Communicated by Jonathan Beckwith, Harvard Medical School, Boston, MA, February 9, 1996 (receivedfor review October 1, 1995). ABSTRACT. The SecY ...
Proc. Natl. Acad. Sci. USA Vol. 93, pp. 5953-5957, June 1996
Microbiology
prlA suppressors in Escherichia coli relieve the proton electrochemical gradient dependency of translocation of wild-type precursors (protein translocation/SecY/proton-motive force) NICO NOUWEN*t$, BEN DE KRUIJFFtt, AND JAN TOMMASSEN*t§ Departments of *Molecular Cell Biology and tBiochemistry of Membranes, Centre for Biomembranes and Lipid Enzymology, and Itnstitute of Biomembranes, Utrecht
University, Padualaan, 8, 3584 CH Utrecht, The Netherlands
Communicated by Jonathan Beckwith, Harvard Medical School,
Boston, MA, February 9, 1996 (received for review October 1, 1995)
ABSTRACT The SecY protein of Escherichia coli is an integral membrane component of the protein export apparatus. Suppressor mutations in the secY gene (prIA alleles) have been isolated that restore the secretion of precursor proteins with defective signal sequences. These mutations have never been shown to affect the translocation of wild-type precursor proteins. Here, we report that pr4A suppressor mutations relieve the proton-motive force (pmf) dependency of the translocation of wild-type precursors, both in vivo and in vitro. Furthermore, the proton-motive force dependency of the translocation of a precursor with a stably folded domain in the mature region was suppressed by prlA mutations in vitro. These data show that prlA mutations cause a general relaxation of the export apparatus rather than a specific change that results in bypassing of the recognition of the signal sequence. In addition, these results are indicative for a mechanism in which the proton-motive force stimulates translocation by altering the conformation of the translocon.
(pmf). Energy from ATP-binding and hydrolysis is probably used to confer conformational changes in the SecA molecule, which lead to a cycle of insertion and deinsertion of SecA in the membrane and the movement of the precursor across the membrane (9-11). The mechanism by which the pmf stimulates translocation is less clear, since the requirement for the pmf differs with the precursor protein investigated (12, 13). Interestingly, the requirement for the pmf can be altered by single point mutations in the signal sequence (14) or by mutations just after the signal peptidase cleavage site (15, 16). These observations suggest, as one explanation, that the process of insertion of the signal sequence into the membrane is stimulated by the pmf. Because prl suppressors have been isolated as mutations that can bypass the signal sequence function, we wondered whether these mutations are also able to suppress the pmf dependency of the translocation of wildtype precursors. Both in vivo and in vitro experiments revealed that prlA suppressors relieve the pmf dependency of the translocation of wild-type precursor proteins.
Most proteins that reside in the periplasm or the outer membrane of Escherichia coli are transported across the inner membrane by the general secretion pathway. The components of this pathway have primarily been identified via two different genetic approaches. The first approach implicated the isolation of conditionally lethal mutations that conferred generalized secretion defects. This strategy resulted in the identification of the secA, secB, secD, secE, secF, and secY genes. An additional component of the export machinery, SecG, was identified after reconstitution of the transport process in vitro (1). The second method involved the selection of suppressors of signal sequence mutations and yielded prl (protein localization) genes. In three cases, the identified prl genes were found to be allelic to sec genes (secA/prlD, secE/prlG, and secY/prlA) (for reviews, see refs. 2 and 3). Originally, it was believed that prl suppressor mutations function by altering or expanding the recognition of the signal sequences by the corresponding Sec protein (4). However, this mechanism predicts allele specificity, which has not been observed for knownprlA,prlG, andprlD suppressors. In addition, such a mechanism would not explain the suppression of export defects caused by complete signal sequence deletions as has been observed in prIG and prlA suppressor strains (5, 6). Therefore, it has been proposed that prlA andpriG suppression functions by preventing the rejection of defective precursor proteins from the export pathway (6, 7). The energetics of the translocation of the precursor of outer membrane protein OmpA (proOmpA) have been studied in vitro (8). Initial stages of proOmpA translocation are driven by the binding and hydrolysis of ATP by SecA, whereas late stages of translocation can be driven by the proton-motive force
