec-dependent proteins in vivo.19 To verify our results, we used the E. coli strain JP325,13 to deplete SecDF. In JP325, the chromosomal secDF operon is under control of the araBAD promoter. As a consequence, YajC, SecD and SecF are synthesized only when grown in the presence of arabinose. For the cysteine mutant I68C, gel mobility-shifts were observed irrespective of whether the cells were depleted of SecDF (Figure 2(c), lanes 2 and 4). In the
997 untreated samples, no shift was observed for I68C (Figure 2(c), lanes 1 and 3), indicating that MscL insertion is independent of SecDF. As a control, the processing of OmpA was analysed in a parallel culture (Figure 2(d)). A decrease in the rate of cleavage of OmpA was noticeable under SecDF depleting conditions. As an additional control, efficient SecDF depletion was demonstrated by the inhibitory effect on signal peptide cleavage of the procoat lep mutant -3MPClep (Figure 2(e)), as observed previously.13 An alternative procedure was applied, in which the pulse–chased cells were first converted into spheroplasts and then treated with AMS. By performing this assay, we can demonstrate that AMS does not inhibit membrane insertion. The results are shown in Figure 2(f) with and without sodium azide, which has been shown to inhibit SecA activity at a concentration of 2 mM.20 To address the role of SecA in MscL membrane insertion, bacteria were treated with 2 mM sodium azide for 5 min before the addition of [35S]Met and conversion into spheroplasts. The results show again that two radiolabelled bands were observed when the cells carrying the cysteine mutant I68C were treated with or without sodium azide in the presence of AMS (Figure 2(f), lanes 2 and 4) after a 10 min chase. Similar results were obtained after a 2 min chase (data not shown). The upper band corresponds to derivatized protein and the lower band to underivatized protein. These results further verify that MscL integration is not dependent on SecA. As expected, proOmpA was converted rapidly to OmpA in the absence of sodium azide (Figure 2(g), left panel). In the presence of sodium azide, the Secdependent proOmpA accumulated in its precursor form in the cells (Figure 2(g), right panel). Insertion of the MscL protein requires YidC The role of YidC in the membrane insertion of MscL was examined in the depletion strain JS7131, where YidC expression is under the control of an araBAD promoter and operator.3 YidC expression is induced with arabinose and repressed tightly in the presence of glucose. To deplete YidC, the cells were grown for 3 h with glucose and then assayed for derivatization with AMS (Figure 3(a), lanes 2, 6 and 10). When the cells were grown with arabinose (YidC+), a gel mobility-shift was observed for the cysteine mutant I68C (Figure 3(a), lanes 8 and 12), indicating MscL was readily inserted into the membrane. In YidC-deficient cells (grown with glucose), no shift was observed with AMS after a 2 min (Figure 3(a), lane 6) or a 10 min chase (Figure 3(a), lane 10), indicating no translocation occurred. This demonstrates that MscL requires YidC for insertion. As expected, no shift was observed for the cysteine mutant S136C, which contains a cytoplasmic-localized cysteine residue (Figure 3(a), left panel). Furthermore, processing of the Sec-dependent protein OmpA was not affected under these conditions, demonstrating that indirect inactivation of the Sec
998
MscL Insertion
Figure 2. The translocation of the periplasmic domain of MscL is independent of SecE, SecDF and SecA. (a) Strain CM124 expressing the MscL cysteine mutants was grown in M9 minimal medium containing arabinose (lanes 3, 4, 7 and 8). For depletion of SecE (lanes 1, 2, 5 and 6), the cells were grown in the presence of glucose and the absence of arabinose for 8 h. IPTG (1 mM) was added for 10 min to induce expression. Cells expressing the mutant proteins were incubated in the presence (+) or in the absence of AMS (−) for 1 min, pulse-labelled with [35S]Met for 30 s, then chased with nonradioactive Met for 2 min. After quenching with 20 mM DTT, the radiolabelled samples were acid-precipitated and immunoprecipitated with an anti-His antibody and then subjected to SDS-PAGE (17% (w/v) polyacrylamide gel). d and u denote the AMS-derivatized and underivatized protein, respectively. (b) As a control, translocation and processing of the Sec-dependent protein OmpA was monitored in parallel to verify the depletion of SecE. Samples were immunoprecipitated with antiserum to OmpA. (c) JP325 cells bearing the MscL cysteine mutant I68C were back-diluted (1:100) in M9 minimal medium containing arabinose and glucose (SecDF+, lanes 3 and 4) or only glucose (SecDF–, lanes 1 and 2). After induction with IPTG for 10 min, cells were pulse-labelled with [35S]Met for 30 s in the presence (+) or in the absence (−) of AMS and then chased with excess cold Met for 2 min. After quenching with DTT, samples were immunoprecipitated, subjected to SDS-PAGE and analysed by phosphorimaging. (d) As a control, the processing of the Sec-dependent protein OmpA was monitored in parallel after 30 s pulse-labelling and a 10 s, 1 min, and 2 min chase. Samples were immunoprecipitated with antiserum to OmpA. (e) Efficient SecDF depletion was demonstrated by the inhibitory effect on signal peptide cleavage of the procoat mutant –3MPClep (left panel). After induction with IPTG for 10 min and labelling with [35S]Met for 30 s, the cells were chased with non-radioactive Met for 10 s, 1 min, and 2 min. p and m denote the precursor form and mature form, respectively. (f) The MscL cysteine mutant I68C was expressed in the E. coli strain C41 (DE3) in the absence (lanes 1 and 2) or in the presence (lanes 3 and 4) of sodium azide. Cells were radiolabelled with [35S] Met for 30 s, chased with non-radioactive Met for 10 min and then converted to spheroplasts as described in Materials and Methods. After spheroplasting, samples were treated with or without AMS for 30 min and analysed as described in Figure 2(a). (g) OmpA accumulated in its precursor form (proOmpA) in the azide-treated cells (right-hand panel).
translocon had not occurred (Figure 3(b)). YidC depletion was confirmed by analysing the YidC content in a cell sample taken before induction with IPTG by immunoblotting using YidC antiserum (Figure 3(c) and (e)). For a control, processing of a YidC-dependent protein M13 procoat was monitored in parallel. M13 procoat was pulse-labelled for 30 s and chased for 1 min and for 2 min, and then analysed for translocation and processing (Figure 3(d)). When the cells were grown in glucose (YidC–), M13 procoat processing was largely inhibited and the protein accumulated in a non-translocated form. In
contrast, when YidC was present, M13 procoat was readily inserted into the membrane. MscL is targeted by the SRP to the inner membrane The bacterial SRP, which includes the 54 homologue protein (Ffh), the 4.5 S RNA and the SRP receptor FtsY, selectively recognizes inner membrane proteins via their long hydrophobic transmembrane segments.21 To investigate the involvement of SRP in the biogenesis of MscL, we transformed
MscL Insertion
999 the Ffh depletion E. coli strain WAM121 with the plasmids encoding the MscL cysteine mutants. Cells expressing the mutant proteins were either grown in medium with arabinose (Ffh+, Figure 4(a), lanes 3, 4, 7, 8, 11 and 12) or with glucose (Ffh–, Figure 4(a), lanes 1, 2, 5, 6, 9 and 10) and then assayed for translocation with AMS. When Ffh was present, the cysteine mutant I68C was readily inserted into the membrane and derivatized (Figure 4(a), lanes 8 and 12). In contrast, in Ffh-depleted cells, MscL insertion was largely inhibited after a 2 min (Figure 4(a), lane 6) and a 10 min chase (Figure 4(a), lane 10). As for the control (S136C), only underivatized protein was observed in the presence and in the absence of AMS (Figure 4(a), left panel). Depletion of Ffh was verified by
Figure 3. YidC is required for membrane insertion of MscL. (a) JS7131 cells expressing the MscL cysteine mutants S136C and I68C were pulse-labelled with [35S] Met for 30 s and chased with non-radioactive Met for 2 min (lanes 1–8) or 10 min (lanes 9–12), either in the presence of arabinose to induce expression of YidC (lanes 3, 4, 7, 8, 11 and 12) or in medium lacking arabinose to deplete YidC (lanes 1, 2, 5, 6, 9 and 10). Before labelling, cells were treated in the presence (+) or in the absence (−) of AMS. Samples were immunoprecipitated with an antiHis antibody and analysed by SDS-PAGE. d, derivatized; u, underivatized. (b) A sample of cells was immunoprecipitated with OmpA antiserum and then analysed by SDS-PAGE and phosphorimaging, showing that OmpA translocation was not affected. (c) Immunoblot analysis was used to determine the levels of YidC in the JS7131 strain expressing the MscL cysteine mutants under glucose (YidC–) and arabinose (YidC+) conditions. Before induction by IPTG, 1 ml of cells was removed from the glucose and the arabinose sample. The cells were precipitated with TCA, resuspended in SDS loading buffer, and resolved by SDS-PAGE. Immunoblotting was done using antiserum against the C terminus of YidC. (d) For a control, the translocation and processing of the M13 procoat protein was analysed under YidC+ and YidC– conditions. After induction with IPTG for 10 min, cells were pulse-labelled with [35S]Met for 30 s and chased with non-radioactive Met for 1 min and 2 min. All samples were precipitated with 20% (w/v) TCA, immunoprecipitated with anti-M13 serum and visualized by phosphorimaging. (e) Immunoblot analysis of the levels of YidC in the JS7131 strain expressing M13 procoat under glucose (YidC–) and arabinose (YidC+) conditions.
