Complementary Impact of Paralogous Oxa1-like Proteins of Bacillus subtilis on Post-translocational Stages in Protein Secretion*

Harold TjalsmaDagger, Sierd Bron§, and Jan Maarten van Dijl§||

From the Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, P. O. Box 14, 9750 AA Haren, The Netherlands and the || Department of Pharmaceutical Biology, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands

Received for publication, February 4, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In mitochondria, chloroplasts, and Gram-negative eubacteria, Oxa1p(-like) proteins are critical for the biogenesis of membrane proteins. Here we show that the Gram-positive eubacterium Bacillus subtilis contains two functional Oxa1p orthologues, denoted SpoIIIJ and YqjG. The presence of either SpoIIIJ or YqjG is required for cell viability. Whereas SpoIIIJ is required for sporulation, YqjG is dispensable for this developmental process. The stability of two membrane proteins was found to be mildly affected upon SpoIIIJ limitation in the absence of YqjG. Surprisingly, the topology and stability of other membrane proteins remained unaffected under these conditions. In contrast, SpoIIIJ- and YqjG-limiting conditions resulted in a strong post-translocational defect in the stability of secretory proteins. Together, these data indicate that SpoIIIJ and YqjG of B. subtilis are involved in both membrane protein biogenesis and protein secretion. However, the reduced stability of secretory proteins seems to be the most prominent phenotype of SpoIIIJ/YqjG-depleted B. subtilis cells. In conclusion, our observations show that SpoIIIJ and YqjG have different, but overlapping functions in B. subtilis. Most importantly, it seems that different members of the Oxa1p protein family have acquired at least partly distinct, species-specific, functions that are essential for life.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In recent years, not only interesting similarities, but also striking differences between protein transport mechanisms in eubacteria, Archaea, eukaryotes, and eukaryotic organelles have been documented (1-3). Insights in the extents of conservation and divergence in these mechanisms were particularly increased by the availability of many complete genome sequences. Unfortunately, the biological significance of such insights is often difficult to test as the majority of organisms with sequenced genomes is poorly amenable to biochemical or genetic approaches. In this respect, the Gram-positive eubacterium Bacillus subtilis, the complete genome sequence of which was published by Kunst et al. (4), turned out to be a very useful exception. First, because this organism has a natural system for genetic transformation and second, because it has a huge capacity for the secretion of proteins directly into the growth medium (5).

The functional genomic approach to dissect the protein secretion process in B. subtilis has yielded a number of remarkable surprises. These include striking differences in the composition of the general secretion (Sec) and twin-arginine translocation (Tat) pathways for the transport of secretory pre-proteins across the membranes of B. subtilis and Escherichia coli (6-10). Specifically, translocated pre-proteins of B. subtilis with Sec-type or twin-arginine signal peptides were shown to be subject to processing by the largest number of type I signal peptidases (SPases)1 known in any organism. In addition to five chromosomally encoded SPases (SipS, -T, -U, -V, and -W; 11), some B. subtilis strains even contain plasmid-encoded SPases (12). Furthermore, SipW was the first known eubacterial SPase of a type that is mainly encountered in Archaea and the eukaryotic endoplasmic reticular membrane (11, 13). Another highly remarkable finding was that the unique type II SPase (Lsp) of B. subtilis (14), which specifically catalyzes the maturation of lipid-modified pre-proteins, turned out to be required for the secretion of non-lipoproteins, such as alpha -amylase, chitosanase, and lipase (15, 16).2

When the negative effect of an SPase II (lsp) mutation on non-lipoprotein secretion was first observed for the alpha -amylase AmyQ, it was largely attributed to a possible malfunctioning of the lipoprotein PrsA, which is essential for the proper folding of various translocated proteins, such as AmyQ. An unexplained observation was, however, that B. subtilis cells lacking SPase II accumulated significantly increased levels of pre-AmyQ (15). As the latter effect was never observed in prsA mutant strains (17, 18), it was concluded that the increased pre-AmyQ accumulation in lsp mutant strains was because of the malfunction of one or more lipoproteins other than PrsA. This formed the incentive to search for lipoproteins of B. subtilis with an as yet unknown role in protein secretion. For this purpose, the amino acid sequences of all 114 (predicted) lipoproteins of B. subtilis (15) were used for similarity searches in public databases. Strikingly, the predicted lipoproteins SpoIIIJ and YqjG both showed significant similarity to the Oxa1 protein of yeast mitochondria (Fig. 1). This inner membrane protein has been implicated in the export of the amino and carboxyl termini of the mitochondrially encoded precursor of cytochrome c oxidase subunit II from the mitochondrial matrix (21, 22). Furthermore, it was shown that the E. coli orthologue of Oxa1p, denoted YidC, is associated with the Sec translocase (23). Thus, it was conceivable that SpoIIIJ and YqjG are involved in protein secretion by B. subtilis. On the other hand, Samuelson and co-workers (24) clearly demonstrated that YidC of E. coli is of major importance for the biogenesis of several membrane proteins, whereas the export of periplasmic and outer membrane proteins was hardly affected in YidC-depleted cells. Therefore, the present studies were aimed at the identification of possible roles of SpoIIIJ and YqjG in protein secretion and/or membrane protein biogenesis. The results show that, even though the phenotype of SpoIIIJ- and YqjG-depleted cells is quite different from that of SPase II mutant cells, SpoIIIJ and YqjG have an important impact on the accumulation of secretory proteins in the growth medium. Consistent with an important role in secretion, the presence of at least one of these two Oxa1p orthologues is essential for cell growth. Notably, under SpoIIIJ- and YqjG-limiting conditions that strongly affect the stability of secretory proteins, the stability of various membrane proteins is not or only mildly affected. Also, whereas SpoIIIJ is essential for sporulation (25), its paralogue YqjG is not required for this developmental process. Taken together, these data imply that SpoIIIJ and YqjG have acquired functions that are at least partly different from those of other members of the Oxa1 family.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Plasmids, Bacterial Strains, and Media-- Table I lists the plasmids and bacterial strains used. TY medium (tryptone/yeast extract) contained Bacto-tryptone (1%), Bacto-yeast extract (0.5%), and NaCl (1%). Minimal medium for B. subtilis was prepared as described by Tjalsma et al. (11). Schaeffer's sporulation medium was prepared as described by Schaeffer et al. (26). When required, medium for E. coli was supplemented with kanamycin (40 µg/ml), ampicillin (50 µg/ml), or erythromycin (100 µg/ml); media for B. subtilis were supplemented with chloramphenicol (5 µg/ml), kanamycin (10 µg/ml), tetracyclin (Tc; 6 µg/ml), or erythromycin (Em; 1 µg/ml).


