An ExeAB complex in the type II secretion pathway of Aeromonas hydrophila: effect of ATP-binding cassette mutations on complex formation and function.
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.
Department of Biology, University of Regina, Saskatchewan, Canada.
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Schoenhofen
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Stratilo
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Howard
D000255:Adenosine Triphosphate / Q000235:genetics / Q000378:metabolism*; D016980:Aeromonas hydrophila / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism*; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents; D019281:Dimerization; D010450:Endopeptidases / Q000378:metabolism; D004795:Enzyme Stability; D006460:Hemolysin Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D052899:Pore Forming Cytotoxic Proteins; D011233:Precipitin Tests; D011494:Protein Kinases / Q000378:metabolism; D012995:Solubility
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; C090402:ExeA protein, Aeromonas hydrophila; C090403:ExeB protein, Aeromonas hydrophila; D006460:Hemolysin Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D052899:Pore Forming Cytotoxic Proteins; C032676:aerolysin; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D010450:Endopeptidases