Direct Interaction of the EpsL and EpsM Proteins of the General Secretion Apparatus in Vibrio cholerae

Abstract

The general secretion pathway of gram-negative bacteria is responsible for extracellular secretion of a number of different proteins, including proteases and toxins. This pathway supports secretion of proteins across the cell envelope in two distinct steps, in which the second step, involving translocation through the outer membrane, is assisted by at least 13 different gene products. Two of these components, the cytoplasmic membrane proteins EpsL and EpsM ofVibrio cholerae, have been purified and characterized. Based on gel filtration analysis, both purified EpsM(His)6 and wild-type EpsL present in anEscherichia coli Triton X-100 extract are dimeric proteins. EpsL and EpsM were also found to interact directly and form a Triton X-100 stable complex that could be precipitated with either anti-EpsL or anti-EpsM antibodies. In addition, when the L and M proteins were coexpressed in E. coli, they formed a stable complex and protected each other from proteolytic degradation, indicating that these two proteins interact in vivo and that no other Eps protein is required for their association. Since EpsL is predicted to contain a large cytoplasmic domain, while EpsM is predominantly exposed on the periplasmic side, we speculate that these components might be part of a structure that is involved in bridging the inner and outer membranes. Furthermore, since EpsL has previously been shown to interact with the autophosphorylating cytoplasmic membrane protein EpsE, we hypothesize that this trimolecular complex might be involved in regulating the opening and closing of the secretion pore and/or transducing energy to the site of outer membrane translocation.

Characterization of the PilN, PilO and PilP type IVa pilus subcomplex

“…The stoichiometry of the three proteins in vivo remains to be determined. In the T2SS, the PilN and PilO homologues (GspL and GspM respectively) have been reported to form homodimers, heterodimers and heterodimers of homodimers (Sandkvist et al, 1999;Py et al, 2001;Robert et al, 2002;Abendroth et al, 2004). In vitro we found evidence that the PilN/PilO/PilP proteins exist in either a 1:1:1 or a 2:2:2 complex.…”

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

“…Recent studies have proposed that XcpP and its homologues are involved in interactions with the inner membrane platform of the secreton and, more particularly, with the XcpY-XcpZ components or their homologues (GspL-GspM families) (Possot et al, 2000;Py et al, 2001;Robert et al, 2002). It is know that XcpZ stabilizes XcpY in a pairwise combination and such an observation has also been made in other organisms (Sandkvist et al, 1999). Recently, Robert and collaborators identified XcpZ derivatives that are not able to confer XcpY stability when co-produced in E. coli, thus in the absence of the other Xcp components (Robert et al, 2002).…”

Biology of type II secretion

“…When OutL was analysed in the yeast two-hybrid system, it was also found to oligomerize (Py et al, 1999). Finally, EpsL and EpsM each migrated as dimers during gel filtration analysis (Sandkvist et al, 1999).…”