                              
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Table I
Plasmids and bacterial strains

DNA Techniques-- Procedures for DNA purification, restriction, ligation, agarose gel electrophoresis, and transformation of E. coli were carried out as described by Sambrook et al. (32). Enzymes were from Roche Molecular Biochemicals. B. subtilis was transformed as described by Tjalsma et al. (11). The PCR was carried out with Pwo DNA polymerase (Roche Molecular Biochemicals) as described by van Dijl et al. (33).

To construct B. subtilis Delta spoIIIJ, first an internal fragment of the spoIIIJ gene was amplified with the primers spoIIIJ-1 (5'-GAGAATTCGACGGGAGATAACTACGGGC-3') and spoIIIJ-2 (5'-ATGGATCCTATGCTCTGAAATCGCCTGGG-3'). The amplified fragment was cleaved with EcoRI and BamHI, and ligated into the corresponding sites of pMutin2, resulting in pMutin-Delta spoIIIJ. Next, B. subtilis Delta spoIIIJ was obtained by a single crossover (Campbell-type) integration of pMutin-Delta spoIIIJ into the spoIIIJ gene of B. subtilis 168, in such a way that the spoIIIJ gene was disrupted and the spoVG-lacZ reporter gene of pMutin2 was placed under the transcriptional control of the spoIIIJ promoter region (Fig. 2). Simultaneously, the jag gene located downstream of spoIIIJ was placed under the control of the isopropyl-beta -D-thiogalacto-pyranoside (IPTG)-dependent Pspac promoter.

To construct B. subtilis Delta yqjG, first an internal fragment of the yqjG gene was amplified with primers yqjG-3 (5'-TGAAGCTTGCCGGGCTGTTTCACGG-3') and yqjG-2 (5'-ATGGATCCATCGTCATCATCACAGGGAAGATG-3'). The amplified fragment was cleaved with HindIII and BamHI, and ligated into the corresponding sites of pMutin2, resulting in pMutin-Delta yqjG. Next, B. subtilis Delta yqjG was obtained by a single crossover integration of pMutin-Delta yqjG into the yqjG gene of B. subtilis 168, in such a way that the yqjG gene was disrupted and the spoVG-lacZ reporter gene of pMutin2 was placed under the transcriptional control of the yqjG promoter region (Fig. 2).

To construct B. subtilis Delta yqjG-Tc, first a fragment comprising the yqjG gene and upstream and downstream sequences was amplified with the primers yqjG-1 (5'-GCTTTGGATTTCTTTTGCCGTCTC-3') and yqjG-4 (5'-GGTTCGTGAGCATAAAGGGAAGC-3'). The amplified fragment was cleaved with XbaI and KpnI, and ligated into the corresponding sites of pUK21. Next, the 714-bp EcoRI and PstI fragment, containing the 5' sequences of the yqjG gene, was replaced with a tetracyclin resistance marker resulting in plasmid pUKyqjG-Tc. Finally, the chromosomal yqjG gene of B. subtilis 168 was largely deleted by a double crossover recombination event with linearized pUKyqjG-Tc, resulting in B. subtilis Delta yqjG-Tc (see Fig. 2).

To construct B. subtilis Delta yqjG-IspoIIIJ, first a fragment comprising the ribosome binding site, start codon, and the 5' region of the spoIIIJ gene, but not the spoIIIJ promoter(s), was amplified with primers spoIIIJ-3 (5'-GGAATTCTAGAGTGTAAAGATTAATTATAGGAGGAAATGTTG-3') and spoIIIJ-2. The amplified fragment was cleaved with EcoRI and BamHI, and ligated into the corresponding sites of pMutin2, resulting in pMutin-IspoIIIJ. Finally, B. subtilis Delta yqjG-IspoIIIJ was obtained by Campbell-type integration of pMutin-IspoIIIJ into the spoIIIJ gene of B. subtilis Delta yqjG-Tc, in such a way that the spoIIIJ gene and the downstream jag gene were placed under the control of the IPTG-dependent Pspac promoter, whereas the spoVG-lacZ reporter gene of pMutin2 was placed under the transcriptional control of the spoIIIJ promoter region (Fig. 2).

B. subtilis Delta yqjG-IspoIIIJ containing a xylose-inducible secDF-Myc gene was obtained by transformation of B. subtilis Delta yqjG-IspoIIIJ with chromosomal DNA of the xSecDF-Myc strain (6). All constructed strains were selected on plates with the proper antibiotics, and checked by PCR analyses for correct integration of plasmids into the chromosome.

Growth and Maintenance of IPTG-dependent B. subtilis Delta yqjG-IspoIIIJ Strains-- The IPTG-dependent strain B. subtilis Delta yqjG-IspoIIIJ, and derivatives thereof, were grown in media containing 500 nM IPTG, Em (1 µg/ml), and Tc (6 µg/ml). It should be noted that in the absence of IPTG, such strains sometimes start to grow again after a lag period. This is probably because of the occurrence of a point mutation in the Pspac promoter, causing constitutive expression of the downstream genes (34). To avoid this potential problem, prior to each experiment, individual colonies were replica-plated on plates without IPTG and colonies displaying no growth on the latter plates were used for SpoIIIJ/YqjG depletion experiments. To this purpose, B. subtilis Delta yqjG-IspoIIIJ was grown overnight in TY medium supplemented with 500 nM IPTG, Em (1 µg/ml), and Tc (6 µg/ml). Cells were washed in fresh TY medium without IPTG and diluted 1:20 in fresh TY medium without, or with limiting concentrations (50 nM) of IPTG. After 3 h of growth, cells were harvested. Delta yqjG-IspoIIIJ strains stop growing after about 2-3 h in the absence of IPTG (Fig. 3).

Assay for Spore Development-- The efficiency of sporulation was determined by overnight growth in Schaeffer's sporulation medium, killing of cells with 0.1 volume chloroform, and subsequent plating.

beta -Galactosidase Activity Assay-- Overnight cultures were diluted 100-fold in fresh medium and samples were taken at hourly intervals for optical density (OD) readings and beta -galactosidase activity determinations. The assays and the calculations of beta -galactosidase units (expressed as units per A600) were carried out as described by Miller (35).