MATERIALS AND METHODS
Enzymes and Chemicals. Carbonylcyanide m-chlorophenylhydrazone (CCCP), carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), phenylmethylsulfonyl fluoride, sodium succinate, oxonol V, 9-amino-6-chloro-2-methoxyacridine (ACMA), gramicidin, and potassium ferricyanide were purchased from Sigma. The reduced form of NADH was obtained from Boehringer Mannheim, proteinase K and sodium azide were from Merck, and restriction enzymes were from Pharmacia. Strains and Plasmids. The E. coli strains used in this study are listed in Table 1. Strains CE1437 and CE1438 are unc mutant derivatives of NT1060 and NT1004, respectively, and were constructed by P1 transduction by using strain K003 as donor. Outer membrane protein PhoE was expressed from plasmid pJP29 (19) by growing cells under low phosphate conditions (20). Plasmid pTacompA was constructed by subcloning a BamHI/HindIII fragment from plasmid pTRComp9 (21) into pJF118 E/H (22). In the resulting plasmid, the E. coli ompA gene is under tac promoter control. Pulse-Labeling and Pulse-Chase Experiments. Cells, grown under phosphate limitation to induce the expression of PhoE protein, were labeled with [35S]methionine (10 ,tCi/ml; 1 Ci = 37 GBq) for 30 s at 37°C and subsequently chased with an excess of nonradioactive methionine (19). To test the dependence of protein translocation on the membrane potential, cells were treated as in a standard pulse-chase experiment, except that 45 s before labeling, CCCP was added to a final 9-amino-6-chloro-2-methoxyacridine; CCCP, FCCP, carbonylcyanide carbonylcyanide m-chlorophenylhydrazone; inner membrane vesicle; IMV, p-trifluoromethoxyphenylhydrazone; pmf, proton-motive force. §To whom reprint requests should be addressed.
Abbreviations: ACMA,
publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
The
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Proc. Natl. Acad. Sci. USA 93 (1996)
Table 1. Bacterial strains Strain Properties NT1060 F-, AlacU169 araD139 rpsL thi relA ptsF25 deoCI lamBA60 prlA4 derivative of NT1060 NT1004 AF82 F-, AlacU169 araD139 rpsL thi relA ptsF25 deoCI malBAl zjb::TnlO prlD2 AF161 F-, AacU169 araD139 rpsL thi relA ptsF25 deoCI lamB14D prlA208 zhc::TnlO AF164 F-, lacU169 araD139 rpsL thi relA ptsF25 deoC1 lamB14D priG1 zij::TnlO MM52 F-, AlacU169 araD139 rpsL thi relA ptsF25 deoCI secA51 K002 Ipp zid::TnlO K003 A(uncB-C) derivative of K002 CE1437 A(uncB-C) zid::TnlO derivative of NT1060 CE1438 A(uncB-C) zid::TnlO derivative of NT1004
concentration of 100 JLM and that twice as much [35S]methionine was used (23). To test the SecA dependence of translocation, sodium azide (2 mM end concentration) was added 1 min before labeling and cells were pulse labeled for 30 s with [35S]methionine (10 ,uCi/ml). Radiolabeled proteins were analyzed by SDS/PAGE (24) followed by autoradiography. Quantifications were performed using the program IMAGE QUANT after scanning the autoradiograms in an optical densitometer (Molecular Dynamics). In Vitro Transcription, Translation, and Translocation Reactions. [35S]Methionine-labeled prePhoE and proOmpA were synthesized from plasmids pJP29 and pTRComp9, respectively, using an S-135 lysate from E. coli strain K002. Isolation of S-135 lysates and of inner membrane vesicles (IMVs) and the in vitro transcription, translation, and translocation reactions were performed as described (25). Oxidation of proOmpA with potassium ferricyanide, and following translocation reactions were performed as described (26). Buffer A, which was used in the translocation experiments using purified proteins (27), contains 40 mM Tris-HAc, 28 mM KOAc, 10 mM MgOAc (pH 8.0), and 2 mM dithiothreitol. Quantification of protease-protected material was performed with the program IMAGE QUANT after exposing the SDS/ PAGE gel in a PhosphorImager (Molecular Dynamics). Proteins. [35S]proOmpA was