Figure 4. MscL is dependent on Ffh for membrane insertion. (a) E. coli WAM121 cells were transformed with the plasmids coding for the MscL cysteine mutants. The cells were depleted of Ffh by growth in the presence of 0.4% glucose (lanes 1, 2, 5, 6, 9 and 10), as described in Materials and Methods. Plasmid-encoded proteins were induced with 1 mM IPTG for 10 min, incubated in the presence (+) or in the absence (−) of AMS for 1 min, pulselabelled with [35S]Met for 30 s and chased for 2 min (lanes 1–8) or 10 min (lanes 9–12) with non-radioactive Met. Samples were immunoprecipitated with an anti-His antibody and analysed by SDS-PAGE. d and u denote the derivatized and underivatized form of MscL. (b) Immunoblot analysis of Ffh levels in the WAM121 strain expressing the MscL cysteine mutants under glucose (Ffh–) and arabinose (Ffh+) conditions. Immunoblotting was done using antiserum against Ffh. (c) As a control, translocation and processing of the secretory protein OmpA was monitored in parallel after a 30 s pulselabelling and a 2 min chase. (d) Translocation of the periplasmic loop of the YidC-dependent M13 coat protein was monitored by pulse-labelling with [35S]Met for 1 min and 5 min. Samples were immunoprecipitated with antiserum to M13 coat protein.
MscL Insertion
1000 analysing the Ffh content in a cell sample taken before induction with IPTG by immunoblotting (Figure 4(b)). To address the possibility that SRP depletion is causing an indirect effect, e.g. inhibiting the insertion of YidC and thereby inhibiting membrane protein insertion of MscL, the processing of a secretory protein OmpA (Figure 4(c)) and a YidCdependent protein M13 procoat (Figure 4(d)) were analysed in a parallel culture. The wild-type M13 procoat protein does not require the SRP homologue Ffh for membrane insertion,21 but requires YidC. Signal peptide processing of both M13 procoat and proOmpA were largely unaffected in cells depleted of Ffh, indicating normal insertion of M13 and export of OmpA. Therefore, the membrane translocation of MscL is affected directly by depletion of Ffh. To verify this, MscL targeting was investigated in the FtsY depletion strain IY26. Cells of strain IY26 in which ftsY expression is under control of an arabinose-inducible promoter were grown in the presence (FtsY+, Figure 5(a), lanes 3, 4, 7 and 8) or in the absence of arabinose (FtsY–, Figure 5(a), lanes 1, 2, 5 and 6) and then assayed for translocation with AMS. In accordance with the effects of depletion of Ffh, depletion of FtsY inhibits membrane insertion of MscL (Figure 5(a), lane 6). Together, the in vivo
Figure 6. MscL does not require the electrochemical membrane potential for membrane insertion. (a) C41(DE3) cells expressing the MscL cysteine mutant I68C were analysed with (+) and without (−) CCCP, which was added 1 min before treatment of the cells with AMS at a final concentration of 50 μM. Cells were then labelled with [35S]Met for 30 s, chased with non-radioactive Met for 10 min and analysed as described for Figure 2(a). d, derivatized; u, underivatized. (b) OmpA accumulated in its precursor form (proOmpA) in CCCP treated cells.
results suggest strongly that MscL requires SRP for efficient membrane targeting. Depletion of FtsY was verified by analysing the FtsY level in a cell sample in parallel by immunoblotting with FtsY antiserum (Figure 5(b)). In this experiment, FtsY levels were reduced dramatically in the FtsYdepleted cells (FtsY–) as judged by immunoblot analysis, whereas YidC levels were not affected (Figure 5(c)). Electrochemical membrane potential is not required for insertion of MscL into the membrane
Figure 5. The SRP receptor FtsY is required for efficient targeting of MscL to the membrane. (a) The FtsY depletion strain IY26 bearing the MscL cysteine mutants were grown under either FtsY depletion or FtsY expression conditions. After induction with IPTG for 10 min, cells were incubated in the presence (+) or absence (−) of AMS for 1 min, pulse-labelled with [35S]Met for 30 sec, then chased with non-radioactive Met for 2 min. Samples were prepared and processed as described for Figure 2(a). (b) Immunoblot analysis of FtsY levels in the IY26 strain expressing the MscL cysteine mutants under glucose (FtsY–) and arabinose (FtsY+) conditions. Immunoblotting was done using antiserum against FtsY. (c) Immunoblot analysis of the levels of YidC in the IY26 strain expressing the MscL cysteine mutants under glucose (FtsY–) and arabinose (FtsY+) conditions. Antibodies against the C terminus of YidC were used for immunoblotting.
To assess the effect of the proton motive force on the membrane insertion of MscL, the cells were treated with CCCP, a protonophore that dissipates the proton motive force.22 The proton motive force was collapsed by adding 50 μM CCCP, 1 min before treatment of the cells with AMS and labelling of the cells with [35S]Met. Figure 6(a) shows the AMS derivatization of the MscL mutant I68C in the absence (lane 2) and in the presence (lane 4) of CCCP. In both cases, gel shifts were observed, indicating that MscL does not require an electrochemical membrane potential for membrane assembly. Samples radiolabelled in the absence of AMS (lanes 1 and 3) yielded a single band corresponding to underivatized protein. The export of OmpA is blocked completely by CCCP, since proOmpA accumulates in the treated cells (Figure 6(b)).
Discussion AMS derivatization is very useful in analysing the biosynthesis of small membrane proteins in vivo. It has been used in the analysis of other membrane proteins as a membrane-impermeable reagent.23,24 Using this technology, we have shown here that the 136 residue long MscL protein is a further substrate
MscL Insertion
of the YidC pathway. Besides MscL, the subunit c of the ATP synthase,10 and the major coat proteins of bacteriophage M13 and Pf3 have been characterized as YidC substrates.3,4 All these inner membrane proteins use the YidC protein without the Sec translocase. In contrast to the bacteriophage coat proteins, the MscL protein inserts independently of the electrochemical membrane potential. So far, only small hydrophilic regions have been shown to be translocated by the YidC-only pathway. In the Pf3 coat protein the N-terminal tail of 18 residues that includes two negatively charged aspartyl groups is translocated, whereas for the csubunit of the F1F0 ATP synthase both terminal tails of seven and three residues are translocated. In contrast to the bacteriophage coat proteins, insertion of the c-subunit of the F1F0 ATP synthase does not depend on the electrochemical membrane potential.10 For the Pf3 protein, it has been shown that the negatively charged residues are required for membrane insertion and determine the orientation of the protein in the membrane.25 The hydrophilic loop region of the M13 procoat protein of 20 residues has five charged residues resulting in a − 3 net charge. For this protein, the negative charge of the periplasmic region is correlated directly with the involvement of the transmembrane potential for membrane insertion. A mutant that lacks the charged residues in the periplasmic domain of the M13 procoat protein was independent of the electrochemical membrane potential.26 In contrast, the hydrophilic loop region of the MscL protein has 29 residues that include five charged residues. The independence of MscL for a membrane potential might be due to the fact that the periplasmic loop has a net charge of only − 1. Previously, it was believed that the bacterial SRP specifically targets proteins to the Sec translocase.16 Later, Fröderberg and co-workers27 showed that an artificial M13 procoatlep construct and a ProW construct required SRP and YidC. Interestingly, our data show that MscL requires also the bacterial SRP and YidC. Taken together, this underlines that membrane targeting and membrane insertion are two separate processes operating by distinct modules.27,28 The YidC-dependent proteins M13 procoat,21 and Pf3 coat4 do not require SRP. The involvement of SRP in targeting of the c-subunit of the F 1F0 ATP synthase is controversial. 10,11,29 Recently, it has been shown that the CyoA subunit II of the cytochrome o oxidase is severely affected upon depletion of YidC. 30,31 CyoA spans the membrane twice and is synthesized with a cleavable signal peptide. It was demonstrated that the Nterminal domain of CyoA consisting of the first transmembrane segment and the small periplasmic domain of 26 residues requires YidC and SRP for targeting and insertion into the membrane, whereas translocation of the large C-terminal periplasmic domain of CyoA requires the Sec translocon and SecA. These results show that two distinct mechanisms are required in translocating the different domains of CyoA.