Western Blot Analysis and Immunodetection-- Western blotting was performed as described by Kyhse-Andersen (36). After separation by SDS-PAGE, proteins were transferred to Immobilon polyvinylidene difluoride membranes (Millipore Corporation). To detect LipA, PhoA, AmyQ(-PSBT), carboxyl-terminal Myc-tagged SecDF, SipS, PrsA, FtsH, CtaC, or QoxA, B. subtilis cells were separated from the growth medium. Cells were resuspended in lysis buffer (20 mM potassium phosphate, pH 7.5, 15 mM MgCl2, 20% sucrose, 0.5 mg/ml lysozyme) and incubated for 15 min at 37 °C. Next, 1 volume of SDS sample buffer (100 mM Tris-HCl, pH 6, 4% SDS, 10% 2-mercaptoethanol, 30% glycerol, 0.005% bromophenol blue, and 1% Triton X-100) was added and the incubation was prolonged for 15 min at 37 °C in the presence of CompleteTM protease inhibitors (Roche Molecular Biochemicals). In general, proteins were visualized with the ECL detection system, using rabbit/mouse sera and horseradish peroxidase anti-rabbit/mouse-IgG conjugates (Amersham Biosciences). Carboxyl-terminal Myc-tagged SecDF was visualized with monoclonal c-Myc antibodies (Clontech), and biotinylated AmyQ-PSBT was visualized with a streptavidin-horseradish peroxidase conjugate (Amersham Biosciences).

Pulse-Chase Protein Labeling, Immunoprecipitation, SDS-PAGE, and Fluorography-- B. subtilis Delta yqjG-IspoIIIJ was grown overnight in S7 medium supplemented with 500 nM IPTG, Em, and Tc. Cells were washed with fresh S7 medium without IPTG and diluted 1:10 in fresh S7 medium containing Em, with or without IPTG. After 2 h of growth, cells were resuspended in methionine and cysteine-free S7 (S7 starvation) medium with or without 500 nM IPTG and grown for another hour prior to pulse-chase labeling with [35S]methionine/cysteine ([35S]Pro-Mix; Amersham Biosciences). Immunoprecipitation, SDS-PAGE, and fluorography were performed as described previously (37, 38).

Protease Accessibility-- Protoplasts were prepared from late exponentially growing cells of B. subtilis. Cells were resuspended in protoplast buffer (20 mM potassium phosphate, pH 7.5, 15 mM MgCl2, 20% sucrose) and incubated for 30 min with 1 mg/ml lysozyme (37 °C). Protoplasts were collected by centrifugation, resuspended in fresh protoplast buffer, and incubated at 37 °C in the presence of 1 mg/ml trypsin (Sigma) for 30 min. The reaction was terminated by the addition of CompleteTM protease inhibitors (Roche Molecular Biochemicals) and protoplasts were used for SDS-PAGE and Western blotting. In parallel, protoplasts were incubated without trypsin, or in the presence of trypsin and 1% Triton X-100.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

YqjG Is Not Required for Sporulation-- The yidC gene of E. coli, specifying a SpoIIIJ/YqjG orthologue (Fig. 1), is essential for cell viability (24). In contrast, the spoIIIJ gene of B. subtilis is not essential for growth. In fact, spoIIIJ was originally identified as the gene containing the spo-87 mutation that blocks sporulation of B. subtilis cells at stage III, after the completion of prespore engulfment (25). To search for possible functions of the YqjG protein, a yqjG disruption strain was constructed with the integration vector pMutin2 (Fig. 2). The fact that B. subtilis Delta yqjG could be obtained showed that YqjG, like SpoIIIJ, is not essential for growth (Fig. 3A). Notably, disruption of the yqjG gene did not detectably affect sporulation, whereas a Delta spoIIIJ control strain constructed with pMutin2 was unable to develop viable spores (data not shown). Thus, YqjG has no essential function in the sporulation process, in contrast to SpoIIIJ.


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Fig. 1.   Conservation of Oxa1p(-like) proteins in B. subtilis, E. coli, and mitochondria of the yeast S. cerevisiae. A, the amino acid sequences of SpoIIIJ and YqjG, and the partial sequences of YidC and Oxa1p, comprising the conserved part as depicted in part B of this figure. Identical residues are indicated in bold. The conserved transmembrane segments I-V (marked in gray shading) were predicted as described by Sipos and von Heijne (19). The putative SPase II cleavage sites (lipoboxes) in SpoIIIJ and YqjG are underlined. Notably, SpoIIIJ/YqjG orthologues with putative lipoprotein signal peptides have been found in several Gram-positive eubacteria, such as Bacillus anthracis, Bacillus halodurans, Bacillus stearothermophilus, Lactococcus lactis, and Staphylococcus aureus (H. Tjalsma, S. Bron, and J. M. van Dijl, unpublished observations). Numbers refer to the position of amino acids in the corresponding protein sequence. B, predicted membrane topologies of SpoIIIJ and YqjG of B. subtilis, YidC of E. coli, and Oxa1p of S. cerevisiae. Only the membrane topology of YidC has been verified experimentally (20). Note that the YidC protein has a large amino-terminal loop located in the periplasm that is absent from SpoIIIJ, YqjG, and Oxa1p. C, carboxyl terminus; the cytoplasmic, cell wall, periplasmic, matrix, or inter membrane space (IMS) sides of the membranes, and the "conserved" parts of the Oxa1p(-like) proteins are indicated. Note that the amino termini of SpoIIIJ and YqjG are most likely lipid-modified.


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Fig. 2.   Construction of spoIIIJ and/or yqjG mutant strains of B. subtilis. B. subtilis Delta spoIIIJ and B. subtilis Delta yqjG were, respectively, constructed by the single crossover integration of pMutin-Delta spoIIIJ and pMutin-Delta yqjG into the chromosome B. subtilis 168. In these strains, the respective spoIIIJ or yqjG genes are disrupted while the lacZ gene of pMutin2 is placed under the transcriptional control of the promoter regions of these genes. B. subtilis Delta yqjG-Tc was constructed by replacement of the 5' part of the yqjG gene with a tetracyclin resistance marker by double crossover recombination. B. subtilis Delta yqjG-IspoIIIJ was constructed by integration of pMutin-IspoIIIJ into the spoIIIJ region of B. subtilis Delta yqjG-Tc. By this approach, the spoIIIJ gene was placed under the control of the IPTG-dependent Pspac promoter. Notably, growth and viability of the latter strain is dependent on the presence of IPTG. The relative positions of open reading frames in the spoIIIJ and yqjG regions are shown. PCR-amplified DNA fragments that were used to direct the integration of pMutin2 into the B. subtilis chromosome are indicated with black bars. Restriction sites relevant for the construction are indicated: B, BamHI; dco, double crossover; E, EcoRI; H, HindIII; jag, gene of unknown function specifying a predicted cytoplasmic protein; P, PstI. Ori pBR322, replication functions of pBR322; Emr, erythromycin resistance marker; sco, single crossover; T1T2, transcriptional terminators on pMutin2; spoIIIJ', 3' truncated spoIIIJ gene; 'spoIIIJ, 5' truncated spoIIIJ gene; yqjG', 3' truncated yqjG gene; 'yqjG, 5' truncated yqjG gene.