purified by using a small-scale purification protocol as developed for prePhoE (27) with some modifications. Overexpression of proOmpA was achieved in secA mutant strain MM52 containing plasmid pTacompA. Cells were grown at 30°C in minimal medium (28), supplemented with 0.5% glucose and 0.5% methionine assay medium (Difco) to an OD660 of 0.5. Then, OmpA expression was induced by the addition of isopropyl 3-D-thiogalactoside (1 mM end concentration). After 15 min at 30°C, cells were labeled with Trans-35S label (ICN; 500 liCi/10 ml cells). After 1 hr, cells were harvested and proOmpA was purified as described for prePhoE (27) with the modification that 2 mM dithiothreitol was added to the buffers and that proOmpA was purified batch-wise by using Q-Sepharose column material, instead of the MonoQ column. The protein was >95% pure as estimated from gel. SecA (9) and SecB (29) were purified as described. Determination of Ai and ApH. The generation of Ai and ApH in IMVs of unc mutant strains was monitored by following the fluorescence quenching of oxonol V and ACMA, (total volume, 1 ml) conrespectively. The reaction mixture tained buffer A, IMVs (OD28o = 750 per ml), and 1 ,LM oxonol V or ACMA and was kept at 25°C. To generate a pmf, NADH (5 mM end concentration) was added to the reaction mixture. The fluorescence emission of oxonol V was measured at 634 nm with excitation at 599 nm and that of ACMA at 500 nm with excitation at 420 nm using a Perkin-Elmer fluorimeter.
RESULTS Effect of CCCP on Protein Translocation in Vivo. In E. coli, suppressor mutations that alleviate the translocation defects
Ref. source T. J. Silhavy T. J. Silhavy T. J. T. J. T. J.
Silhavy Silhavy Silhavy 17 18 18
This This
study study
caused by signal sequence mutations have been found in three known sec genes-i.e., prlA (secY), prlD (secA), and prlG (secE). To investigate ifprl suppressors are able to relieve the pmf dependency of the translocation of a wild-type precursor in vivo, pulse-chase experiments were performed after dissipation of the pmf with 100 ,uM CCCP. Two different prIA suppressor alleles were included in these studies. In the wild-type strain, addition of CCCP resulted in the accumulation of the precursor of PhoE, followed by a slow conversion of the precursor into the mature form (Fig. 1 A and D). In contrast, in both prlA mutant strains, processing of PhoE was much less affected by CCCP (Fig. 1 B and D). Only a slight retardation in the maturation of prePhoE was observed. Processing kinetics in the prlD2 strain were similar to those in the wild-type strain, whereas theprlGl strain appeared to be even more sensitive to the addition of CCCP than the wild-type strain. To investigate whether SecA function is still required for protein translocation in theprlA mutants, we treated these cells with 2 mM sodium azide before pulse-labeling, a treatment that inhibits the ATPase activity of SecA (30). In both prlA mutant strains, prePhoE was found to accumulate in the presence of sodium azide (Fig. 1C). In conclusion, prlA suppressor mutations are not only able to suppress signal sequence mutations, but they are also able to relieve the pmf dependency of the translocation of a wild-type precursor protein. Translocation of prePhoE into PrIA Vesicles Is Independent of the pmf. To determine whether a prlA suppressor mutation is also able to relieve the pmf dependency of translocation of prePhoE in vitro, we constructed aprlA4 strain that also lacked the FiFo ATPase. IMVs from this strain cannot generate a pmf by ATP hydrolysis, but they do generate a pmf when a substrate for the electron transport chain, such as NADH, is added to the vesicles (Fig. 2A). As reported previously (14), the generation of a pmf resulted in a 5- to 10-fold increase in the translocation kinetics of prePhoE into wild-type vesicles (Fig. 2B). In contrast, the translocation kinetics of prePhoE into IMVs from the prlA4 strain were not increased by the generation of a pmf. However, in the presence as well as in the absence of a pmf, the translocation kinetics into prlA4 IMVs were comparable with those into wild-type vesicles in the presence of a pmf (Fig. 2B). Thus, like in vivo, the prlA4 suppressor mutation also relieves the pmf dependency of translocation of prePhoE in vitro. Translocation of proOmpA into PrIA Vesicles Is Solely Dependent