1001 Our data suggest that SRP plays a prominent role in the targeting of MscL to the membrane. Depletion of Ffh or the SRP receptor FtsY, had a pronounced effect on the insertion of MscL in vivo. It is unclear whether SRP or FtsY play a direct role in delivering MscL to YidC, or simply act to target MscL to the membrane to promote interaction with YidC. In Arabidopsis thaliana, experimental evidence shows that cpSRP and cpFtsY interact with the Alb3 component independent of a substrate.32 Alb3 is the homologue of YidC in the thylakoid membrane and is required for the assembly of the lightharvesting chlorophyll-binding proteins (LHCP) into the thylakoid membrane, whereby the targeting of LHCP to the thylakoid membrane is mediated by cpSRP.32,33 Little is known about the folding and assembly of membrane proteins in the membrane. It has been suggested for Sec-dependent proteins that YidC may be involved in the lateral movement of membrane proteins into the lipid bilayer after leaving the Sec channel.34 On the basis of photocrosslinking studies, it was demonstrated that in vitro the hydrophobic domain of FtsQ first contacts SecY, and then interacts with YidC.2 Similar results were observed for the membrane insertion of mannitol permease (MtlA).5 These findings suggest that YidC functions as an assembly site for transmembrane regions and may play a role in the three-dimensional arrangement of the helices within the membrane. Recently, it was shown that YidC can function on its own as a membrane insertase. 9,10 In contrast to the Sec-dependent proteins, the YidC-dependent substrates all have short periplasmic regions. YidC may have only a limited translocation ability, since procoat mutants with alterations in the periplasmic region have been found to require YidC as well as SecA and SecYEG for efficient insertion. 35 It would be interesting to make alterations in the periplasmic region of MscL to investigate the requirements that specify proteins for the Sec or YidC specific pathways. Also, it would be interesting to study whether YidC is required for the assembly of the MscL pentamer. Mechanosensitive ion channels play a key role in allowing a cell to sense physical stress within its environment. On the basis of the resolved channel structure of the MscL homologue from Mycobacterium tuberculosis determined by X-ray crystallography, the periplasmic loop region between the two transmembrane helices creates a flap that forms the extracellular surface of the pore.15 The authors noted that the amino acid residues 58–64 in the loop region (which corresponds to residues 62–68 in E. coli) are found deep in the pore of the channel near Lys33 (Ser35 in E. coli) of transmembrane helix 1. This might explain why the cysteine mutant I68C is not fully accessible to AMS, because not all the cysteine residues are well exposed to the aqueous phase. It has been implicated that the periplasmic loop region is important for MscL gating.36–39
MscL Insertion
1002
Materials and Methods Genetic manipulations MscL was amplified from E. coli K12 using the primers 5′-CAT ATG AGC ATT ATT AAA GAA TTT CGC G-3′ (forward) and 5′-TGC TCA GCT TAA GAG CGG TTA TTC TGC TCT TT-3′ (reverse). The PCR product was then ligated into the pT-Adv vector (Clontech). The NdeI/ Bpu1102I fragment from the resulting plasmid (pT-AdvMscL) was cloned into the phage T7 expression vector pET16b (Novagen), which placed a His10 tag at the N terminus. The DNA encoding MscL was then subcloned into plasmid pMS119 using XbaI and HindIII, and into plasmid pEH1 using NcoI and HindIII to allow expression in strains of different genetic background. Native MscL has no endogenous cysteine residue. Single cysteine substitutions at positions 68 and 136 were constructed using the QuikChange site-directed mutagenesis method (Stratagene) and verified by sequencing. Bacterial strains, plasmids and growth conditions SecE and YidC depletion was carried out using strains CM124,18 and JS7131,3 respectively, as described.40 The SecE depletion strain CM12418 was cultured in M9 minimal medium supplemented with 0.4% (w/v) glucose and 0.2% (w/v) arabinose. To deplete cells for SecE, overnight cultures were washed once with M9 medium and backdiluted 1:20 (v/v) into fresh M9 medium in the presence of 0.4% (w/v) glucose and the absence of arabinose. Cells were grown for 8 h. To deplete cells for YidC, overnight cultures were grown in 0.2% (w/v) arabinose and then washed twice with LB to remove cells of arabinose and back-diluted 1:50 (v/v) into fresh LB medium with 0.2% (w/v) glucose. Cells were grown for 3 h in glucose. To deplete SecDFYajC, cells of E. coli strain JP325,13 which is a RecA+ version of the SecDF depletion strain JP352,41 were grown in M9 medium containing 0.2% (w/v) arabinose and 0.2% (w/v) glucose. After overnight growth, the cells were harvested, washed in medium without arabinose and back-diluted 1:100 (v/v) in M9 medium containing 0.2% (w/v) glucose. For depletion, cells were further grown until an absorbance at 600 nm (A600) of 0.6 and diluted twofold with M9 medium containing 0.2% (w/v) glucose. This process was repeated for a total growth time of 6.5 h, as described.41 For Ffh depletion, cells of E. coli strain WAM12121 were grown overnight in LB medium containing 0.1% (w/v) arabinose and 0.4% (w/v) glucose, washed in medium lacking arabinose and back-diluted 1:20 (v/v) in LB medium containing glucose. When cultures reached an A600 of 0.4 (∼ 4 h), the cells were transferred to M9 minimal medium for 30 min before pulse–chase labelling. The FtsY depletion strain IY26 (BW25113-Kan-AraCPftsY) was obtained from E. Bibi. FtsY is under the control of the araBAD promoter and operator. The FtsY depletion strain IY26 was grown in LB medium supplemented with 0.2% (w/v) arabinose. For FtsY depletion, cultures grown overnight were first washed twice with LB medium and then back-diluted 1:40 (v/v) and were grown to an A600 of 0.4 (∼ 4 h) in LB medium with 0.2% (w/v) glucose. Medium was switched to M9 minimal medium containing glucose and the cells were grown for an additional 30 min before labelling. The MscL cysteine mutants S136C and I68C were expressed by induction with isopropyl thio-β-D-galacto-
side (IPTG) from the vectors pET16b and pEH1,42 in strains C41(DE3),43 and CM124, respectively, and from the pMS119 vector,44 in strains JS7131, WAM121 and IY26. Mutant –3MPClep13 is a procoat lep (PClep) mutant in which the soluble domain P2 of leader peptidase is attached to the M13 procoat. The mutant consists of three uncharged residues at positions +2, + 4 and +5 replacing negatively charged residues. In addition, three negatively charged residues were introduced after residue +10 in the periplasmic loop. Pulse–chase and immunoprecipitation Cells expressing the MscL cysteine mutants were induced for 10 min with IPTG (final concentration 1 mM) and incubated in 4-acetamido-4′-maleimidylstilbene-2, 2′-disulfonic acid disodium salt (AMS; final concentration 2.5 mM; from Molecular Probes) for 1 min. Cells were then radiolabelled with [35S]Met (10 μCi/ml) for 30 s, whereupon non-radioactive Met was added (final concentration 500 μg/ml) for 2 min or for 10 min. After labelling, cells were combined with 20 mM DTT to quench the AMS reaction. After quenching, samples were acid-precipitated, resuspended in 10 mM Tris, 2% (w/v) SDS, and immunoprecipitated with antiHis antibodies. In experiments where the membrane potential was depleted, carbonyl cyanide p-chlorophenylhydrazone (CCCP; final concentration 50 μM) was added 1 min before treatment with AMS and labelling with [35S]Met. For the azide studies, the cells were treated by the addition of 2 mM sodium azide for 5 min before labelling of the cells. For spheroplasting, cells were centrifuged at 12 000g and resuspended in 500 μl of ice-cold spheroplast buffer (33 mM Tris–HCl (pH 8.0), 40% (w/v) sucrose). Lysozyme (final concentration 5 mg/ml) and EDTA (final concentration 1 mM) were added for 15 min. Samples of the spheroplast suspension were incubated on ice for 30 min either in the presence or in the absence of AMS (final concentration 2.5 mM). After quenching with DTT, samples were precipitated as described above. The samples were then subjected to SDS-PAGE in 40 cm long, 17% polyacrylamide gels at constant voltage (200 V) at 4 °C. Gels were examined by Phosphorimager analysis (Fuji BAS1500).
Acknowledgements We thank Ross Dalbey for E. coli JP325, Chris Murphy for E. coli CM124 and Jan-Willem de Gier for providing E. coli WAM121 and the plasmid pEH1. We thank Eitan Bibi for E. coli IY26 and for anti-Ffh serum, Hans-Georg Koch for anti-FtsY serum, and Christian Hartmann for the construction of plasmid pET16b/MscL. This work was supported by the Deutsche Forschungsgemeinschaft Sonderforschungsbereich 495.
