Biomedical subjects

Jörg P Müller

Publications and source records attributed to Jörg P Müller.

9 recordsLinked to original sources

PubMed2006

Affinity of TatCd for TatAd elucidates its receptor function in the Bacillus subtilis twin arginine translocation (Tat) translocase system.

Twin arginine translocation (Tat) systems catalyze the transport of folded proteins across the bacterial cytosolic membrane or the chloroplast thylakoid membrane. In the Tat systems of Escherichia coli and many other species TatA-, TatB-, and TatC-like proteins have been identified as essential translocase components. In contrast, the Bacillus subtilis phosphodiesterase PhoD-specific system consists only of a pair of TatA(d)/TatC(d) proteins and involves a TatA(d) protein engaged in a cytosolic and a membrane-embedded localization. Because soluble TatA(d) was able to bind the twin arginine signal peptide of prePhoD prior to membrane integration it could serve to recruit its substrate to the membrane via the interaction with TatC(d). By analyzing the distribution of TatA(d) and studying the mutual affinity with TatC(d) we have shown here that TatC(d) assists the membrane localization of TatA(d). Besides detergent-solubilized TatC(d), membrane-integrated TatC(d) showed affinity for soluble TatA(d). By using a peptide library-specific binding of TatA(d) to cytosolic loops of membrane protein TatC(d) was demonstrated. Depletion of TatC(d) in B. subtilis resulted in a drastic reduction of TatA(d), indicating a stabilizing effect of TatC(d) for TatA(d). In addition, the presence of the substrate prePhoD was the prerequisite for appropriate localization in the cytosolic membrane of B. subtilis as demonstrated by freeze-fracture experiments.

Amino Acid Sequence↗

PubMed2006

The TatAd component of the Bacillus subtilis twin-arginine protein transport system forms homo-multimeric complexes in its cytosolic and membrane embedded localisation.

The twin arginine translocation (Tat) system has the capacity to transfer completely folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. The most abundant TatA protein of this system has been suggested to form the protein conducting channel. Here, the molecular organisation of soluble and membrane embedded Bacillus subtilis TatAd was analysed using negative contrast and freeze-fractured electron microscopy. In both compartments, the protein showed homo-oligomerisation. In aqueous solution, TatAd formed homo-multimeric micelle-like complexes. Freeze-fracture analysis of proteoliposomes revealed self association of membrane-integrated TatAd independent from TatCd, the second component of this transport system. Immunogold labelling demonstrated that the substrate prePhoD was co-localised with membrane-integrated TatAd complexes.

Bacillus subtilis↗

PubMed2004

Tat dependent export of E. coli phytase AppA by using the PhoD-specific transport system of Bacillus subtilis.

It has been shown recently that the twin-arginine signal peptide of Bacillus subtilis phosphodiesterase PhoD (SPPhoD) can mediate Tat dependent transport of proteins via its specific Tat-transport components. In order to test the use of Tat dependent transport signals for heterologous product synthesis, Escherichia coli phytase AppA was expressed under control of PhoD-specific export signals in B. subtilis. Induction of Tat components TatAd/TatCd was mediated by using a functionally altered PhoR/PhoP signal transduction system which regulates the expression of these components. AppA was highly susceptible to host specific extracellular proteases. Expression of appA in B. subtilis wprA strain resulted in the stable production of AppA. A fusion protein consisting of SPPhoD and mature AppA remained unprocessed, while introduction of the AppA signal peptidase cleavage site resulted in efficient processing of the fusion protein.

6-Phytase↗

PubMed2004

Subcellular sites for bacterial protein export.

Most bacterial proteins destined to leave the cytoplasm are exported to extracellular compartments or imported into the cytoplasmic membrane via the highly conserved SecA-YEG pathway. In the present studies, the subcellular distributions of core components of this pathway, SecA and SecY, and of the secretory protein pre-AmyQ, were analysed using green fluorescent protein fusions, immunostaining and/or immunogold labelling techniques. It is shown that SecA, SecY and (pre-)AmyQ are located at specific sites near and/or in the cytoplasmic membrane of Bacillus subtilis. The localization patterns of these proteins suggest that the Sec machinery is organized in spiral-like structures along the cell, with most of the translocases organized in specific clusters along these structures. However, this localization appears to be independent of the helicoidal structures formed by the actin-like cytoskeletal proteins, MreB or Mbl. Interestingly, the specific localization of SecA is dynamic, and depends on active translation. Moreover, reducing the phosphatidylglycerol phospholipids content in the bacterial membrane results in delocalization of SecA, suggesting the involvement of membrane phospholipids in the localization process. These data show for the first time that, in contrast to the recently reported uni-ExPortal site in the coccoïd Streptococcus pyogenes, multiple sites dedicated to protein export are present in the cytoplasmic membrane of rod-shaped B. subtilis.

Adenosine Triphosphatases↗

PubMed2003

Characterisation of preYvaY export reveals differences in the substrate specificities of Bacillus subtilis and Escherichia coli leader peptidases.

Translocation, processing and secretion of YvaY, a Bacillus subtilis protein of unknown function, were characterised both in B. subtilis and in Escherichia coli. In its natural host B. subtilis, YvaY was transiently synthesised at the end of the exponential growth phase. It was efficiently secreted into the culture supernatant in spite of a calculated membrane spanning domain in the mature part of the protein. In E. coli, despite the high conservation of Sec-dependent transport components, processing of preYvaY was strongly impaired. To uncover which elements of E. coli and B. subtilis translocation systems are responsible for the observed substrate specificity, components of the B. subtilis Sec-system were co-expressed besides yvaY in E. coli. Expression of B. subtilis secA or secYEG genes did not affect processing, but expression of B. subtilis signal peptidase genes significantly enhanced processing of preYvaY in E. coli. While the major signal peptidases SipS or SipT had a strong stimulatory effect on preYvaY processing, the minor signal peptidases SipU, SipV or SipW had a far less stimulatory effect in E. coli. These results reveal that targeting and translocation of preYvaY is mediated by the E. coli Sec proteins but processing of preYvaY is not performed by E. coli signal peptidase LepB. Thus, differences in substrate specificities of E. coli LepB and the B. subtilis Sip proteins provide the bottleneck for export of YvaY in E. coli. Significant slower processing of preYvaY in absence of SecB indicated that SecB mediates targeting of the B. subtilis precursor.