on the ATP Concentration. To investigate the relationship between the role of ATP, pmf, andprlA mutations in more detail, we studied the initial rate of translocation of another well-studied precursor protein, proOmpA, as a function of these parameters. In the presence of a pmf, proOmpA translocation into both wild-type andprlA4 IMVs was linear up to 10 min at the highest ATP concentration (500 ,M) tested (data not shown). Both in the presence and absence of a pmf, the initial rate of proOmpA translocation increased with the ATP -concentration for both types of vesicles (Fig. 3). However, whereas the initial rate of translocation into wild-type vesicles was 2- to 3-fold stimulated by the pmf, the transloca-
Proc. Natl. Acad. Sci. USA 93
Microbiology: Nouwen et al. A precursor
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mM (data not shown). PrIA Suppressor Mutations Relieve the pmf Dependency of the Translocation of a Stably Folded Domain in the Mature Moiety of proOmpA. pr4A suppressors have been isolated as mutants that can export precursors with defective signal sequences. Next, we investigated whether a prlA suppressor mutation is also able to suppress a translocation defect caused by a stably folded domain in the mature moiety of the precursor. proOmpA contains two cysteines near the C terminus in the mature domain. These two cysteines can be oxidized into an intramolecular disulfide bridge, thus forming a stable loop structure comprising 12 amino acids. In vitro translocation of this "looped" precursor into wild-type vesicles is strictly dependent on the pmf and results in the accumulation of translocation intermediates (ref. 26; see also Fig. 4). Translocation into prlA4 IMVs, however, took place even in the absence of a pmf, although a 2-fold stimulation by the presence of a pmf was observed (Fig. 4). Similar results were obtained when IMVs from the prlA208 mutant strain were used after dissipation of the pmf with CCCP (data not shown). Thus,prL4 suppression is not restricted to translocation of precursors with mutant signal sequences, but it also facilitates translocation of precursors that are translocation defective due to an alteration in the mature domain.
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tion of proOmpA into prlA4 IMVs was independent of the presence of a pmf. Apparently, translocation of proOmpA into IMVs of the prlA4 strain is solely dependent on the ATP concentration. Remarkably, at low ATP concentrations (i.e., below 0.1 mM) and in the presence of a pmf, the initial rate of proOmpA translocation was more than 2-fold higher in the IMVs from the prlA4 strain than in those from the wild-type strain. This suggests that the mutant SecY protein in the prL4 strain not only relieves the pmf dependency of translocation but also affects the SecA activity, possibly by an altered interaction between SecA and SecY. Therefore, we tested the sensitivity of prlA suppressor strains for sodium azide, an inhibitor of the translocation ATPase activity of SecA. In agreement with an altered activity of SecA, growth tests on Luria-Bertani plates revealed that sodium azide concentrations above 0.4 mM were lethal to theprlA208 andprlA4 strains AF161 and NT1004, respectively, whereas the wild-type strain NT1060 was resistent to sodium azide concentrations up to 0.6
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(1996)
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FIG. 1. In vivo processing kinetics of pJP29-encoded prePhoE protein in various prl strains. Cells grown under phosphate limitation were pulse-labeled for 30 s with [35S]methionine and chased with unlabeled methionine for the indicated periods. The proteins were analyzed by SDS/PAGE and autoradiography. (A) Processing kinetics in wild-type strain NT1060 in the absence and presence of 100 zM CCCP. (B) Processing kinetics in variousprl strains in the absence and presence of 100 jzM CCCP. (C) Processing inprlA strains during 30-s pulse-labeling in the presence of 2 mM sodium azide. Lanes: 1 and 2,
wild-type strain NT1060; lane 3, prlA4 strain NT1004; lane 4, prlA208 strain AF161. (D) Quantification of the processing kinetics. Percent precursor of the total amount of PhoE was determined by scanning of the autoradiograms shown in A and B and plotted against the chase time. 0, -CCCP; *, +CCCP.