References 1. Bonnefoy, N., Chalvet, F., Hamel, P., Slonimski, P. P. & Dujardin, G. (1994). OXA1, a Saccharomyces cerevisiae
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nuclear gene whose sequence is conserved from prokaryotes to eukaryotes controls cytochrome oxidase biogenesis. J. Mol. Biol. 239, 201–212. Urbanus, M. L., Scotti, P. A., Fröderberg, L., Sääf, A., de Gier, J.-W. L., Brunner, J. et al. (2001). Secdependent membrane protein insertion: sequential interaction of nascent FtsQ with SecY and YidC. EMBO Rep. 2, 524–529. Samuelson, J. C., Chen, M., Jiang, F., Möller, I., Wiedmann, M., Kuhn, A. et al. (2000). YidC mediates membrane protein insertion in bacteria. Nature, 406, 637–641. Chen, M., Samuelson, J. C., Jiang, F., Müller, M., Kuhn, A. & Dalbey, R. E. (2002). Direct interaction of YidC with the Sec-independent Pf3 coat protein during its membrane protein insertion. J. Biol. Chem. 277, 7670–7675. Beck, K., Eisner, G., Trescher, D., Dalbey, R. E., Brunner, J. & Müller, M. (2001). YidC, an assembly site for polytopic Escherichia coli membrane proteins located in immediate proximity to the SecYE translocon and lipids. EMBO Rep. 2, 709–714. Dalbey, R. E. & Kuhn, A. (2004). YidC family members are involved in the membrane insertion, lateral integration, folding, and assembly of membrane proteins. J. Cell Biol. 166, 769–774. Nouwen, N. & Driessen, A. J. (2002). SecDFyajC forms a heterotetrameric complex with YidC. Mol. Microbiol. 44, 1397–1405. Urbanus, M. L., Fröderberg, L., Drew, D., Björk, P., de Gier, J.-W. L., Brunner, J. et al. (2002). Targeting, insertion and localization of Escherichia coli YidC. J. Biol. Chem. 277, 12718–12723. Serek, J., Bauer-Manz, G., Struhalla, G., van den Berg, L., Kiefer, D., Dalbey, R. & Kuhn, A. (2004). Escherichia coli YidC is a membrane insertase for Sec-independent proteins. EMBO J. 23, 294–301. van der Laan, M., Bechtluft, P., Kol, S., Nouwen, N. & Driessen, A. J. M. (2004). F1F0 ATP synthase subunit c is a substrate of the novel YidC pathway for membrane protein biogenesis. J. Cell Biol. 165, 213–222. Yi, L., Celebi, N., Chen, M. & Dalbey, R. E. (2004). Sec/ SRP requirements and energetics of membrane insertion of subunits a, b, and c of the Escherichia coli F1F0 ATP synthase. J. Biol. Chem. 279, 39260–39267. Kuhn, A. (1988). Alterations in the extracellular domain of M13 procoat protein make its membrane insertion dependent on secA and secY. Eur. J. Biochem. 177, 267–271. Chen, M., Xie, K., Yuan, J., Yi, L., Facey, S. J., Pradel, N. et al. (2005). Involvement of SecDF and YidC in the membrane insertion of M13 procoat mutants. Biochemistry, 44, 10741–10749. Booth, I. R. & Louis, P. (1999). Managing hypoosmotic stress: aquaporins and mechanosensitive channels in Escherichia coli. Curr. Opin. Microbiol. 2, 166–169. Chang, G., Spencer, R. H., Lee, A. T., Barclay, M. T. & Rees, D. C. (1998). Structure of the MscL homolog from Mycobacterium tuberculosis: a gated mechanosensitive ion channel. Science, 282, 2220–2226. Valent, Q. A., Scotti, P. A., High, S., de Gier, J.-W. L., von Heijne, G., Lentzen, G. et al. (1998). The Escherichia coli SRP and SecB targeting pathways converge at the translocon. EMBO J. 17, 2504–2512. Uchida, K., Mori, H. & Mizushima, S. (1995). Stepwise movement of preproteins in the process of translocation across the cytoplasmic membrane of Escherichia coli. J. Biol. Chem. 270, 30862–30868.
1003 18. Traxler, B. & Murphy, C. (1996). Insertion of the polytopic membrane protein MalF is dependent on the bacterial secretion machinery. J. Biol. Chem. 271, 12394–12400. 19. Pogliano, J. A. & Beckwith, J. (1994). SecD and SecF facilitate protein export in Escherichia coli. EMBO J. 13, 554–561. 20. Oliver, D. B., Cabelli, R. J., Dolan, K. M. & Jarosik, G. P. (1990). Azide-resistant mutants of Escherichia coli alter the SecA protein, an azide-sensitive component of the protein export machinery. Proc. Natl Acad. Sci. USA, 87, 8227–8231. 21. de Gier, J.-W. L., Scotti, P. A., Sääf, A., Valent, Q. A., Kuhn, A., Luirink, J. & von Heijne, G. (1998). Differential use of the signal recognition particle translocase targeting pathway for inner membrane protein assembly in Escherichia coli. Proc. Natl Acad. Sci. USA, 95, 14646–14651. 22. Daniels, C. J., Bole, D. G., Quay, S. C. & Oxender, D. L. (1981). Role for membrane potential in the secretion of protein into the periplasm of Escherichia coli. Proc. Natl Acad. Sci. USA, 78, 5396–5400. 23. Long, J. C., Wang, S. & Vik, S. B. (1998). Membrane topology of subunit a of the F1F0 ATP synthase as determined by labeling of unique cysteine residues. J. Biol. Chem. 273, 16235–16240. 24. Dale, H. & Krebs, M. P. (1999). Membrane insertion kinetics of a protein domain in vivo. The bacterioopsin N terminus inserts co-translationally. J. Biol. Chem. 274, 22693–22698. 25. Kiefer, D. & Kuhn, A. (1999). Hydrophobic forces drive spontaneous membrane insertion of the bacteriophage Pf3 coat protein without topological control. EMBO J. 18, 6299–6306. 26. Cao, G., Kuhn, A. & Dalbey, R. E. (1995). The translocation of negatively charged residues across the membrane is driven by the electrochemical potential: evidence for an electrophoresis-like membrane transfer mechanism. EMBO J. 14, 866–875. 27. Fröderberg, L., Houben, E., Samuelson, J. C., Chen, M., Park, S.-K., Phillips, G. J. et al. (2003). Versatility of inner membrane protein biogenesis in Escherichia coli. Mol. Microbiol. 47, 1015–1027. 28. Facey, S. J. & Kuhn, A. (2004). Membrane integration of E. coli model membrane proteins. Biochim. Biophys. Acta, 1694, 55–66. 29. van Bloois, E., Haan, G. J., de Gier, J.-W., Oudega, B. & Luirink, J. (2004). F1F0 ATP synthase subunit c is targeted by the SRP to YidC in the E. coli inner membrane. FEBS Letters, 576, 97–100. 30. Celebi, N., Yi, L., Facey, S. J., Kuhn, A. & Dalbey, R. E. (2006). Membrane biogenesis of subunit II of cytochrome bo oxidase: contrasting requirements for insertion of N-terminal and C-terminal domains. J. Mol. Biol. 357, 1428–1436. 31. van Bloois, E., Haan, G.-J., de Gier, J.-W., Oudega, B. & Luirink, J. (2006). Distinct requirements for translocation of the N-tail and C-tail of the Escherichia coli inner membrane protein CyoA. J. Biol. Chem. 281, 10002–10009. 32. Moore, M., Goforth, R. L., Mori, H. & Henry, R. (2003). Functional interaction of chloroplast SRP/FtsY with the ALB3 translocase in thylakoids: substrate not required. J. Cell Biol. 162, 1245–1254. 33. Moore, M., Harrison, M. S., Peterson, E. C. & Henry, R. (2000). Chloroplast Oxa1p homolog albino3 is required for post-translational integration of the light harvesting chlorophyll-binding protein into thylakoid membranes. J. Biol. Chem. 275, 1529–1532.
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1004 34. Mitra, K., Schaffitzel, C., Shaikh, T., Tama, F., Jenni, S., Brooks, C. L., III et al. (2005). Structure of the E. coli protein-conducting channel bound to a translating ribosome. Nature, 438, 318–324. 35. Roos, T., Kiefer, D., Hugenschmidt, S., Economou, A. & Kuhn, A. (2001). Indecisive M13 procoat protein mutants bind to SecA but do not activate the translocation ATPase. J. Biol. Chem. 276, 37909–37915. 36. Blount, P., Sukharev, S. I., Schroeder, M. J., Nagle, S. K. & Kung, C. (1996). Single residue substitutions that change the gating properties of a mechanosensitive channel in Escherichia coli. Proc. Natl Acad. Sci. USA, 93, 11652–11657. 37. Maurer, J. A., Elmore, D. E., Lester, H. A. & Dougherty, D. A. (2000). Comparing and contrasting Escherichia coli and Mycobacterium tuberculosis mechanosensitive channels (MscL). New gain of function mutations in the loop region. J. Biol. Chem. 275, 22238–22244. 38. Maurer, J. A. & Dougherty, D. A. (2003). Generation and evaluation of a large mutational library from the Escherichia coli mechanosensitive channel of large conductance, MscL: implications for channel gating and evolutionary design. J. Biol. Chem. 278, 21076–21082. 39. Ajouz, B., Berrier, C., Besnard, M., Martinac, B. & Ghazi, A. (2000). Contributions of the different
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extramembranous domains of the mechanosensitive ion channel MscL to its response to membrane tension. J. Biol. Chem. 275, 1015–1022. Facey, S. J. & Kuhn, A. (2003). The sensor protein KdpD inserts into the Escherichia coli membrane independent of the Sec translocase and YidC. Eur. J. Biochem. 270, 1724–1734. Economou, A., Pogliano, J. A., Beckwith, J., Oliver, D. B. & Wickner, W. (1995). SecA membrane cycling at SecYEG is driven by distinct ATP binding and hydrolysis events and is regulated by SecD and SecF. Cell, 83, 1171–1181. Hashemzadeh-Bonehi, L., Mehraein-Ghomi, F., Mitsopoulos, C., Jacob, J. P., Hennessey, E. S. & BroomeSmith, J. K. (1998). Importance of using lac rather than ara promoter vectors for modulating the levels of toxic gene products in Escherichia coli. Mol. Microbiol. 30, 676–678. Miroux, B. & Walker, J. E. (1996). Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J. Mol. Biol. 260, 289–298. Balzer, D., Ziegelin, G., Pansegrau, W., Kruft, V. & Lanka, E. (1992). KorB protein of promiscuous plasmid RP4 recognizes inverted sequence repetitions in regions essential for conjugative plasmid transfer. Nucl. Acids Res. 20, 1851–1858.
