<i>Bacillus subtilis</i> PrsA is required <i>in vivo</i> as an extracytoplasmic chaperone for secretion of active enzymes synthesized either with or without pro‐sequences

118

The PrsA protein of Bacillus subtilis is an essential membrane-bound lipoprotein that is assumed to assist post-translocational folding of exported proteins and stabilize them in the compartment between the cytoplasmic membrane and cell wall. This folding activity is consistent with the homology of a segment of PrsA with parvulin-type peptidyl-prolyl cis/trans isomerases (PPIase). In this study, molecular modeling showed that the parvulin-like region can adopt a parvulin-type fold with structurally conserved active site residues. PrsA exhibits PPIase activity in a manner dependent on the parvulin-like domain. We constructed deletion, peptide insertion, and amino acid substitution mutations and demonstrated that the parvulin-like domain as well as flanking N-and C-terminal domains are essential for in vivo PrsA function in protein secretion and growth. Surprisingly, none of the predicted active site residues of the parvulin-like domain was essential for growth and protein secretion, although several active site mutations reduced or abolished the PPIase activity or the ability of PrsA to catalyze proline-limited protein folding in vitro. Our results indicate that PrsA is a PPIase, but the essential role in vivo seems to depend on some non-PPIase activity of both the parvulin-like and flanking domains.

mentioning

confidence: 74%

Structure-Function Analysis of PrsA Reveals Roles for the Parvulin-like and Flanking N- and C-terminal Domains in Protein Folding and Secretion in Bacillus subtilis

Vitikainen

Lappalainen

Seppälä

et al. 2004

118

“…Consequently, prsA mutations result in significantly reduced amounts of cell-associated and secreted mature AmyQ, but they do not result in the accumulation of pre-AmyQ in the cell (32,33). As a first approach to identify the stage in the secretion pathway in which TepA is involved, we investigated whether TepA is active before or after PrsA.…”

Section: Resultsmentioning

confidence: 99%

Signal Peptide Peptidase- and ClpP-like Proteins of Bacillus subtilis Required for Efficient Translocation and Processing of Secretory Proteins

Bolhuis¹,

Matzen

Hyyryläinen

et al. 1999

Signal peptides direct the export of secretory proteins from the cytoplasm. After processing by signal peptidase, they are degraded in the membrane and cytoplasm. The resulting fragments can have signaling functions. These observations suggest important roles for signal peptide peptidases. The present studies show that the Gram-positive eubacterium Bacillus subtilis contains two genes for proteins, denoted SppA and TepA, with similarity to the signal peptide peptidase A of Escherichia coli. Notably, TepA also shows similarity to ClpP proteases. SppA of B. subtilis was only required for efficient processing of pre-proteins under conditions of hyper-secretion. In contrast, TepA depletion had a strong effect on pre-protein translocation across the membrane and subsequent processing, not only under conditions of hyper-secretion. Unlike SppA, which is a typical membrane protein, TepA appears to have a cytosolic localization, which is consistent with the observation that TepA is involved in early stages of the secretion process. Our observations demonstrate that SppA and TepA have a role in protein secretion in B. subtilis. Based on their similarity to known proteases, it seems likely that SppA and TepA are specifically required for the degradation of proteins or (signal) peptides that are inhibitory to protein translocation.

“…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.…”

mentioning

confidence: 99%

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

Tjalsma

Bron

Dijl

2003

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 YqjGlimiting 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.