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Proc. Natl. Acad. Sci. USA 93 (1996)
Microbiology: Nouwen et al.
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FIG. 3. In vitro translocation of purified proOmpA into IMVs from strain CE1437 (wild type) and strain CE1438 (prlA4). The initial rate of proOmpA translocation was assayed at 1-500 /LM ATP in the absence (e) or presence (0) of 5 mM NADH to generate a pmf. Reactions were carried out in buffer A supplemented with SecA (34 ,ug/ml), SecB (6 ,g/ml), MgATP, IMVs (40 0lOD280 = 100 per ml), creatine phosphate (10 mM), creatine kinase (10 /Lg/ml), and purified [35S]proOmpA (80,000 cpm per sample). Five minutes after initiation of translocation, the translocation reactions were terminated on ice by treatment with proteinase K. Protease-protected material was analyzed by SDS/PAGE followed by fluorography and quantified after exposing the gel in a PhosphorImager (Molecular Dynamics).
0
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FIG. 2. Translocation kinetics of wild-type prePhoE into IMVs in the absence or presence of a pmf. (A) Generation of Aqk and ApH by membrane vesicles from unc mutant strain CE1437 (wild type) and from uncprlA mutant strain CE1438 (prIA4). The generation of Ami and ApH was determined by monitoring the fluoresence quenching of oxonol V and ACMA, respectively. Where specified, ATP (5 mM end concentration), NADH (5 mM end concentration), FCCP (1 ,uM end concentration), or gramicidin (1 ,iM end concentration) were added to the vesicles. (B) In vitro synthesized prePhoE was translocated into IMVs from strains CE1437 and CE1438 in the absence (-) or presence (+) of 5 mM NADH. Translocation reactions were initiated by the addition of IMVs. At the indicated times after initiation of translocation, samples were withdrawn and the translocation reactions were terminated on ice by treatment with proteinase K. Protease-protected material representing translocated PhoE protein was analyzed by SDS/PAGE followed by fluorography. [In addition to the mature PhoE form, translocated but unprocessed precursor is generally found in prokaryotic in vitro translocation systems (25, 31)]. (C) Quantification of the results as shown in B: *, translocation in the absence of a pmf; o, translocation in the presence of a pmf. ture in the mature domain of proOmpA, could be suppressed by the prIA mutation. Previously, it has been suggested that prlA suppressors bypass a "proofreading" function of SecY that normally rejects precursors with a defective signal sequence (6, 7). Our results could indicate that this proofreading function is more general in that it extends beyond the signal sequence to defects in mature sequences. However, the observed pmf independency of protein translocation in the prlA mutants would be difficult to explain within this concept. Although our results do not
the initiation of the translocation process. The last few years, evidence for the concept that translocation across the inner membrane occurs through a channel involving the membrane components SecY/E/G has accumulated (32-34). This channel should be gated, since a constantly "open" channel would be lethal. Upon interaction with the signal sequence and under influence of the pmf, the closed channel could change conformation, resulting in a "relaxed" state of the translocon. When the translocon is in this relaxed state, SecA can open it and push the mature protein through. In this concept, we propose that theprL4 suppressor mutations in secYresult in the relaxed conformation of the translocon. An alternative interpretation of our results is that the SecA protein is more active in pr4A strains. In support of this view, the initial rates of proOmpA translocation into IMVs from the prlA4 strain were at low ATP concentrations more than 2-fold higher as compared with those into IMVs from a wild-type strain (Fig. 3). In priA4