Edited by I. B. Holland (Received 12 September 2006; received in revised form 18 October 2006; accepted 25 October 2006) Available online 28 October 2006
doi:10.1016/j.jmb.2006.10.083
The Mechanosensitive Channel Protein MscL Is Targeted by the SRP to The Novel YidC Membrane Insertion Pathway of Escherichia coli Sandra J. Facey, Stella A. Neugebauer, Susanne Krauss and Andreas Kuhn⁎ Institute of Microbiology, University of Hohenheim, 70599 Stuttgart, Germany
The mechanosensitive channel MscL in the inner membrane of Escherichia coli is a homopentameric complex involved in homeostasis when cells are exposed to hypo-osmotic conditions. The E. coli MscL protein is synthesized as a polypeptide of 136 amino acid residues and uses the bacterial signal recognition particle (SRP) for membrane targeting. The protein is inserted into the membrane independently of the Sec translocon. Mutants affected in the Sec-components are competent for MscL assembly. Translocation of the periplasmic domain was detected using a membrane-impermeant, sulfhydryl-specific gel-shift reagent. The modification of a single cysteine residue at position 68 indicated its translocation across the inner membrane. From these in vivo experiments, it is concluded that the electrical chemical membrane potential is not necessary for membrane insertion of MscL. However, depletion of the membrane insertase YidC inhibits translocation of the protein across the membrane. We show here that YidC is essential for efficient membrane insertion of the MscL protein. YidC is a component of a recently identified membrane insertion pathway that is evolutionarily conserved in bacteria, mitochondria and chloroplasts. © 2006 Elsevier Ltd. All rights reserved.
*Corresponding author
Keywords: MscL; proton motive force; Sec translocase; SRP; YidC
Introduction YidC is a 61 kDa protein in the inner membrane of Escherichia coli. It was first identified as a homolog of the Oxa1 protein, which is involved in assembly of the cytochrome oxidase of Saccharomyces cerevisiae.1 YidC was found to be associated with nascent chains of bacterial membrane proteins, suggesting that it is involved in the membrane assembly as well.2 The proof that YidC is involved directly in the membrane insertion process came from a mutant E. coli strain in which YidC was depleted within 3 h of growth, accumulating the non-inserted membrane proteins in the cytoplasm, whereas exported proteins were not affected.3 Intriguingly, Sec-independent proteins, such as the major coat proteins of Abbreviations used: AMS, 4-acetamido-4′maleimidylstilbene-2, 2′-disulfonic acid disodium salt; CCCP, carbonyl cyanide p-chlorophenylhydrazone; SRP, signal recognition particle. E-mail address of the corresponding author: [email protected]
bacteriophage M13 and Pf3, were inhibited dramatically, whereas the Sec-dependent proteins were only retarded for membrane insertion.3,4 The Sec-dependent proteins contact YidC either shortly after they bind to SecYEG,2 or to both simultaneously.5 Most likely, YidC forms together with the Sec complex, a collection site for the transmembrane regions of multispanning proteins by shielding the protein chain from lipids and allowing the three-dimensional folding.6 In accordance with this, YidC has been found to be bound to the Sec translocase, specifically with the SecDFYajC complex.7 The SecDFYajC complex connects YidC to the SecYEG machinery. Since YidC is present in the E. coli membrane in molecular excess, only a fraction of YidC is bound to the Sec translocase and most of the YidC forms an independent entity.8 Biochemical purification and reconstitution into proteoliposomes showed that YidC functions per se as a membrane insertase and catalyses the insertion of Sec-independent proteins into the membrane.9,10 Therefore, YidC by itself resembles a novel membrane insertion pathway that can operate independent of the Sec translocon. It is
0022-2836/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.
996 not known how many membrane proteins use the YidC or the Sec pathway. The fact that YidC is essential for growth suggests that phage proteins are not the only substrates. Recently, subunit c from the F1F0 ATP synthase complex was shown to use the YidC-only pathway, which could explain the lethal effect of YidC depletion.10,11 The YidC substrates are limited to small periplasmic regions with only a few charged residues, since mutations that change these features switch to the Sec-dependent pathway.12,13 The MscL protein of the mechanosensitive channel with large conductance appears to play a primary role in protecting cells exposed to osmotic downshock.14 MscL is believed to open in response to high levels of tension within the membrane, allowing ions and other solutes to be released from the cell and thus preventing rupture of the cell membrane. The crystal structure of the MscL homolog from Mycobacterium tuberculosis revealed a homopentameric channel.15 Each subunit spans the inner membrane twice, and has a small periplasmic region of 29 residues that includes five charged residues. The structure of MscL shows that the N-terminal transmembrane helix forms an inner ring in the pentameric structure, whereas the Cterminal transmembrane helix is located at the periphery of the channel. The periplasmic regions of each subunit form the extracellular surface of the pore. We show here that the MscL protein is targeted to the membrane by the signal recognition particle (SRP) pathway, and inserted into the inner membrane of E. coli independently of SecA, SecE and SecDF, but uses the membrane insertase YidC. Previously, it was thought that the bacterial SRP targets proteins specifically to the Sec translocon.16 Since MscL uses SRP and YidC, this underlines that the membrane targeting, insertion and the translocation components operate as individual modules.
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immunoprecipitated. Derivatized and underivatized proteins were then separated by SDS-PAGE and examined by phosphorimaging. We chose the AMS derivatization method to analyse the topology of the MscL protein instead of the conventional protease mapping assay, because the periplasmic loop of MscL was mostly resistant to digestion by proteinase K. To apply the assay, several single-cysteine mutants of MscL were prepared at positions 59, 64 and 68 in the periplasmic loop, because it was not known which proteins would be expressed or would exhibit a gel mobility-shift with AMS. In all cases, a shift was observed for the cysteine mutants (data not shown), suggesting that all of the cysteine residues in the mutant proteins are derivatizable to AMS. The cysteine mutant I68C was selected for further analysis (Figure 1). In this mutant, cysteine is substituted for isoleucine at position 68 in the periplasmic loop. As expected, the cysteine mutant I68C, which is located in the periplasmic loop, is labelled by AMS in whole and disrupted cells (Figure 1(b), middle panel). In contrast, wild-type MscL, which contains no endogenous cysteine residue, shows no reaction with AMS (Figure 1(b), left panel). In the S136C mutant, serine was substituted for cysteine at position 136 in the C terminus of MscL, which is located in the cytoplasm.
Results Membrane topology analysed by AMS derivatization Insertion of MscL into the cytoplasmic membrane was monitored by rapid derivatization of a unique cysteine residue in the periplasmic loop of the protein with AMS, a membrane-impermeable, sulfhydryl reagent that shifts protein mobility on SDS/ polyacrylamide gels. 17 Derivatization of free cysteine residues retards the mobility of the protein on a SDS/polyacrylamide gel by an amount corresponding roughly to the added mass of AMS (∼ 0.5 kDa). Thus, the derivatized form of MscL will display a higher molecular mass than the underivatized form. Cells expressing the membrane protein were incubated in AMS and then pulselabelled and chased with non-radioactive Met. After radiolabelling, proteins were acid-precipitated and
Figure 1. Topological model of the E. coli MscL protein and location of cysteine substitutions and AMS derivatization of MscL mutants. (a) MscL is a 136 amino acid residue membrane protein that consists of two transmembrane α-helices, which are connected by a periplasmic loop of 29 residues. Arrows indicate the positions of the cysteine mutations introduced for AMS derivatization. (b) Cells expressing the mutant proteins were pulse-labelled with [35S]Met for 30 s, then chased with non-radioactive Met for 2 min. After pulse-chase labelling, the cells were incubated in the presence (+) or absence (−) of AMS or disrupted by sonification and then treated with AMS for 10 min. The AMS-treated aliquots were combined with an equal volume of 20 mM DTT in medium salts to quench the AMS reaction. The proteins were then acid-precipitated and immunoprecipitated with anti-His antiserum, and subjected to SDS-PAGE (17% (w/v) polyacrylamide). d and u denote the AMS-derivatized and underivatized protein, respectively.