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Fig. 3.   Properties of spoIIIJ and/or yqjG mutant strains of B. subtilis. A, IPTG-dependent growth of B. subtilis Delta yqjG-IspoIIIJ on plates at 15 and 37 °C. Individual colonies of B. subtilis Delta yqjG-IspoIIIJ were transferred to fresh TY-agar plates containing 1000, 500, 100, 50, or 0 nM IPTG. Next, the plates were incubated overnight at 37 °C (upper panel), or for 5 days at 15 °C (lower panel). B, IPTG-dependent growth of B. subtilis Delta yqjG-IspoIIIJ. Overnight cultures of B. subtilis 168 (parental; triangle ), B. subtilis Delta spoIIIJ (black-triangle), B. subtilis Delta yqjG (open circle ), and B. subtilis Delta yqjG-IspoIIIJ (black-square), grown in TY medium at 37 °C, were washed and diluted 20-fold in fresh TY medium without IPTG and incubated at 37 °C. In addition, B. subtilis Delta yqjG-IspoIIIJ was diluted in fresh TY medium containing 50 () or 500 () nM IPTG. Zero time (t = 0) indicates the transition point between the exponential and post-exponential growth phases. C, transcription profiles of the spoIIIJ and yqjG genes. Time courses of the transcription of the spoIIIJ-lacZ and yqjG-lacZ gene fusions in B. subtilis Delta spoIIIJ (open circle ) and B. subtilis Delta yqjG (black-square) were determined in cells growing at 37 °C in TY medium. beta -Galactosidase activities were determined in units per A600. Zero time (t = 0) indicates the transition point between the exponential and post-exponential growth phases.

The Presence of Either SpoIIIJ or YqjG Is Required for Growth of B. subtilis-- To determine whether the combined activities of SpoIIIJ and YqjG are required for the viability of B. subtilis cells, a conditional (IPTG-dependent) yqjG-spoIIIJ double mutant strain was constructed in two steps. First, the largest part of the yqjG gene was replaced with a tetracyclin resistance marker via double crossover recombination. Second, the spoIIIJ gene of the latter strain was placed under the control of the IPTG-inducible Pspac promoter, present on pMutin2. This was achieved by single crossover of pMutin2 into the spoIIIJ region of the chromosome (Fig. 2). Notably, the resulting B. subtilis Delta yqjG-IspoIIIJ strain could be obtained only in the presence of IPTG, indicating that at least the spoIIIJ gene had to be transcribed for the growth of this strain. In fact, about 500 nM IPTG was required for this strain to display unimpaired growth on TY agar plates at 37 °C (Fig. 3A). Upon dilution in fresh TY medium without IPTG, B. subtilis Delta yqjG-IspoIIIJ stopped growing after 2-3 h of incubation (Fig. 3B). Similar to the IPTG-dependent growth of this strain on TY plates, the presence of 500 nM IPTG was required to support wild-type growth of B. subtilis Delta yqjG-IspoIIIJ in liquid TY medium at 37 °C. In contrast, growth of B. subtilis Delta yqjG-IspoIIIJ in the presence of 50 nM IPTG was significantly reduced as compared with that of the parental strain 168, indicating that SpoIIIJ was synthesized at limiting levels. Importantly, growth of B. subtilis Delta yqjG-IspoIIIJ in the absence of IPTG could be restored by the ectopic expression of the spoIIIJ gene,3 showing that the growth inhibition of this strain is because of SpoIIIJ limitation, and not polar effects on the expression of the downstream located jag gene. Together with the observation that spoIIIJ and yqjG single mutants are viable, these findings show that the presence of either SpoIIIJ or YqjG is required for growth of B. subtilis. Notably, the IPTG dependence of B. subtilis Delta yqjG-IspoIIIJ was strongly increased at 15 °C (Fig. 3A). As protein transport via the Sec pathways of B. subtilis and E. coli is intrinsically cold-sensitive (6, 39, 40), the observed temperature effect suggests that SpoIIIJ and YqjG have a role in Sec-dependent protein transport.

Maximal spoIIIJ and yqjG Transcription in the Exponential Growth Phase-- Previous studies showed that the spoIIIJ gene is transcribed during the exponential growth phase and that this transcription is shut down at about the onset of sporulation (25). To investigate whether transcription of the yqjG gene is regulated in a similar manner, we used the transcriptional yqjG-lacZ and spoIIIJ-lacZ gene fusions that are present in B. subtilis Delta yqjG and Delta spoIIIJ, respectively (Fig. 2). Both strains were grown in TY medium, minimal medium, or sporulation medium, and samples withdrawn at hourly intervals were assayed for beta -galactosidase activity. The results showed that, irrespective of the growth medium, the beta -galactosidase levels in both strains reached a maximum in the exponential phase (Fig. 3C, only the results obtained with cells grown in TY medium are documented). The beta -galactosidase levels were strongly decreased upon the transition (t = 0) into the post-exponential growth phase. As the transcription profiles of the yqjG-lacZ gene fusion were very similar to those of the spoIIIJ-lacZ fusion under all conditions tested (Fig. 3C; data not shown), it seems highly unlikely that the lack of effect of a yqjG mutation on sporulation is because of differences in the transcription of these genes.

SpoIIIJ and YqjG Limitation Affects a Post-translocational Stage in Protein Secretion-- To evaluate the importance of YqjG and SpoIIIJ function for protein secretion, B. subtilis Delta yqjG, Delta spoIIIJ, and Delta yqjG-IspoIIIJ, as well as parental strain 168, were transformed with plasmid pLip2031 for the secretion of the B. subtilis esterase LipA (29), pPSPhoA5 for the secretion of the alkaline phosphatase PhoA of E. coli fused to the prepro-region of the lipase gene from Staphylococcus hyicus (7), or pKTH10 for the secretion of the alpha -amylase AmyQ (30). As no secretion defects were detectable in the single mutant strains (data not shown), we focused our attention on the Delta yqjG-IspoIIIJ double mutant. To deplete B. subtilis Delta yqjG-IspoIIIJ of SpoIIIJ, this strain was grown for 3 h in TY medium without IPTG, as described under "Experimental Procedures." As a control, TY medium with 50 nM IPTG (limiting amounts of SpoIIIJ) or 500 nM IPTG (full induction of SpoIIIJ) was used (see Fig. 3, A and B). Next, the secretion of LipA, PhoA, and AmyQ was analyzed by Western blotting. As shown in Fig. 4A (lower panels), the levels of LipA, PhoA, and AmyQ in the medium of SpoIIIJ-depleted cells of B. subtilis Delta yqjG-IspoIIIJ (no IPTG) were significantly reduced compared with those in the media of the fully induced double mutant (500 nM IPTG), or the parental strain 168. Moreover, SpoIIIJ-depleted double mutant cells containing pPSPhoA5 or pKTH10 also contained significantly decreased levels of mature PhoA or AmyQ, respectively (Fig. 4A, upper panels). In contrast, the cellular levels of mature LipA, and the precursor forms of LipA and AmyQ were not affected by SpoIIIJ depletion in the absence of YqjG (Fig. 4A). Interestingly, the levels of LipA and PhoA in the media of Delta yqjG-IspoIIIJ strains that were fully induced with IPTG (500 nM) were higher than those in the media of the parental control strains. The latter finding suggests that overexpression of the spoIIIJ gene can result in improved protein secretion in B. subtilis.


