Institute for Molecular Biology, Jena University, Winzerlaer Strasse 10, 07745 Jena, Germany1
Department of Genetics, Center for Biological Sciences, Kerklaan 30, 9751 NN Haren, The Netherlands2
Department of Pharmaceutical Biology, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands3
Author for correspondence: J. P. Müller. Tel: +49 3641 65 7577. Fax: +49 3641 65 7520. e-mail: jmueller{at}imb-jena.de
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ABSTRACT |
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Keywords: Bacillus subtilis, CsaA, protein export, protein targeting, chaperone
Abbreviations: 6H-CsaA, hexa-histidine-tagged CsaA
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INTRODUCTION |
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Cytoplasmic proteins, like SecB (Kumamoto & Beckwith, 1985 ), GroEL, GroES (Altman et al., 1991
; Kusukawa et al., 1989
), DnaK, DnaJ and GrpE (Altman et al., 1991
; Wild et al., 1992
, 1993
), denoted as chaperones, are important for the export competence of pre-proteins, either by stabilizing an unfolded conformation, or by preventing their aggregation (Hendrick & Hartl, 1993
). In addition, SecB is involved in the targeting of pre-proteins to the membrane-bound pre-protein translocase complex (Fekkes et al., 1997
; Hartl et al., 1990
). A second targeting factor, which is homologous to the eukaryotic signal recognition particle (SRP), assists the export of a subset of pre-proteins (De Gier et al., 1997
; Luirink et al., 1992
; Phillips & Silhavy, 1992
; Wolin, 1994
).
The pre-protein translocase consists of the peripheral membrane protein SecA (Oliver & Beckwith, 1982 ), which acts as a force generator for protein translocation, and a complex of the integral membrane proteins SecD, SecE, SecF, SecG, SecY and YajC, which form the translocation channel (for recent reviews, see Fekkes & Driessen, 1999
; Duong et al., 1997
; Pohlschröder et al., 1997
). SecA plays a crucial role in protein translocation. First, it can function as a receptor for pre-proteinSecB complexes (de Cock & Randall, 1998
; Fekkes et al., 1997
; Hartl et al., 1990
). Second, SecA drives protein transport through cycles of pre-protein binding, membrane insertion, pre-protein release and deinsertion from the membrane (Economou et al., 1995
; Economou & Wickner, 1994
; Kim & Oliver, 1994
). The cycling of SecA is regulated by ATP binding and hydrolysis, which causes major conformational changes in this protein (den Blaauwen & Driessen, 1996
; van der Does et al., 1998
). During or shortly after the translocation of the pre-protein across the membrane, the signal peptide is removed by signal peptidase (also known as leader peptidase), which is a prerequisite for the release of the mature protein from the membrane (Dalbey et al., 1997
).
Compared to E. coli, the protein export apparatus of Bacillus subtilis has been studied in less detail. So far, homologues of GroEL, GroES (Schmidt et al., 1992 ), DnaK, DnaJ and GrpE (Wetzstein et al., 1992
), SRP (Honda et al., 1993
; Struck et al., 1988
), SecA (Overhoff et al., 1991
; Sadaie et al., 1991
), SecY (Nakamura et al., 1990
; Suh et al., 1990
), SecE (Jeong et al., 1993
), SecDF and YajC (Bolhuis et al., 1998
) and several signal peptidases have been identified (Bolhuis et al., 1996
; Meijer et al., 1995
; Tjalsma et al., 1997
, 1998
; van Dijl et al., 1992
). In an early attempt to clone the B. subtilis secA gene by complementation of the E. coli secA51(Ts) mutation, we identified the B. subtilis csaA gene (Müller et al., 1992
). The deduced amino acid sequence of CsaA showed no similarities to that of SecA or other known components of the E. coli protein export apparatus. Nevertheless, the expression of the complete csaA gene resulted in a suppression of the growth and protein export defects associated with the secA51 mutation. Unlike other previously identified suppressors of secA51 (Brickman et al., 1984
; Lee & Beckwith, 1986
; Oliver, 1985
; Overhoff-Freundlieb & Freudl, 1991
), csaA showed no pleiotropic effects on mutations in other sec genes with the exception of the secB::Tn5 mutation (Müller et al., 1992
). The present studies were aimed at the elucidation of the mechanism by which csaA suppresses secretion defects in E. coli. The results show that the CsaA protein has chaperone-like activities in vivo and in vitro, suggesting that these activities are responsible for the suppression of the secA51 mutation.
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METHODS |
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Purification of hexa-histidine-tagged CsaA.
To provide CsaA with an amino-terminal hexa-histidine tag, the csaA gene was amplified by PCR with the primers csa-5' (5'-GGAGTTATTGGATCCGCAGTTATTGATGAC-3'; containing a BamHI site) and csa-3' (5'-GCCGATCTGCAGGCCTTTACGGCACACACG-3'; containing a PstI site) and cloned into plasmid pQE9, resulting in plasmid pQE9csaA. The sequence of the amplified fragment was verified by DNA sequencing. Hexa-histidine-tagged CsaA (6H-CsaA) was purified from E. coli M15 (pREP4, pQE9csaA) using Ni2+-nitrilotriacetic acid agarose (Qiagen) under non-denaturing conditions, according to the manufacturers instructions.