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Mutant S136C is our negative control, and exhibits no gel mobility-shift in the presence of AMS, which is membrane-impermeable (Figure 1(b), right panel). However, disruption of the cells by sonification should permit labelling of the available SH group upon addition of AMS. Figure 1(b) shows this was indeed the case. Mutant S136C shows a shift with AMS when the membrane is disrupted, indicating that AMS is membrane-impermeable under the assay conditions, and that derivatization of the periplasmic-localized cysteine residue is evidence for translocation. Depletion of the Sec translocase components does not affect the membrane insertion of MscL We investigated whether the SecYEG/DF translocase mediates the membrane insertion of MscL. Depletion strains that control the expression of SecE and SecDF were used to investigate the translocation of MscL. The role of SecE was examined in the depletion strain E. coli CM124.18 Expression of SecE is induced with arabinose and repressed tightly in the presence of glucose. Cells carrying the cysteine mutants were either grown in medium with arabinose (Figure 2(a), lanes 3, 4, 7 and 8) or with glucose for 8 h (Figure 2(a), lanes 1, 2, 5 and 6). To examine translocation, CM124 cells expressing the MscL mutant I68C were pulse-labelled with [35S] Met for 30 s and chased with non-radioactive Met for 2 min in the presence or in the absence of AMS. The results show that two major radiolabelled bands were observed when the cells carrying the cysteine mutant I68C were depleted of SecE (Figure 2(a), lane 6). Samples radiolabelled in the absence of AMS with or without SecE (lanes 5 and 7) yielded a single band that co-migrated with the lower band of lanes 6 and 8, indicating that the band corresponds to underivatized protein. The upper band in lanes 6 and 8 corresponds to derivatized protein. This demonstrates that MscL has inserted into the membrane without SecE (lane 6). In contrast, translocation and signal peptide cleavage of the Sec-dependent outer membrane protein (OmpA) was blocked completely by SecE depletion (Figure 2(b)). To ensure that only the translocated cysteine residues were derivatized, cells expressing a MscL mutant containing a cytoplasmic-localized cysteine residue (S136C) were radiolabelled in the presence of AMS (Figure 2(a), lanes 2 and 4). As expected, S136C exhibited no gel mobility-shift in the presence of AMS. Previously, it was shown that SecDF facilitates the insertion of Sec-dependent proteins in vivo.19 To verify our results, we used the E. coli strain JP325,13 to deplete SecDF. In JP325, the chromosomal secDF operon is under control of the araBAD promoter. As a consequence, YajC, SecD and SecF are synthesized only when grown in the presence of arabinose. For the cysteine mutant I68C, gel mobility-shifts were observed irrespective of whether the cells were depleted of SecDF (Figure 2(c), lanes 2 and 4). In the
997 untreated samples, no shift was observed for I68C (Figure 2(c), lanes 1 and 3), indicating that MscL insertion is independent of SecDF. As a control, the processing of OmpA was analysed in a parallel culture (Figure 2(d)). A decrease in the rate of cleavage of OmpA was noticeable under SecDF depleting conditions. As an additional control, efficient SecDF depletion was demonstrated by the inhibitory effect on signal peptide cleavage of the procoat lep mutant -3MPClep (Figure 2(e)), as observed previously.13 An alternative procedure was applied, in which the pulse–chased cells were first converted into spheroplasts and then treated with AMS. By performing this assay, we can demonstrate that AMS does not inhibit membrane insertion. The results are shown in Figure 2(f) with and without sodium azide, which has been shown to inhibit SecA activity at a concentration of 2 mM.20 To address the role of SecA in MscL membrane insertion, bacteria were treated with 2 mM sodium azide for 5 min before the addition of [35S]Met and conversion into spheroplasts. The results show again that two radiolabelled bands were observed when the cells carrying the cysteine mutant I68C were treated with or without sodium azide in the presence of AMS (Figure 2(f), lanes 2 and 4) after a 10 min chase. Similar results were obtained after a 2 min chase (data not shown). The upper band corresponds to derivatized protein and the lower band to underivatized protein. These results further verify that MscL integration is not dependent on SecA. As expected, proOmpA was converted rapidly to OmpA in the absence of sodium azide (Figure 2(g), left panel). In the presence of sodium azide, the Secdependent proOmpA accumulated in its precursor form in the cells (Figure 2(g), right panel). Insertion of the MscL protein requires YidC The role of YidC in the membrane insertion of MscL was examined in the depletion strain JS7131, where YidC expression is under the control of an araBAD promoter and operator.3 YidC expression is induced with arabinose and repressed tightly in the presence of glucose. To deplete YidC, the cells were grown for 3 h with glucose and then assayed for derivatization with AMS (Figure 3(a), lanes 2, 6 and 10). When the cells were grown with arabinose (YidC+), a gel mobility-shift was observed for the cysteine mutant I68C (Figure 3(a), lanes 8 and 12), indicating MscL was readily inserted into the membrane. In YidC-deficient cells (grown with glucose), no shift was observed with AMS after a 2 min (Figure 3(a), lane 6) or a 10 min chase (Figure 3(a), lane 10), indicating no translocation occurred. This demonstrates that MscL requires YidC for insertion. As expected, no shift was observed for the cysteine mutant S136C, which contains a cytoplasmic-localized cysteine residue (Figure 3(a), left panel). Furthermore, processing of the Sec-dependent protein OmpA was not affected under these conditions, demonstrating that indirect inactivation of the Sec
998
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Figure 2. The translocation of the periplasmic domain of MscL is independent of SecE, SecDF and SecA. (a) Strain CM124 expressing the MscL cysteine mutants was grown in M9 minimal medium containing arabinose (lanes 3, 4, 7 and 8). For depletion of SecE (lanes 1, 2, 5 and 6), the cells were grown in the presence of glucose and the absence of arabinose for 8 h. IPTG (1 mM) was added for 10 min to induce expression. Cells expressing the mutant proteins were incubated in the presence (+) or in the absence of AMS (−) for 1 min, pulse-labelled with [35S]Met for 30 s, then chased with nonradioactive Met for 2 min. After quenching with 20 mM DTT, the radiolabelled samples were acid-precipitated and immunoprecipitated with an anti-His antibody and then subjected to SDS-PAGE (17% (w/v) polyacrylamide gel). d and u denote the AMS-derivatized and underivatized protein, respectively. (b) As a control, translocation and processing of the Sec-dependent protein OmpA was monitored in parallel to verify the depletion of SecE. Samples were immunoprecipitated with antiserum to OmpA. (c) JP325 cells bearing the MscL cysteine mutant I68C were back-diluted (1:100) in M9 minimal medium containing arabinose and glucose (SecDF+, lanes 3 and 4) or only glucose (SecDF–, lanes 1 and 2). After induction with IPTG for 10 min, cells were pulse-labelled with [35S]Met for 30 s in the presence (+) or in the absence (−) of AMS and then chased with excess cold Met for 2 min. After quenching with DTT, samples were immunoprecipitated, subjected to SDS-PAGE and analysed by phosphorimaging. (d) As a control, the processing of the Sec-dependent protein OmpA was monitored in parallel after 30 s pulse-labelling and a 10 s, 1 min, and 2 min chase. Samples were immunoprecipitated with antiserum to OmpA. (e) Efficient SecDF depletion was demonstrated by the inhibitory effect on signal peptide cleavage of the procoat mutant –3MPClep (left panel). After induction with IPTG for 10 min and labelling with [35S]Met for 30 s, the cells were chased with non-radioactive Met for 10 s, 1 min, and 2 min. p and m denote the precursor form and mature form, respectively. (f) The MscL cysteine mutant I68C was expressed in the E. coli strain C41 (DE3) in the absence (lanes 1 and 2) or in the presence (lanes 3 and 4) of sodium azide. Cells were radiolabelled with [35S] Met for 30 s, chased with non-radioactive Met for 10 min and then converted to spheroplasts as described in Materials and Methods. After spheroplasting, samples were treated with or without AMS for 30 min and analysed as described in Figure 2(a). (g) OmpA accumulated in its precursor form (proOmpA) in the azide-treated cells (right-hand panel).
translocon had not occurred (Figure 3(b)). YidC depletion was confirmed by analysing the YidC content in a cell sample taken before induction with IPTG by immunoblotting using YidC antiserum (Figure 3(c) and (e)). For a control, processing of a YidC-dependent protein M13 procoat was monitored in parallel. M13 procoat was pulse-labelled for 30 s and chased for 1 min and for 2 min, and then analysed for translocation and processing (Figure 3(d)). When the cells were grown in glucose (YidC–), M13 procoat processing was largely inhibited and the protein accumulated in a non-translocated form. In
contrast, when YidC was present, M13 procoat was readily inserted into the membrane. MscL is targeted by the SRP to the inner membrane The bacterial SRP, which includes the 54 homologue protein (Ffh), the 4.5 S RNA and the SRP receptor FtsY, selectively recognizes inner membrane proteins via their long hydrophobic transmembrane segments.21 To investigate the involvement of SRP in the biogenesis of MscL, we transformed
MscL Insertion
999 the Ffh depletion E. coli strain WAM121 with the plasmids encoding the MscL cysteine mutants. Cells expressing the mutant proteins were either grown in medium with arabinose (Ffh+, Figure 4(a), lanes 3, 4, 7, 8, 11 and 12) or with glucose (Ffh–, Figure 4(a), lanes 1, 2, 5, 6, 9 and 10) and then assayed for translocation with AMS. When Ffh was present, the cysteine mutant I68C was readily inserted into the membrane and derivatized (Figure 4(a), lanes 8 and 12). In contrast, in Ffh-depleted cells, MscL insertion was largely inhibited after a 2 min (Figure 4(a), lane 6) and a 10 min chase (Figure 4(a), lane 10). As for the control (S136C), only underivatized protein was observed in the presence and in the absence of AMS (Figure 4(a), left panel). Depletion of Ffh was verified by
Figure 3. YidC is required for membrane insertion of MscL. (a) JS7131 cells expressing the MscL cysteine mutants S136C and I68C were pulse-labelled with [35S] Met for 30 s and chased with non-radioactive Met for 2 min (lanes 1–8) or 10 min (lanes 9–12), either in the presence of arabinose to induce expression of YidC (lanes 3, 4, 7, 8, 11 and 12) or in medium lacking arabinose to deplete YidC (lanes 1, 2, 5, 6, 9 and 10). Before labelling, cells were treated in the presence (+) or in the absence (−) of AMS. Samples were immunoprecipitated with an antiHis antibody and analysed by SDS-PAGE. d, derivatized; u, underivatized. (b) A sample of cells was immunoprecipitated with OmpA antiserum and then analysed by SDS-PAGE and phosphorimaging, showing that OmpA translocation was not affected. (c) Immunoblot analysis was used to determine the levels of YidC in the JS7131 strain expressing the MscL cysteine mutants under glucose (YidC–) and arabinose (YidC+) conditions. Before induction by IPTG, 1 ml of cells was removed from the glucose and the arabinose sample. The cells were precipitated with TCA, resuspended in SDS loading buffer, and resolved by SDS-PAGE. Immunoblotting was done using antiserum against the C terminus of YidC. (d) For a control, the translocation and processing of the M13 procoat protein was analysed under YidC+ and YidC– conditions. After induction with IPTG for 10 min, cells were pulse-labelled with [35S]Met for 30 s and chased with non-radioactive Met for 1 min and 2 min. All samples were precipitated with 20% (w/v) TCA, immunoprecipitated with anti-M13 serum and visualized by phosphorimaging. (e) Immunoblot analysis of the levels of YidC in the JS7131 strain expressing M13 procoat under glucose (YidC–) and arabinose (YidC+) conditions.