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Fig. 4.   SpoIIIJ and YqjG are required for extracellular accumulation of secretory proteins. A, precultures of B. subtilis Delta yqjG-IspoIIIJ and the parental strain B. subtilis 168, both transformed with plasmid pLip2031 (specifying LipA), pPSPhoA5 (specifying PhoA), or pKTH10 (specifying AmyQ), were prepared by overnight growth at 37 °C in TY medium containing 500 nM IPTG. Next, cells were washed with fresh TY medium without IPTG, diluted 20-fold in fresh TY medium containing 500, 50 nM, or no (0) IPTG, and incubated for 3 h at 37 °C before sampling for SDS-PAGE and Western blotting. Specific antibodies were used to detect the cellular (pre-)LipA, PhoA, or (pre-)AmyQ levels (upper panels), and the levels of secreted LipA, PhoA, or AmyQ in the growth medium (lower panels). The positions of pre-LipA and pre-AmyQ (open circle ), mature LipA, PhoA, and AmyQ (), or degradation products of PhoA (star ) are indicated. B, processing of pre-AmyQ in B. subtilis Delta yqjG-IspoIIIJ was analyzed by pulse-chase labeling at 37 °C in S7 medium with (500 nM; upper panel) or without (0) IPTG (lower panel), subsequent immunoprecipitation, SDS-PAGE, and fluorography. Cells were labeled with [35S]methionine/cysteine for 1 min prior to chase with excess non-radioactive methionine/cysteine. Samples were withdrawn after the chase at the times indicated. Because the incorporation of label into (pre-)AmyQ cannot be stopped instantaneously by the addition of non-radioactive methionine, samples withdrawn at t = 0, and t = 1 contain lower amounts of labeled AmyQ than the sample withdrawn at t = 5. The positions of pre-AmyQ (open circle ) and mature AmyQ () are indicated. C, translocation of pre-AmyQ-PSBT in SpoIIIJ-depleted cells lacking YqjG. To analyze pre-AmyQ translocation, cells of B. subtilis Delta yqjG-IspoIIIJ, B. subtilis Delta secDF (positive control), and the parental strain B. subtilis 168 (negative control) were transformed with plasmid pKTH10-BT (specifying AmyQ-PSBT), and grown as described for panel A of this figure. Cellular (biotinylated) AmyQ-PSBT was visualized by SDS-PAGE and Western blotting using AmyQ-specific antibodies (upper panel), or a streptavidin-horseradish peroxidase conjugate (lower panel). Precursor (open circle ) and mature () forms of AmyQ-PSBT are indicated.

To investigate the nature of the secretion defect of B. subtilis Delta yqjG-IspoIIIJ, further experiments were performed with AmyQ and an AmyQ variant (AmyQ-PSBT) as described previously by Bolhuis et al. (6). As shown by pulse-chase labeling experiments, within the period of labeling (zero time), the pre-AmyQ synthesis and processing in cells of B. subtilis Delta yqjG-IspoIIIJ depleted of SpoIIIJ (no IPTG) was not significantly different from that observed in cells of this strain in which SpoIIIJ was fully induced (500 nM IPTG; Fig. 4B), or the parental strain 168 (data not shown). However, in particular after 5 min of chase, significantly reduced amounts of mature AmyQ were detectable in the Delta yqjG-IspoIIIJ cells depleted of SpoIIIJ. This suggests that these cells have a defect in post-translocational protein folding into a stable conformation rather than a defect in protein translocation. To verify that SpoIIIJ-depleted cells lacking YqjG have no translocation defect, experiments were performed with AmyQ-PSBT, which contains a carboxyl-terminal biotin-accepting domain (PSBT) of a transcarboxylase from Propionibacterium shermannii (11). The rationale of this experiment is that pre-AmyQ-PSBT can only be biotinylated by the cytoplasmic biotin ligase when the PSBT domain folds into its native three-dimensional structure prior to translocation. Consequently, biotinylation of pre-AmyQ-PSBT occurs at significantly increased levels when pre-AmyQ-PSBT translocation is slowed-down, for example, by the disruption of the secDF gene (6). As shown in Fig. 4C, SpoIIIJ-depleted cells lacking YqjG did not accumulate increased amounts of biotinylated pre-AmyQ-PSBT as compared with cells in which SpoIIIJ synthesis was induced with IPTG, or the parental strain 168. In contrast, B. subtilis cells with a disrupted secDF gene (positive control) accumulated strongly increased amounts of biotinylated AmyQ-PSBT (Fig. 4C). Notably, the biotinylated AmyQ-PSBT in the Delta secDF cells was present both in the precursor and mature forms. Taken together, these observations support the view that the translocation and processing of pre-AmyQ is not affected in SpoIIIJ-depleted cells lacking YqjG. Instead, SpoIIIJ and YqjG appear to be important for the post-translocational folding stages in protein secretion.

SpoIIIJ and YqjG Are Required for the Stability or Cell Wall Passage of Translocated Secretory Proteins-- To further investigate the possibility of a post-translocational secretion defect in SpoIIIJ-depleted cells lacking YqjG, combined pulse-chase labeling and fractionation experiments were performed. For this purpose, pulse-labeled cells were separated from their growth medium by centrifugation. Next, the collected cells were treated with lysozyme, and the resulting protoplasts were separated from the cell wall fraction (i.e. the protoplast supernatant) by centrifugation. Finally, radiolabeled reporter proteins were recovered from lysed protoplasts, the cell wall fraction, and the growth medium by immunoprecipitation. As shown in Fig. 5A, the kinetics of synthesis and processing of pre-AmyQ (upper panel), and release of mature AmyQ into the cell wall (middle panel) in cells of B. subtilis Delta yqjG-IspoIIIJ depleted of SpoIIIJ (no IPTG), or the parental strain, were not significantly different. However, the amount of mature AmyQ released into the growth medium after 10 min of chase (lower panel) was about 3-fold reduced in the case of Delta yqjG-IspoIIIJ cells depleted of SpoIIIJ. Moreover, a specific AmyQ degradation product was detectable in the medium fractions of pulse-labeled Delta yqjG-IspoIIIJ cells depleted of SpoIIIJ, but not in the corresponding fractions of the parental strain. Similarly, the prepro-PhoA synthesis and processing (Fig. 5B, upper panel), as well as the release of pro-PhoA into the cell wall (not shown) was not significantly different in cells of B. subtilis Delta yqjG-IspoIIIJ depleted of SpoIIIJ, or the parental strain. Importantly, significantly reduced amounts of (pro-)PhoA were detected in all medium fractions of pulse-labeled B. subtilis Delta yqjG-IspoIIIJ depleted of SpoIIIJ (Fig. 5B, lower panel). Also, a specific PhoA degradation product was present in the medium of Delta yqjG-IspoIIIJ cells depleted of SpoIIIJ, but not in the medium of the parental strain (lower panel). These observations imply that mature AmyQ and (pro-)PhoA are subject to proteolysis upon their release from SpoIIIJ/YqjG-depleted cells into the growth medium. This suggests that SpoIIIJ and YqjG are (directly or indirectly) required for the folding of AmyQ and PhoA into a stable, protease-resistant conformation that allows the accumulation of these proteins in the growth medium.