Bacillus subtilis↗

PubMed2003

Interaction of the Bacillus subtilis chaperone CsaA with the secretory protein YvaY.

Bacillus subtilis CsaA was previously characterised as a molecular chaperone with export-related activities. In order to elucidate the functionality of CsaA further, interaction with its postulated substrate YvaY was investigated. Similar binding to carrier immobilised mature and preYvaY revealed that the interaction was not mediated via the signal peptide of preYvaY. Higher affinity to denatured peptides compared to native peptides indicated preferred binding to unfolded proteins. To characterise affinity of CsaA more detailed, binding to preYvaY derived peptides was analysed. CsaA showed affinity to multiple peptides in the scan, mainly correlated to a positive net charge. Affinity of export-specific Escherichia coli chaperone SecB to the carrier immobilised peptides indicated partially overlapping binding characteristics of SecB and CsaA.

Bacillus subtilis↗

PubMed2003

Sequence-specific binding of prePhoD to soluble TatAd indicates protein-mediated targeting of the Tat export in Bacillus subtilis.

The Tat (twin-arginine protein translocation) system initially discovered in the thylakoid membrane of chloroplasts has been described recently for a variety of eubacterial organisms. Although in Escherichia coli four Tat proteins with calculated membrane spanning domains have been demonstrated to mediate Tat-dependent transport, a specific transport system for twin-arginine signal peptide containing phosphodiesterase PhoD of Bacillus subtilis consists of one TatA/TatC (TatAd/TatCd) pair of proteins. Here, we show that TatAd was found beside its membrane-integrated localization in the cytosol were it interacted with prePhoD. prePhoD was efficiently co-immunoprecipitated by TatAd. Inefficient co-immunoprecipitation of mature PhoD and missing interaction to Sec-dependent and cytosolic peptides by TatAd demonstrated a particular role of the twin-arginine signal peptide for this interaction. Affinity of prePhoD to TatAd was interfered by peptides containing the twin-arginine motif but remained active when the arginine residues were substituted. The selective binding of TatAd to peptides derived from the signal peptide of PhoD elucidated the function of the twin-arginine motif as a target site for pre-protein TatAd interaction. Substitution of the binding motif demonstrated the pivotal role of basic amino acid residues for TatA binding. These features suggest that TatA interacts prior to membrane integration with its pre-protein substrate and could therefore assist targeting of twin-arginine pre-proteins.

Amino Acid Motifs↗

PubMed2001

The twin-arginine signal peptide of PhoD and the TatAd/Cd proteins of Bacillus subtilis form an autonomous Tat translocation system.

The bacterial twin-arginine translocation (Tat) pathway has been recently described for PhoD of Bacillus subtilis, a phosphodiesterase containing a twin-arginine signal peptide. The expression of phoD is co-regulated with the expression of tatA(d) and tatC(d) genes localized downstream of phoD. To characterize the specificity of PhoD transport further, translocation of PhoD was investigated in Escherichia coli. By using gene fusions, we analyzed the particular role of the signal peptide and the mature region of PhoD in canalizing the transport route. A hybrid protein consisting of the signal peptide of beta-lactamase and mature PhoD was transported in a Sec-dependent manner indicating that the mature part of PhoD does not contain information canalizing the selected translocation route. Pre-PhoD, as well as a fusion protein consisting of the signal peptide of PhoD (SP(PhoD)) and beta-galactosidase (LacZ), remained cytosolic in the E. coli. Thus, SP(PhoD) is not recognized by E. coli transport systems. Co-expression of B. subtilis tatA(d)/C(d) genes resulted in the processing of SP(PhoD)-LacZ and periplasmic localization of LacZ illustrating a close substrate specificity of the TatA(d)/C(d) transport system. While blockage of the Sec-dependent transport did not affect the localization of SP(PhoD)-LacZ, translocation and processing was dependent on the pH gradient of the cytosolic membrane. Thus, the minimal requirement of a functional Tat-dependent protein translocation system consists of a twin-arginine signal peptide-containing Tat substrate, its specific TatA/C proteins, and the pH gradient across the cytosolic membrane.

Arginine↗

PubMed2000

Chaperone-like activities of the CsaA protein of Bacillus subtilis.

The growth and protein export defects of Escherichia coli secA51(Ts) strains can be suppressed by the CsaA protein of Bacillus subtilis. The present studies indicate that this effect can be attributed to chaperone-like activities of CsaA. First, CsaA stimulated protein export in secB, groES and dnaJ mutant strains of E. coli. Second, CsaA suppressed the growth defects of dnaK, dnaJ and grpE mutants of E. coli. Third, and most importantly, CsaA exhibited chaperone-like properties by stimulating the reactivation of heat-denatured firefly luciferase in groEL, groES, dnaK and grpE mutant strains of E. coli, and by preventing the aggregation of heat-denatured luciferase in vitro. Thus, it seems that CsaA suppresses the growth and secretion defects of E. coli secA(Ts) strains either by improving the translocation competence of exported pre-proteins, thereby making them better substrates for mutant SecA proteins, or by stimulating the translocation activity of mutant SecA proteins.

Adenosine Triphosphatases↗