Figure 4. MscL is dependent on Ffh for membrane insertion. (a) E. coli WAM121 cells were transformed with the plasmids coding for the MscL cysteine mutants. The cells were depleted of Ffh by growth in the presence of 0.4% glucose (lanes 1, 2, 5, 6, 9 and 10), as described in Materials and Methods. Plasmid-encoded proteins were induced with 1 mM IPTG for 10 min, incubated in the presence (+) or in the absence (−) of AMS for 1 min, pulselabelled with [35S]Met for 30 s and chased for 2 min (lanes 1–8) or 10 min (lanes 9–12) with non-radioactive Met. Samples were immunoprecipitated with an anti-His antibody and analysed by SDS-PAGE. d and u denote the derivatized and underivatized form of MscL. (b) Immunoblot analysis of Ffh levels in the WAM121 strain expressing the MscL cysteine mutants under glucose (Ffh–) and arabinose (Ffh+) conditions. Immunoblotting was done using antiserum against Ffh. (c) As a control, translocation and processing of the secretory protein OmpA was monitored in parallel after a 30 s pulselabelling and a 2 min chase. (d) Translocation of the periplasmic loop of the YidC-dependent M13 coat protein was monitored by pulse-labelling with [35S]Met for 1 min and 5 min. Samples were immunoprecipitated with antiserum to M13 coat protein.
MscL Insertion
1000 analysing the Ffh content in a cell sample taken before induction with IPTG by immunoblotting (Figure 4(b)). To address the possibility that SRP depletion is causing an indirect effect, e.g. inhibiting the insertion of YidC and thereby inhibiting membrane protein insertion of MscL, the processing of a secretory protein OmpA (Figure 4(c)) and a YidCdependent protein M13 procoat (Figure 4(d)) were analysed in a parallel culture. The wild-type M13 procoat protein does not require the SRP homologue Ffh for membrane insertion,21 but requires YidC. Signal peptide processing of both M13 procoat and proOmpA were largely unaffected in cells depleted of Ffh, indicating normal insertion of M13 and export of OmpA. Therefore, the membrane translocation of MscL is affected directly by depletion of Ffh. To verify this, MscL targeting was investigated in the FtsY depletion strain IY26. Cells of strain IY26 in which ftsY expression is under control of an arabinose-inducible promoter were grown in the presence (FtsY+, Figure 5(a), lanes 3, 4, 7 and 8) or in the absence of arabinose (FtsY–, Figure 5(a), lanes 1, 2, 5 and 6) and then assayed for translocation with AMS. In accordance with the effects of depletion of Ffh, depletion of FtsY inhibits membrane insertion of MscL (Figure 5(a), lane 6). Together, the in vivo
Figure 6. MscL does not require the electrochemical membrane potential for membrane insertion. (a) C41(DE3) cells expressing the MscL cysteine mutant I68C were analysed with (+) and without (−) CCCP, which was added 1 min before treatment of the cells with AMS at a final concentration of 50 μM. Cells were then labelled with [35S]Met for 30 s, chased with non-radioactive Met for 10 min and analysed as described for Figure 2(a). d, derivatized; u, underivatized. (b) OmpA accumulated in its precursor form (proOmpA) in CCCP treated cells.
results suggest strongly that MscL requires SRP for efficient membrane targeting. Depletion of FtsY was verified by analysing the FtsY level in a cell sample in parallel by immunoblotting with FtsY antiserum (Figure 5(b)). In this experiment, FtsY levels were reduced dramatically in the FtsYdepleted cells (FtsY–) as judged by immunoblot analysis, whereas YidC levels were not affected (Figure 5(c)). Electrochemical membrane potential is not required for insertion of MscL into the membrane
Figure 5. The SRP receptor FtsY is required for efficient targeting of MscL to the membrane. (a) The FtsY depletion strain IY26 bearing the MscL cysteine mutants were grown under either FtsY depletion or FtsY expression conditions. After induction with IPTG for 10 min, cells were incubated in the presence (+) or absence (−) of AMS for 1 min, pulse-labelled with [35S]Met for 30 sec, then chased with non-radioactive Met for 2 min. Samples were prepared and processed as described for Figure 2(a). (b) Immunoblot analysis of FtsY levels in the IY26 strain expressing the MscL cysteine mutants under glucose (FtsY–) and arabinose (FtsY+) conditions. Immunoblotting was done using antiserum against FtsY. (c) Immunoblot analysis of the levels of YidC in the IY26 strain expressing the MscL cysteine mutants under glucose (FtsY–) and arabinose (FtsY+) conditions. Antibodies against the C terminus of YidC were used for immunoblotting.
To assess the effect of the proton motive force on the membrane insertion of MscL, the cells were treated with CCCP, a protonophore that dissipates the proton motive force.22 The proton motive force was collapsed by adding 50 μM CCCP, 1 min before treatment of the cells with AMS and labelling of the cells with [35S]Met. Figure 6(a) shows the AMS derivatization of the MscL mutant I68C in the absence (lane 2) and in the presence (lane 4) of CCCP. In both cases, gel shifts were observed, indicating that MscL does not require an electrochemical membrane potential for membrane assembly. Samples radiolabelled in the absence of AMS (lanes 1 and 3) yielded a single band corresponding to underivatized protein. The export of OmpA is blocked completely by CCCP, since proOmpA accumulates in the treated cells (Figure 6(b)).
Discussion AMS derivatization is very useful in analysing the biosynthesis of small membrane proteins in vivo. It has been used in the analysis of other membrane proteins as a membrane-impermeable reagent.23,24 Using this technology, we have shown here that the 136 residue long MscL protein is a further substrate
MscL Insertion
of the YidC pathway. Besides MscL, the subunit c of the ATP synthase,10 and the major coat proteins of bacteriophage M13 and Pf3 have been characterized as YidC substrates.3,4 All these inner membrane proteins use the YidC protein without the Sec translocase. In contrast to the bacteriophage coat proteins, the MscL protein inserts independently of the electrochemical membrane potential. So far, only small hydrophilic regions have been shown to be translocated by the YidC-only pathway. In the Pf3 coat protein the N-terminal tail of 18 residues that includes two negatively charged aspartyl groups is translocated, whereas for the csubunit of the F1F0 ATP synthase both terminal tails of seven and three residues are translocated. In contrast to the bacteriophage coat proteins, insertion of the c-subunit of the F1F0 ATP synthase does not depend on the electrochemical membrane potential.10 For the Pf3 protein, it has been shown that the negatively charged residues are required for membrane insertion and determine the orientation of the protein in the membrane.25 The hydrophilic loop region of the M13 procoat protein of 20 residues has five charged residues resulting in a − 3 net charge. For this protein, the negative charge of the periplasmic region is correlated directly with the involvement of the transmembrane potential for membrane insertion. A mutant that lacks the charged residues in the periplasmic domain of the M13 procoat protein was independent of the electrochemical membrane potential.26 In contrast, the hydrophilic loop region of the MscL protein has 29 residues that include five charged residues. The independence of MscL for a membrane potential might be due to the fact that the periplasmic loop has a net charge of only − 1. Previously, it was believed that the bacterial SRP specifically targets proteins to the Sec translocase.16 Later, Fröderberg and co-workers27 showed that an artificial M13 procoatlep construct and a ProW construct required SRP and YidC. Interestingly, our data show that MscL requires also the bacterial SRP and YidC. Taken together, this underlines that membrane targeting and membrane insertion are two separate processes operating by distinct modules.27,28 The YidC-dependent proteins M13 procoat,21 and Pf3 coat4 do not require SRP. The involvement of SRP in targeting of the c-subunit of the F 1F0 ATP synthase is controversial. 10,11,29 Recently, it has been shown that the CyoA subunit II of the cytochrome o oxidase is severely affected upon depletion of YidC. 30,31 CyoA spans the membrane twice and is synthesized with a cleavable signal peptide. It was demonstrated that the Nterminal domain of CyoA consisting of the first transmembrane segment and the small periplasmic domain of 26 residues requires YidC and SRP for targeting and insertion into the membrane, whereas translocation of the large C-terminal periplasmic domain of CyoA requires the Sec translocon and SecA. These results show that two distinct mechanisms are required in translocating the different domains of CyoA.
