Subcomplexes from the Xcp secretion system ofPseudomonas aeruginosa

Abstract

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.

Interaction domains in the Pseudomonas aeruginosa type II secretory apparatus component XcpS (GspF)

“…However, we cannot exclude a direct interaction between XcpS and XcpZ. The existence of an XcpRSYZ subcomplex is in agreement with a recent publication showing the co-purification of XcpRSY with His-tagged XcpZ after cross-linking (Robert et al, 2005). It should be stressed that XcpS production was substantially higher from a construct carrying xcpR-Z than upon co-expression of only xcpRYZ.…”

“…Knowledge of the role of XcpS in the secreton and its interactions with other Xcp components is rather limited. Recently, the components XcpR, S and Y were shown to co-purify with his-tagged XcpZ (GspM) after cross-linking (Robert et al, 2005), and yeast twohybrid studies with Erw. chrysanthemi T2SS components revealed interactions of the N terminus of OutF, the XcpS homologue, with OutE, the XcpR homologue, and with the cytoplasmic segment of OutL, the XcpY homologue (Py et al, 2001).…”

Cysteine Scanning Mutagenesis and Disulfide Mapping Analysis of Arrangement of GspC and GspD Protomers within the Type 2 Secretion System

“…The T2SS spans the two bacterial membranes and ensures secretion of folded proteins across the outer membrane pore formed by GspD. GspC, GspL, GspM, and GspF constitute together the inner membrane complex (3)(4)(5)(6). The cytoplasmic domains of GspL and GspF interact with an ATPase, GspE.…”

Polar secretion of proteins via the Xcp type II secretion system in Pseudomonas aeruginosa

“…Ball et al (1999) have shown that the XcpY protein is necessary and sufficient for the association of XcpR with the inner membrane, making XcpY the most likely candidate to recruit XcpR to the pole. Since XcpY and XcpR interact with XcpS (Robert et al, 2005b), the XcpS protein may be important to retain XcpY and XcpR to the pole. However, the instability of XcpS in the absence of other Xcp components (Arts et al, 2007) makes it unlikely that XcpS acts as a polar nucleation factor for the other Xcp components.…”

“…These pseudopilins are produced with a leader peptide, which is cleaved off by the prepilin peptidase XcpA (GspO) (Bally et al, 1992;Bleves et al, 1998;Nunn & Lory, 1992). The energy that is required for assembly of the pseudopilus and the extrusion of substrates is in all probability generated in the cytoplasm by the ATPase XcpR (GspE) (Camberg & Sandkvist, 2005;Robien et al, 2003), which is part of the inner-membrane platform further consisting of the integral inner-membrane proteins XcpY (GspL), XcpZ (GspM) and XcpS (GspF) (Py et al, 2001;Robert et al, 2005b). XcpP (GspC) is thought to form a bridge between the secretin and the inner-membrane platform Gérard-Vincent et al, 2002;Robert et al, 2005a).…”