CsaA antiserum.
A CsaA-based peptide with the sequence KAEEFPEARC (residues 1927 of CsaA: the last C was added for experimental reasons; Müller et al., 1992 ) was cross-linked to KLH (Keyhole Limpet Hemocyanin) from Pierce using the m-maleimidobenzoic acid N-hydroxysuccinimide ester, as described by Närvänen (1990)
. Next, the peptideKLH conjugate was used to immunize rabbits.
Protein labelling, immunoprecipitation, SDS-PAGE and fluorography.
Pulse labelling, or pulsechase labelling experiments in E. coli and subsequent immunoprecipitations were performed as described by van Dijl et al. (1991) . SDS-PAGE was performed according to Laemmli (1970)
. [14C]Methylated molecular mass reference markers were from Amersham. Fluorography was performed as described by Skinner & Griswold (1983)
. All pulsechase labelling experiments were repeated at least twice. Relative amounts of radioactivity in gels were determined with a phosphorimager or by scanning of films with a densitometer. Pre-protein processing was calculated as the percentage of the total labelled protein (precursor + mature) present in the mature form at the time of sampling. Differences of more than 1015% pre-protein processing were reproducibly detected in parallel labelling experiments. Differences in pre-protein processing that are smaller than 10% are, in general, not reproducible.
Immunoblotting.
Samples for SDS-PAGE were prepared by boiling of cells (5 min) in sample buffer (Laemmli, 1970 ). Separated proteins were transferred to a nitrocellulose membrane (BA85; Schleicher & Schuell) as described by Towbin et al. (1979)
. Proteins were detected with specific antisera and 125I-labelled protein A. Relative amounts of radioactivity on blots were determined with a phosphorimager.
ß-Galactosidase activity.
ß-Galactosidase activity assays were carried out as described by Miller (1972) .
Luciferase activity and aggregation.
In vivo luciferase activity assays were performed essentially as described by Schröder et al. (1993) . E. coli cells producing luciferase were grown at 30 °C in TY medium. When the cells reached an absorbance (A600) of 0·5, kanamycin (100 µg ml-1) and chloramphenicol (50 µg ml-1) were added to stop further protein synthesis, and samples of 1 ml were incubated for 10 min at 42 °C to inactivate luciferase. Next, the samples were incubated at 30 °C and the reactivation of luciferase was determined in duplicate experiments. In vitro luciferase aggregation assays were performed as described by Schröder et al. (1993)
.
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RESULTS |
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Taken together, our data imply that CsaA either has a direct effect on protein export in E. coli MM54 secA51 and CK1953 secB::Tn5 at 42 °C, or that CsaA induces the synthesis of as yet unidentified proteins which participate in maintaining pre-proteins in an export-competent conformation, thereby reducing (but not bypassing) the requirements for SecA and/or SecB. In what follows, we show that CsaA can prevent protein aggregation, suggesting that CsaA can have a direct effect on protein export.
CsaA-mediated suppression of the temperature-sensitive growth of dnaK, dnaJ and grpE mutants
Since the observed effects of CsaA on protein export are in accordance with the hypothesis that CsaA itself could have a chaperone-like activity (Müller et al., 1992 ), we tested whether this protein can complement the temperature-sensitive growth phenotype of E. coli strains carrying mutations in various chaperone-encoding genes. To this purpose, strains containing the groEL44, groES619, dnaK756, dnaJ259 or grpE280 mutations were transformed with plasmids pBNB8, pTZcsa82 or pBR322 (control). As essentially the same results were obtained with strains containing pBNB8 or pTZcsa82, only the results obtained with strains containing pBNB8 are documented here. As shown in Fig. 4
, at 42 °C, the post-exponential growth in TY medium of strains containing the groEL44 or groES619 mutations was slightly, but reproducibly, impaired due to the expression of CsaA, similar to the wild-type strain MC4100. In contrast, the post-exponential growth of strains containing the dnaK756, dnaJ259 or grpE280 mutations was stimulated by the expression of CsaA, the growth of the dnaJ mutant strain being stimulated most strongly (Fig. 4
). To verify these effects of CsaA, the growth of chaperone mutant strains expressing csaA was analysed on plates (Table 2
). Whilst the expression of csaA neither exacerbated nor improved the ability of strains containing the groEL44 or groES619 mutations to form single colonies at 42 °C, a stimulatory effect could be observed in strains containing the dnaK756, dnaJ259 or grpE280 mutations (Table 2
). Furthermore, the expression of CsaA strongly reduced the formation of long filamentous cells of strains containing the dnaK756, dnaJ259 or grpE280 mutations (data not shown). These findings show that CsaA can suppress growth defects of strains with impaired activity of the DnaKDnaJGrpE chaperone machinery. Nevertheless, CsaA was unable to replace DnaK as the transformation of E. coli BB1553
dnaK52, which lacks the dnaK gene (Hesterkamp & Bukau, 1998
), with pBNB8 or pTZcsa82 did not result in a suppression of the growth defects of this strain at 42 °C (data not shown). Furthermore, at 37 °C and 42 °C, the impaired replication of bacteriophage in the above strains with defective DnaKDnaJGrpE or GroELGroES chaperone machineries (see Polissi et al., 1995
) was not restored by CsaA (data not shown). These observations demonstrate that CsaA can not complement for the malfunction of the major chaperone machineries of E. coli.