1001 Our data suggest that SRP plays a prominent role in the targeting of MscL to the membrane. Depletion of Ffh or the SRP receptor FtsY, had a pronounced effect on the insertion of MscL in vivo. It is unclear whether SRP or FtsY play a direct role in delivering MscL to YidC, or simply act to target MscL to the membrane to promote interaction with YidC. In Arabidopsis thaliana, experimental evidence shows that cpSRP and cpFtsY interact with the Alb3 component independent of a substrate.32 Alb3 is the homologue of YidC in the thylakoid membrane and is required for the assembly of the lightharvesting chlorophyll-binding proteins (LHCP) into the thylakoid membrane, whereby the targeting of LHCP to the thylakoid membrane is mediated by cpSRP.32,33 Little is known about the folding and assembly of membrane proteins in the membrane. It has been suggested for Sec-dependent proteins that YidC may be involved in the lateral movement of membrane proteins into the lipid bilayer after leaving the Sec channel.34 On the basis of photocrosslinking studies, it was demonstrated that in vitro the hydrophobic domain of FtsQ first contacts SecY, and then interacts with YidC.2 Similar results were observed for the membrane insertion of mannitol permease (MtlA).5 These findings suggest that YidC functions as an assembly site for transmembrane regions and may play a role in the three-dimensional arrangement of the helices within the membrane. Recently, it was shown that YidC can function on its own as a membrane insertase. 9,10 In contrast to the Sec-dependent proteins, the YidC-dependent substrates all have short periplasmic regions. YidC may have only a limited translocation ability, since procoat mutants with alterations in the periplasmic region have been found to require YidC as well as SecA and SecYEG for efficient insertion. 35 It would be interesting to make alterations in the periplasmic region of MscL to investigate the requirements that specify proteins for the Sec or YidC specific pathways. Also, it would be interesting to study whether YidC is required for the assembly of the MscL pentamer. Mechanosensitive ion channels play a key role in allowing a cell to sense physical stress within its environment. On the basis of the resolved channel structure of the MscL homologue from Mycobacterium tuberculosis determined by X-ray crystallography, the periplasmic loop region between the two transmembrane helices creates a flap that forms the extracellular surface of the pore.15 The authors noted that the amino acid residues 58–64 in the loop region (which corresponds to residues 62–68 in E. coli) are found deep in the pore of the channel near Lys33 (Ser35 in E. coli) of transmembrane helix 1. This might explain why the cysteine mutant I68C is not fully accessible to AMS, because not all the cysteine residues are well exposed to the aqueous phase. It has been implicated that the periplasmic loop region is important for MscL gating.36–39
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Materials and Methods Genetic manipulations MscL was amplified from E. coli K12 using the primers 5′-CAT ATG AGC ATT ATT AAA GAA TTT CGC G-3′ (forward) and 5′-TGC TCA GCT TAA GAG CGG TTA TTC TGC TCT TT-3′ (reverse). The PCR product was then ligated into the pT-Adv vector (Clontech). The NdeI/ Bpu1102I fragment from the resulting plasmid (pT-AdvMscL) was cloned into the phage T7 expression vector pET16b (Novagen), which placed a His10 tag at the N terminus. The DNA encoding MscL was then subcloned into plasmid pMS119 using XbaI and HindIII, and into plasmid pEH1 using NcoI and HindIII to allow expression in strains of different genetic background. Native MscL has no endogenous cysteine residue. Single cysteine substitutions at positions 68 and 136 were constructed using the QuikChange site-directed mutagenesis method (Stratagene) and verified by sequencing. Bacterial strains, plasmids and growth conditions SecE and YidC depletion was carried out using strains CM124,18 and JS7131,3 respectively, as described.40 The SecE depletion strain CM12418 was cultured in M9 minimal medium supplemented with 0.4% (w/v) glucose and 0.2% (w/v) arabinose. To deplete cells for SecE, overnight cultures were washed once with M9 medium and backdiluted 1:20 (v/v) into fresh M9 medium in the presence of 0.4% (w/v) glucose and the absence of arabinose. Cells were grown for 8 h. To deplete cells for YidC, overnight cultures were grown in 0.2% (w/v) arabinose and then washed twice with LB to remove cells of arabinose and back-diluted 1:50 (v/v) into fresh LB medium with 0.2% (w/v) glucose. Cells were grown for 3 h in glucose. To deplete SecDFYajC, cells of E. coli strain JP325,13 which is a RecA+ version of the SecDF depletion strain JP352,41 were grown in M9 medium containing 0.2% (w/v) arabinose and 0.2% (w/v) glucose. After overnight growth, the cells were harvested, washed in medium without arabinose and back-diluted 1:100 (v/v) in M9 medium containing 0.2% (w/v) glucose. For depletion, cells were further grown until an absorbance at 600 nm (A600) of 0.6 and diluted twofold with M9 medium containing 0.2% (w/v) glucose. This process was repeated for a total growth time of 6.5 h, as described.41 For Ffh depletion, cells of E. coli strain WAM12121 were grown overnight in LB medium containing 0.1% (w/v) arabinose and 0.4% (w/v) glucose, washed in medium lacking arabinose and back-diluted 1:20 (v/v) in LB medium containing glucose. When cultures reached an A600 of 0.4 (∼ 4 h), the cells were transferred to M9 minimal medium for 30 min before pulse–chase labelling. The FtsY depletion strain IY26 (BW25113-Kan-AraCPftsY) was obtained from E. Bibi. FtsY is under the control of the araBAD promoter and operator. The FtsY depletion strain IY26 was grown in LB medium supplemented with 0.2% (w/v) arabinose. For FtsY depletion, cultures grown overnight were first washed twice with LB medium and then back-diluted 1:40 (v/v) and were grown to an A600 of 0.4 (∼ 4 h) in LB medium with 0.2% (w/v) glucose. Medium was switched to M9 minimal medium containing glucose and the cells were grown for an additional 30 min before labelling. The MscL cysteine mutants S136C and I68C were expressed by induction with isopropyl thio-β-D-galacto-
side (IPTG) from the vectors pET16b and pEH1,42 in strains C41(DE3),43 and CM124, respectively, and from the pMS119 vector,44 in strains JS7131, WAM121 and IY26. Mutant –3MPClep13 is a procoat lep (PClep) mutant in which the soluble domain P2 of leader peptidase is attached to the M13 procoat. The mutant consists of three uncharged residues at positions +2, + 4 and +5 replacing negatively charged residues. In addition, three negatively charged residues were introduced after residue +10 in the periplasmic loop. Pulse–chase and immunoprecipitation Cells expressing the MscL cysteine mutants were induced for 10 min with IPTG (final concentration 1 mM) and incubated in 4-acetamido-4′-maleimidylstilbene-2, 2′-disulfonic acid disodium salt (AMS; final concentration 2.5 mM; from Molecular Probes) for 1 min. Cells were then radiolabelled with [35S]Met (10 μCi/ml) for 30 s, whereupon non-radioactive Met was added (final concentration 500 μg/ml) for 2 min or for 10 min. After labelling, cells were combined with 20 mM DTT to quench the AMS reaction. After quenching, samples were acid-precipitated, resuspended in 10 mM Tris, 2% (w/v) SDS, and immunoprecipitated with antiHis antibodies. In experiments where the membrane potential was depleted, carbonyl cyanide p-chlorophenylhydrazone (CCCP; final concentration 50 μM) was added 1 min before treatment with AMS and labelling with [35S]Met. For the azide studies, the cells were treated by the addition of 2 mM sodium azide for 5 min before labelling of the cells. For spheroplasting, cells were centrifuged at 12 000g and resuspended in 500 μl of ice-cold spheroplast buffer (33 mM Tris–HCl (pH 8.0), 40% (w/v) sucrose). Lysozyme (final concentration 5 mg/ml) and EDTA (final concentration 1 mM) were added for 15 min. Samples of the spheroplast suspension were incubated on ice for 30 min either in the presence or in the absence of AMS (final concentration 2.5 mM). After quenching with DTT, samples were precipitated as described above. The samples were then subjected to SDS-PAGE in 40 cm long, 17% polyacrylamide gels at constant voltage (200 V) at 4 °C. Gels were examined by Phosphorimager analysis (Fuji BAS1500).
Acknowledgements We thank Ross Dalbey for E. coli JP325, Chris Murphy for E. coli CM124 and Jan-Willem de Gier for providing E. coli WAM121 and the plasmid pEH1. We thank Eitan Bibi for E. coli IY26 and for anti-Ffh serum, Hans-Georg Koch for anti-FtsY serum, and Christian Hartmann for the construction of plasmid pET16b/MscL. This work was supported by the Deutsche Forschungsgemeinschaft Sonderforschungsbereich 495.
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Edited by I. B. Holland (Received 12 September 2006; received in revised form 18 October 2006; accepted 25 October 2006) Available online 28 October 2006