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FIG. 4. Translocation of proOmpA with an element of tertiary the mature domain into IMVs from prlA4 strain CE1438 is hardly dependent on a pmf. In vitro synthesized proOmpA was pretreated with 10 mM ferricyanide for 2 min to create an intramolecular disulfide bridge, followed by dilution in an equal volume of buffer B (40 mM Tris-HAc, pH 8.0/28 mM KOAc). This mixture was used for translocation into IMVs from strain CE1437 (wild type) and strain CE1438 (prlA4) in the absence and presence of 5 mM succinate to generate a pmf. At the indicated times, samples were withdrawn and the translocation reactions were terminated on ice by treatment with SDS/ proteinase K. Protease-protected material was analyzed bymature PAGE followed by fluorography. In addition to translocated OmpA and proOmpA, bands of lower molecular weight are visible, which probably represent translocation intermediates (8, 26). structure in
Microbiology: Nouwen et al. addition, such an altered activity of SecA could explain the observation that prlA strains are more sensitive to sodium azide than a wild-type strain. The increased activity of SecA in this model should be caused by an altered interaction with the mutant SecY protein. Actually, if it is the relaxed state of the translocon, induced by the prlA mutation, that increases the SecA activity, than the two proposed models for PrlA suppression are not entirely different. One could argue that a relaxed mutant form of the translocon would imply a functional defect (i.e., a less well-gated channel), whereas prIA strains are apparently as viable as wild-type strains. However, it should be noted that severalprlA strains contain two distinct mutations in SecY, where only one of them is responsible for the suppression (7). For one double mutant, it has been reported that the mutation that is responsible for the suppression causes a growth defect, which is restored by the second mutation (35). These two observations
during the screening for suppressors of signal sequence mutations, there was a second dominant selection for cell viability. Due to this additional selection pressure, the negative effects of priA mutations could have been obscured, and only those suppressors were picked up that are viable under standard laboratory conditions. Many studies have been performed to investigate the mechanism by which the pmf stimulates translocation. Some studies suggest that the pmf stimulates translocation by electrophoresis of negatively charged residues in the precursor protein (16, 36, 37). Other in vitro studies have shown that the pmf affects the SecA function (38-40). On the basis of our results obtained with prIA suppressors, we propose a mechanism in which the pmf is involved in the opening of the translocon towards translocating precursor proteins and thereby indirectly affects
Proc. Natl. Acad. Sci. USA 93 10. 11. 12. 13. 14. 15. 16.
17. 18. 19.
20.
could indicate that
the SecA function.
We gratefully acknowledge Drs. Ann Flower and Tom Princeton University for their generous gift of prl strains. 1.
2. 3. 4. 5. 6.
7. 8. 9.
21.
22. 23. 24.
25. 26.
27. 28. 29.
Silhavy at
Nishiyama, IC, Mizushima, S. & Tokuda, H. (1993) EMBOJ. 12, 3409-3415. Bieker, K. L., Philips, G. J. & Silhavy, T. J. (1990) J. Bioenerg. Biomembr. 22, 291-310. Schatz, P. J. & Beckwith, J. (1990)Annu. Rev. Genet. 24,215-248. Emr, S. D., Hanley-Way, S. & Silhavy, T. J. (1981) Cell 5, 79-88. Derman, A. I., Puziss, J. W., Bassford, P. J., Jr., & Beckwith, J. (1993) EMBO J. 12, 879-888. Flower, A. M., Doebele, R. C. & Silhavy, T. J. (1994)J. Bacteriol. 176, 5607-5614. Osborne, R. S. & Silhavy, T. J. (1993) EMBO J. 12, 3391-3398. Schiebel, E., Driessen, A. J. M., Hartl, F.-U. & Wickner, W. (1991) Cell 64, 927-939. Breukink, E., Demel, R. A., de Korte-Kool, G. & de Kruijff, B. (1992) Biochemistry 31, 1119-1124.
30.
31. 32. 33. 34.
35. 36. 37. 38. 39. 40.
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