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DISCUSSION |
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Interestingly, the production of CsaA antagonized the expression of the secB gene specifically, as shown by immunoprecipitation of pulse-labelled SecB and determination of ß-galactosidase activities in a secB'lacZ strain producing different levels of CsaA. As CsaA has chaperone-like activities, this observation is in accordance with the observation that the expression of secB is regulated in response to the cellular chaperone levels: secB expression was increased in groES, groEL, dnaK, dnaJ and grpE mutant strains (Müller, 1996 ) and reduced in strains overexpressing DnaK and DnaJ, or GroEL and GroES (our unpublished observations). Our present findings indicate that secB expression is not only regulated in response to the levels of homologous chaperones, but also in response to the production of a heterologous protein with chaperone-like activities. Notably, the expression of DnaK and GroEL was not affected by the production of CsaA, indicating that these chaperones are not involved in the CsaA-mediated repression of secB. Recently, it has been demonstrated that secB expression is under the control of catabolic repression by cAMP receptor proteincAMP complexes at the transcriptional level (Seoh & Tai, 1997
, 1999
). However, in contrast to catabolic repression, which caused a 1·4-fold reduction of secB expression, the presence of CsaA affected the expression levels of secB about two- to fivefold, depending on the level of CsaA production. This suggests that CsaA does not exert its effects on secB expression via catabolic repression, but we are presently unable to exclude this possibility.
The phenotypes observed upon the production of CsaA in groEL44, groES619, dnaK756, dnaJ259 and grpE280 mutant strains indicate that CsaA can suppress a variety of different defects caused by the malfunction of the corresponding mutant chaperones. Two key observations may, at least in part, provide explanations for the effects of CsaA production in the latter strains. First, 6H-CsaA was shown to prevent the aggregation of luciferase in vitro, but it was unable to reactivate heat-denatured luciferase or to prevent its inactivation. This indicates that CsaA has an aggregation-preventing rather than a folding-promoting chaperone-like function. Second, CsaA was unable to replace DnaK in the dnaK52 mutant strain with respect to temperature-sensitive growth and the in vivo reactivation of heat-inactivated luciferase. In contrast, CsaA was able to suppress these defects in the dnaK756 mutant strain. Taken together, the latter findings show that the presence of CsaA supported the activity of the mutant DnaK756 protein. This could be a direct effect of CsaA on the DnaK756 protein (see above). Alternatively, an indirect effect through the prevention of protein aggregation by CsaA would also be a plausible explanation for these findings, the in vivo reactivation of heat-inactivated luciferase in particular. In fact, if CsaA can indeed prevent the in vivo aggregation of denatured luciferase, as it does in vitro, CsaA would keep this denatured protein in a proper condition for refolding/reactivation by the DnaKDnaJGrpE and/or GroELGroES chaperone machineries. This would explain why CsaA showed a pleiotropic effect with respect to the reactivation of heat-inactivated luciferase in dnaK756, grpE289, groEL44 and groES619 mutant strains. Notably, the production of CsaA did not induce a general stress response as evidenced by the lack of effect of CsaA on the expression of DnaK and GroEL. In fact, CsaA activity seemed to interfere to some extent with chaperone activity in wild-type cells, as evidenced by the reduced reactivation of inactivated luciferase in cells of E. coli MC4100 producing CsaA.
Interestingly, as reflected by the processing of pre-ß-lactamase to the mature form, CsaA only had a significant stimulating effect on ß-lactamase export in those chaperone mutant strains in which the rate of export was drastically slowed down (i.e. groES619 and dnaJ259). Consistent with the above hypothesis that CsaA keeps unfolded luciferase in a proper condition for in vivo refolding by general chaperones, our observations suggest that CsaA keeps pre-ß-lactamase in a proper condition for translocation, for example by preventing its folding and/or aggregation in the cytoplasm. If so, this implies that CsaA has a pre-protein folding-preventing activity, analogous to that of SecB (Topping & Randall, 1997 ), which would explain the CsaA-mediated suppression of protein export defects in secA51 and secB::Tn5 mutant strains as well (see above). This view is consistent with our unpublished observation that purified SecB prevented the aggregation of denatured luciferase in vitro in a similar manner to CsaA.
Finally, the role of CsaA in B. subtilis remains to be defined. First results indicate that CsaA is required for the efficient secretion of at least a subset of proteins and that it interacts specifically with SecA and pre-proteins, suggesting that it could have protein-secretion-specific chaperone-like activities. This is an intriguing hypothesis in view of the fact that B. subtilis lacks a SecB homologue (Kunst et al., 1997 ).
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ACKNOWLEDGEMENTS |
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Received 17 June 1999;
revised 27 September 1999;
accepted 12 October 1999.