Premium
An ExeAB complex in the type II secretion pathway of Aeromonas hydrophila : effect of ATP‐binding cassette mutations on complex formation and function
Author(s) -
Schoenhofen Ian C.,
Stratilo Chad,
Howard S. Peter
Publication year - 1998
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.1998.01011.x
Subject(s) - aerolysin , biology , secretion , aeromonas hydrophila , mutant , immunoprecipitation , biochemistry , wild type , type three secretion system , secretory protein , microbiology and biotechnology , gene , genetics , bacteria
The energy‐dependent secretion of aerolysin by Aeromonas hydrophila requires the ExeA and ExeB proteins. An 85 kDa complex containing the two proteins was identified in wild‐type cells but not in cells producing either protein alone. Radiolabelling followed by cross‐linking, immunoprecipitation and then reduction of the cross‐links confirmed the presence of the two proteins in the same complex. The complex could also be extracted intact from cell membranes with non‐ionic detergents. A G229D substitution in the kinase‐3a motif of ExeA strongly reduced the level of aerolysin secretion, as did the replacement of the invariant Lys of the kinase‐1a motif (K56) with Arg. The G229D mutant contained very little of the ExeA–ExeB complex, but overexpression of the mutant complex until wild‐type levels were achieved allowed normal secretion. In contrast, the K56R mutation had no effect on complex formation, but normal secretion levels occurred only when there was a far greater amount of the complex present. These results are consistent with a model in which binding of ATP by ExeA is required for ExeA–ExeB complex formation, while hydrolysis is required for its function in secretion once established.