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Fig. 5.   SpoIIIJ and YqjG are required for stability or release of secretory proteins. The secretion of AmyQ (A), PhoA (B), and LipA (C) was analyzed in B. subtilis Delta yqjG-IspoIIIJ and the parental strain 168 by pulse-chase labeling in S7 medium at 37 °C, fractionation (protoplasts, cell wall and medium), immunoprecipitation, SDS-PAGE, and fluorography. Cells were labeled with [35S]methionine/cysteine for 1 min prior to chase with excess non-radioactive methionine/cysteine. Samples were withdrawn at 1, 2, and 10 min after the chase. Cells and medium were separated by centrifugation, and protoplast were subsequently prepared as described under "Experimental Procedures." Finally, the cell wall and protoplast fractions were separated by centrifugation. The positions of pre-AmyQ and prepro-PhoA (open circle ), degradation products of (pro-)PhoA and AmyQ (*), and mature AmyQ, PhoA, and LipA () are indicated.

As shown by pulse-chase labeling, the synthesis of LipA in cells of B. subtilis Delta yqjG-IspoIIIJ depleted of SpoIIIJ (no IPTG) was not affected (Fig. 5C, upper panel; note that processing of pre-LipA is complete after 1 min of chase). However, significant amounts of mature LipA were retained in the cell wall of Delta yqjG-IspoIIIJ cells depleted of SpoIIIJ, whereas this protein was rapidly chased from the cell wall of the parental strain (Fig. 5C, middle panel). These findings indicate that the post-translocational release of mature LipA from the cell wall into the growth medium is significantly affected in SpoIIIJ/YqjG-depleted cells. Compared with AmyQ and (pro-)PhoA, the effect of SpoIIIJ depletion on the kinetics of LipA secretion into the growth medium by cells of B. subtilis Delta yqjG-IspoIIIJ was relatively mild (Fig. 5, compare A, B, and C).

Together, these data confirm the view that the secretion of proteins into the growth medium by SpoIIIJ/YqjG-depleted cells of B. subtilis is affected at stages that follow translocation across the cytoplasmic membrane. Within the first 10 min of the "life" of secretory proteins investigated here, the limitation of B. subtilis Oxa1-like proteins may either result in a significantly reduced stability (AmyQ and PhoA) or cell wall retention (LipA).

Specific role of SpoIIIJ and YqjG in Membrane Protein Stability-- As shown by Samuelson et al. (24), the YidC protein of E. coli is very important for the correct insertion of various proteins into the inner membrane, but not the export and processing of Sec-dependent pre-proteins. To investigate whether the SpoIIIJ and YqjG functions could be required for membrane protein biogenesis in B. subtilis, the cellular levels of the BdbB, BdbC, FtsH, SecDF-Myc, SipS, and SPase II proteins in the Delta yqjG-IspoIIIJ strain were monitored by Western blotting. In addition, the lipoprotein PrsA was included in the analyses. These seven membrane(-associated) proteins were primarily selected because they have different membrane topologies and different numbers of transmembrane segments (Fig. 6; not shown for BdbB/C and SPase II, which have four transmembrane segments and an Nin-Cin topology). Furthermore, these proteins, which are known to be involved in protein secretion by B. subtilis (5), were selected to investigate whether the secretion defects of SpoIIIJ-depleted cells lacking YqjG might be indirectly caused by the impaired membrane biogenesis of secretion machinery components. Interestingly, the levels of SecDF-Myc, SipS, and PrsA were not detectably affected in SpoIIIJ-depleted cells lacking YqjG that were grown at 37 °C (Fig. 6), or 15 °C (data not shown). Similarly, the cellular amounts of BdbB, BdbC, and SPase II remained unchanged (data not shown). In fact, FtsH was the only protein involved in protein secretion (41) that was (mildly) affected upon SpoIIIJ depletion in the absence of YqjG. As shown in Fig. 6, low amounts of an FtsH degradation product (lower panel) accumulated in Delta yqjG-IspoIIIJ cells depleted of SpoIIIJ.


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Fig. 6.   Specific role of SpoIIIJ and YqjG in membrane protein biogenesis. Cells of B. subtilis Delta yqjG-IspoIIIJ xSecDF-Myc (Delta yqjG-IspoIIIJ) and the control strain B. subtilis xSecDF-Myc (parental) were grown overnight at 37 °C in TY medium containing 500 nM IPTG. Next, cells were washed with fresh TY medium without IPTG, diluted 20-fold in fresh TY medium containing 1% xylose (production SecDF-Myc) and 500, 50 nM, or no (0) IPTG, and incubated at 37 °C for 3 h. Samples for SDS-PAGE and Western blotting were prepared from cells and specific antibodies were used to detect the cellular levels of SecDF-Myc, SipS, FtsH, CtaC, QoxA, and PrsA. The positions of the native proteins () and their degradation products (*) are indicated. It should be noted that FtsH-derived degradation products (FtsH, lower panel) were only visible after prolonged fluorography. Putative membrane topologies of these proteins are depicted (N, amino terminus; C, carboxyl terminus; in, cytoplasmic side of the membrane; out, cell wall-exposed side of the membrane). Note that the amino termini of CtaC, QoxA, and PrsA are lipid-modified.

The Oxa1p protein of yeast mitochondria was previously shown to be required for the correct assembly of cytochrome c oxidase complexes (42, 43), and the specific export of the amino and carboxyl termini of the precursor of cytochrome c oxidase subunit II from the mitochondrial matrix to the intermembrane space (21, 22, 44). Therefore, a possible role of SpoIIIJ and YqjG in the biogenesis of the transmembrane lipoproteins CtaC and QoxA, which are orthologues of cytochrome c oxidase subunit II (45), was investigated. Indeed, the results showed that, upon SpoIIIJ depletion of cells lacking YqjG, the cellular levels of CtaC were slightly reduced, whereas a specific CtaC degradation product accumulated concomitantly (Fig. 6). However, under the same conditions, the cellular level and stability of QoxA were not detectably affected (Fig. 6).

