Biomedical subjects

Viviane Robert

Publications and source records attributed to Viviane Robert.

5 recordsLinked to original sources

PubMed2006

Assembly factor Omp85 recognizes its outer membrane protein substrates by a species-specific C-terminal motif.

Integral beta-barrel proteins are found in the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. The assembly of these proteins requires a proteinaceous apparatus of which Omp85 is an evolutionary conserved central component. To study its molecular mechanism, we have produced Omp85 from Escherichia coli in inclusion bodies and refolded it in vitro. The interaction of Omp85 with its substrate proteins was studied in lipid-bilayer experiments, where it formed channels. The properties of these channels were affected upon addition of unfolded outer-membrane proteins (OMPs) or synthetic peptides corresponding to their C-terminal signature sequences. The interaction exhibited species specificity, explaining the inefficient assembly of OMPs from Neisseria in E. coli. Accordingly, the in vivo assembly of the neisserial porin PorA into the E. coli outer membrane was accomplished after adapting its signature sequence. These results demonstrate that the Omp85 assembly machinery recognizes OMPs by virtue of their C-terminal signature sequence.

Amino Acid Motifs↗

PubMed2005

Subcomplexes from the Xcp secretion system of Pseudomonas aeruginosa.

In Gram-negative bacteria, most of the sec-dependent exoproteins are secreted via the type II secretion system (T2SS or secreton). In Pseudomonas aeruginosa, T2SS consists of 12 Xcp proteins (XcpA and XcpP to XcpZ) organized as a multiproteic complex within the envelope. In this study, by a co-purification approach using a His-tagged XcpZ as a bait, XcpY and XcpZ were found associated together to constitute the most stable functional unit so far isolated from the P. aeruginosa secreton. This subcomplex was also found to interact with XcpR and XcpS to form a XcpRSYZ complex which was isolated under native conditions. Another component, XcpP was not found to be associated to the complex but the results suggest that it can transiently interact with the XcpYZ subcomplex in vivo.

Bacterial Proteins↗

PubMed2005

Role of XcpP in the functionality of the Pseudomonas aeruginosa secreton.

In gram-negative bacteria, most signal-peptide-dependent exoproteins are secreted via the type II secretion system (T2SS or secreton). In Pseudomonas aeruginosa, T2SS consists of twelve Xcp proteins (XcpA and XcpP to XcpZ) thought to be organized as a multiproteic complex within the envelope. Although well conserved, T2SS are known to be species-specific, namely for distant organisms, and this characteristic was thought to involve XcpP. To check which domain of XcpP could be involved in the species specificity, hybrid proteins were generated using protein domain swapping between P. aeruginosa XcpP and homolog proteins of either Erwinia chrysanthemi or Pseudomonas alcaligenes. The results obtained with hybrid proteins constructed by exchanging the C-terminal domains of P. aeruginosa and E. chrysanthemi suggested that XcpP interacts with XcpQ, probably via its C-terminal domain. More interestingly, the data obtained with a hybrid protein containing the C-terminal part of the P. alcaligenes XcpP homolog, showed that the wild-type C-terminal end plays a very important role in the function of the protein and is required both for a correct interaction with XcpQ and for modulating the opening of the secreton channel.

Bacterial Proteins↗

PubMed2002

Identification of XcpP domains that confer functionality and specificity to the Pseudomonas aeruginosa type II secretion apparatus.

Gram-negative bacteria have evolved several types of secretion mechanisms to release proteins into the extracellular medium. One such mechanism, the type II secretory system, is a widely conserved two-step process. The first step is the translocation of signal peptide-bearing exoproteins across the inner membrane. The second step, the translocation across the outer membrane, involves the type II secretory apparatus or secreton. The secretons are made up of 12-15 proteins (Gsp) depending on the organism. Even though the systems are conserved, heterologous secretion is mostly species restricted. Moreover, components of the secreton are not systematically exchangeable, especially with distantly related microorganisms. In closely related species, two components, the GspC and GspD (secretin) family members, confer specificity for substrate recognition and/or secreton assembly. We used Pseudomonas aeruginosa as a model organism to determine which domains of XcpP (GspC member) are involved in specificity. By constructing hybrids between XcpP and OutC, the Erwinia chrysanthemi homologue, we identified a region of 35 residues that was not exchangeable. We showed that this region might influence the stability of the XcpYZ secreton subcomplex. Remarkably, XcpP and OutC have domains, coiled-coil and PDZ, respectively, which exhibit the same function but that are structurally different. Those two domains are exchangeable and we provided evidence that they are involved in the formation of homomultimeric complexes of XcpP.

Bacterial Outer Membrane Proteins↗

PubMed2002

Identification of XcpZ domains required for assembly of the secreton of Pseudomonas aeruginosa.

Most of the exoproteins secreted by Pseudomonas aeruginosa are transported via the type II secretion system. This machinery, which is widely conserved in gram-negative bacteria, consists of 12 Xcp proteins organized as a multiprotein complex, also called the secreton. We previously reported that the mutual stabilization of XcpZ and XcpY plays an important role in the assembly of the secreton. In this study, we engineered variant XcpZ proteins by using linker insertion mutagenesis. We identified three distinct regions of XcpZ required for both the stabilization of XcpY and the functionality of the secreton. Interestingly, we also demonstrated that another component of the machinery, XcpP, can modulate the stabilizing activity of XcpZ on XcpY.

Amino Acid Sequence↗