Previous studies by de Gier et al. (46) have shown that impaired membrane protein insertion does not necessarily affect the total cellular levels of membrane proteins. To study whether membrane(-associated) proteins were properly inserted in SpoIIIJ-depleted cells lacking YqjG, the membrane topology of SecDF-Myc, SipS, FtsH, CtaC, QoxA, and PrsA was assessed by protoplasting and subsequent protease mapping assays. As shown in Fig. 7, the protease accessibility of none of the tested proteins was detectably affected in Delta yqjG-IspoIIIJ cells upon SpoIIIJ depletion. The cytoplasmic protein GroEL was not degraded by extracellular trypsin, indicating that lysis of protoplasts did not occur during the assay. It should be noted that the degradation product of FtsH, which accumulates in Delta yqjG-IspoIIIJ cells depleted of SpoIIIJ (Fig. 6), was also detectable in the parental strain upon protoplasting. The degradation product of CtaC, which accumulates in Delta yqjG-IspoIIIJ cells depleted of SpoIIIJ (Fig. 6), was not detectable upon protoplasting of these cells.


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Fig. 7.   Protease mapping of membrane proteins in SpoIIIJ-depleted cells lacking YqjG. To analyze the insertion of the membrane proteins SecDF-Myc, SipS, FtsH, CtaC, QoxA, and PrsA, cells of B. subtilis Delta yqjG-IspoIIIJ xSecDF-Myc (Delta yqjG-IspoIIIJ) and the control strain B. subtilis xSecDF-Myc (parental) were grown overnight at 37 °C in TY medium containing 500 nM IPTG. Cells were washed with fresh TY medium without IPTG, diluted 20-fold in fresh TY medium without IPTG, and incubated at 37 °C for 3 h. Next, the production of SecDF-Myc was induced by the addition of 1% xylose 15 min prior to protoplasting. Protoplasts were incubated for 30 min without further additions, in the presence of trypsin (1 mg/ml), or trypsin and Triton X-100 (1%). This procedure was performed in parallel with cells of B. subtilis Delta yqjG-IspoIIIJ and B. subtilis 168 (parental), both containing pKTH10 for production of AmyQ. Samples were used for SDS-PAGE, Western blotting, and specific antibodies were used to detect SecDF-Myc, SipS, FtsH, CtaC, QoxA, PrsA, GroEL, or AmyQ. The positions of intact proteins and pre-AmyQ (), degradation products because of the incubation with trypsin (*), mature AmyQ (open circle ), and trypsin-resistant fragments (right-triangle ) are indicated.

To examine the kinetics of membrane protein insertion, a pulse-chase/protease mapping experiment was performed with cells of the parental B. subtilis 168 strain and Delta yqjG-IspoIIIJ cells upon SpoIIIJ depletion. To this purpose, pulse-labeled cells were subject to protoplasting and, subsequently, protoplasts were incubated in the presence of trypsin, plus or minus Triton X-100. As shown in Fig. 8, the protease accessibility of SipS, QoxA, and PrsA at the surface of intact protoplasts was not detectably affected in SpoIIIJ-depleted Delta yqjG-IspoIIIJ cells after a chase of 1 min. GroEL was not degraded by extracellular trypsin, indicating that no lysis of protoplasts occurred during this assay. Consistent with the lack of effect on the insertion of PrsA in the membrane, the processing of pre-PrsA by SPase II, as verified by pulse-chase labeling, was not affected in Delta yqjG-IspoIIIJ cells upon SpoIIIJ depletion (data not shown). Unfortunately, we could not use SecDF-Myc, CtaC, and FtsH for pulse-chase labeling experiments as we were unable to immunoprecipitate these proteins from labeled cell extracts.


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Fig. 8.   Protease mapping of pulse-labeled membrane proteins in SpoIIIJ-depleted cells lacking YqjG. To analyze the kinetics of SipS, QoxA, and PrsA biogenesis, cells of B. subtilis Delta yqjG-IspoIIIJ and the parental strain 168 were grown in S7 medium at 37 °C, and labeled with [35S]methionine/cysteine for 1 min followed by a chase of 1 min with excess non-radioactive methionine/cysteine. Cells were collected by centrifugation, and protoplasts were subsequently prepared as described under "Experimental Procedures." Protoplasts were incubated for 30 min without further additions, in the presence of trypsin (1 mg/ml), or trypsin and Triton X-100 (1%) prior to immunoprecipitation, SDS-PAGE, and fluorography. Immunoprecipitation with anti-GroEL antibodies was used to check protoplast integrity. The positions of intact proteins () and trypsin-resistant fragments (right-triangle ) are indicated.

Taken together, these findings show that the stability of some membrane proteins is affected in cells with limiting amounts of SpoIIIJ and YqjG. However, the stability, topology, and insertion kinetics of other membrane proteins and the essential lipoprotein PrsA is not detectably affected under these conditions. The fact that the biogenesis of none of the tested protein secretion machinery components is SpoIIIJ/YqjG-dependent suggests that the defect in the secretion of AmyQ, LipA, and PhoA is not caused by impaired membrane assembly of individual secretion machinery components in SpoIIIJ-depleted cells lacking YqjG.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Proteins homologous to Oxa1p are conserved in eubacteria and eukaryotic organelles, where they appear to have important functions in protein transport and membrane protein assembly. In fact, SpoIIIJ of B. subtilis was the first protein of this family to which a function was assigned. Errington and co-workers (25) showed in 1992 that a spoIIIJ mutation blocks sporulation of B. subtilis cells at stage III, after the completion of forespore engulfment. It was thought that this protein was involved in a signal transduction pathway, coupling gene expression in the forespore to concomitant events in the mother cell. However, the exact mechanism of SpoIIIJ function in sporulation was not unraveled.

Our present observations demonstrate that the synthesis of either SpoIIIJ, or its paralogue YqjG, is required for growth of the Gram-positive eubacterium B. subtilis, which is in good agreement with recent data from Murakami and co-workers (47). Furthermore, the accumulation of the mature AmyQ, PhoA, and LipA proteins in the growth medium is strongly impaired under conditions of SpoIIIJ/YqjG-limitation. Notably, the reduction in AmyQ and PhoA levels in the medium is not paralleled by reduced rates of pre-AmyQ or prepro-PhoA translocation and processing, or the cellular accumulation of the respective precursors, as previously documented for secDF, tepA, or sip mutant strains with translocation defects (6, 11, 48, 49). Instead, pulse-chase labeling experiments combined with cell fractionation indicate that AmyQ and PhoA are degraded upon release of these proteins into the growth medium. Thus, the AmyQ and PhoA secretion defects observed for SpoIIIJ-depleted cells lacking YqjG seem to occur during post-translocational stages that involve the folding of secretory proteins into their active and protease-resistant conformation. The latter is of particular importance because the extracytoplasmic side of the membrane, the cell wall, and the growth medium of B. subtilis are highly proteolytic (5, 16).

In a wild-type cell, SpoIIIJ and YqjG might facilitate the folding of translocated proteins in several ways. First, it is conceivable that SpoIIIJ and YqjG have a direct role in the correct folding of mature proteins shortly after their translocation across the membrane, similar to the essential function proposed for the folding catalyst PrsA (15, 17). Alternatively, SpoIIIJ and YqjG might have an indirect role in protein secretion by modulating the activity of folding catalysts, such as PrsA. A third possibility would be that SpoIIIJ and YqjG depletion impacts on the composition of the cell wall. For example, it has been shown that negatively charged teichoic acids can increase the rate of folding and stability of certain exported proteins (50). The observation that the release of mature LipA from the cell wall into the growth medium is affected upon SpoIIIJ/YqjG limitation could point to the direction that the composition of the cell wall is altered under these conditions. However, it is also conceivable that the depletion of SpoIIIJ and YqjG affects the folding of LipA, and that malfolded LipA interacts more strongly with cell wall components than correctly folded LipA (51).

In recent years, the Oxa1p protein of yeast mitochondria was shown to be required for the processing of mitochondrially encoded precursors (52), the export of amino and carboxyl termini from precursor cytochrome c oxidase subunit II synthesized in the mitochondrial matrix (21, 22), and the insertion of transmembrane domains into the mitochondrial inner membrane in a pairwise fashion (53). Considering the facts that yeast mitochondria completely lack Sec components, and that the Oxa1p orthologue in chloroplasts (Albino III) is required for the Sec-independent integration of the light-harvesting chlorophyll-binding protein into the thylakoid membrane (54), members of the Oxa1p family might represent the key components of a novel pathway for protein export or membrane protein assembly (55). Consistent with this view, YidC of E. coli was shown to facilitate the Sec-independent insertion of certain membrane proteins, such as the M13 procoat, being of minor importance for the export of Sec-dependent preproteins (24, 56). Nevertheless, YidC, was also shown to be associated with the Sec machinery, indicating that this protein has a more general role in membrane protein biogenesis in E. coli, for example, by catalyzing the exit of membrane proteins from the Sec translocase (23, 24, 57). Indications for such a lateral movement were obtained by Urbanus and co-workers (58), who showed a sequential interaction of the membrane protein FtsQ with SecY and YidC. Recent studies by Houben et al. (59) showed that YidC can contact a transmembrane domain during biogenesis, even when it is not yet fully exposed on the outside of the ribosome. Thus, YidC seems to have a role in both the reception and lipid partitioning of transmembrane segments. Our present studies support the view that SpoIIIJ and YqjG of B. subtilis are also involved in membrane protein biogenesis as the stability of the membrane proteins FtsH and CtaC is affected under conditions of SpoIIIJ/YqjG limitation. However, pulse-chase labeling and protease mapping experiments indicate that not all membrane proteins of B. subtilis require SpoIIIJ and YqjG for their biogenesis. For example, the insertion kinetics of SipS and QoxA were not significantly affected upon SpoIIIJ/YqjG limitation. In this respect, it is interesting to note that the membrane topology of QoxA of B. subtilis is similar to that of Lep of E. coli for which it has been shown that the rate of membrane insertion is significantly affected in YidC-depleted cells (24). Even though the post-translocational protein secretion defect is presently the most prominent phenotype of SpoIIIJ/YqjG-depleted cells of B. subtilis, it is important to bear in mind that this secretion defect may be indirectly caused by the misassembly of an as yet unidentified membrane protein.

Our present results, together with the well documented requirement of Oxa1p and YidC for membrane protein biogenesis, suggest that SpoIIIJ and YqjG have rather specific roles in membrane protein biogenesis in B. subtilis. It is therefore concluded that different orthologous members of the Oxa1 protein family have acquired different species-specific functions in Sec-dependent and Sec-independent membrane protein biogenesis and protein secretion. Interestingly, not only orthologous Oxa1-like proteins, but also Oxa1-like paralogues within one organism have acquired (partly) distinct functions, as evidenced by the present observation that SpoIIIJ, but not YqjG, is required for spore development. Similarly, only one of the two Oxa1p orthologues of Schizosaccharomyces pombe is essential for respiration (60). As only minor differences in the timing of transcription have been observed for the genes specifying the paralogous Oxa1p proteins of B. subtilis (Fig. 3C) and S. pombe (60), the differences in function of these protein pairs are, most likely, based on differences in their primary structures.

Finally, what is the mechanism underlying SpoIIIJ involvement in the sporulation process? First, in view of the fact that SpoIIIJ and YqjG are important for post-translocational protein folding steps in the secretion process, it is conceivable that SpoIIIJ is specifically required for the folding of certain, as yet unknown, translocated sporulation factors. The importance of protein transport for sporulation is underscored by the observations that SecA, and the type I SPases SipT and SipV, are required for this process (61, 62).3 Second, SpoIIIJ might be involved in the insertion of specific membrane proteins that are essential for the sporulation process after stage III. Possibly, these membrane proteins are involved in the communication between the forespore and the mother cell as originally proposed by Errington and co-workers (25). Our ongoing research is focused on the identification of specific substrates of SpoIIIJ, and determinants for the different specificities of SpoIIIJ and YqjG. This will provide better insights into the molecular mode-of-action of the members of the Oxa1 protein family.

    ACKNOWLEDGEMENTS

We thank Drs. H. Antelmann, R. Dorenbos, A. J. M. Driessen, J. D. H. Jongbloed, J. Swaving, and other members of the Groningen and European Bacillus Secretion Groups (see www.ncl.ac.uk/ebsg) for stimulating discussions, and Drs. M. Sarvas, T. Wiegert, V. P. Kontinen, J. Bengtsson, and A. Kröger for providing specific antibodies against AmyQ, FtsH, PrsA, CtaC, and QoxA, respectively.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported by Genencor International (Leiden, The Netherlands).

§ Supported in part by European Union Grants QLK3-CT-1999-00413 and QLK3-CT-1999-00917.

To whom correspondence should be addressed. Tel.: 31503632105; Fax: 31503632348; E-mail: S.Bron@biol.rug.nl.

Published, JBC Papers in Press, February 13, 2003, DOI 10.1074/jbc.M301205200

2 H. Antelmann, H. Tjalsma, J. M. van Dijl, and M. Hecker, unpublished observations.

3 H. Tjalsma, S. Bron, and J. M. van Dijl, unpublished data.

    ABBREVIATIONS

The abbreviations used are: SPase, signal peptidase; IPTG, isopropyl-beta -D-thiogalactopyranoside; Em, erythromycin; Tc, tetracyclin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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