Colicin V is one of the few proteins that are secreted out of Escherichia coli cells. The excretion of Colicin V requires the products of two accessory genes, cvaA and cvaB. The CvaB is a member of homologous membrane proteins that are involved in the energy-dependent transport of various kinds of molecules, including proteins, peptides, drugs, ions and polysaccharides. The homologies of these proteins are characterized by the presence of several potential membrane-spanning regions and a highly conserved hydrophilic ATP-binding domain, and may share a common mechanism for transporting different molecules. The objective of this project is to understand the mechanism of protein export via the accessory protein- specified pathway, which may differ markedly from the general SecA/SecY export pathway.
The aim i s to elucidate biochemically the molecular mechanism of this export pathway to complement concurrent genetic studies. We will develop an in vitro system for the specific translocation of Colicin V, which has an atypical N-terminal targeting sequence that is cleaved during export. The precursor of Colicin V will be synthesized in a T7-polymerase-directed transcription/translation system. It is anticipated from genetic studies that the active system will require the products of cvaA and cvaB genes. The roles of the CvaA and CvaB proteins will be assessed by using mutants devoid of one or more of these accessory proteins, and by using antibodies against specific regions of the peptides. The ATP-binding domain of CvaB that shares extensive homology with that of P-glycoprotein will be labeled by ATP analogs and changed by site-specific mutagenesis and its role in CvaB function will be examined. We will determine the requirements for the translocation and assess whether SecA, SecY/PrlA, general signal peptidases and ATP are involved in the process. For comparison to the requirements of general protein translocation, we will use precursors of fusions of N-terminal CvaC targeting sequence 39 amino acid residues) to the mature region of alkaline phosphatase and/or OmpA. Chemical crosslinking with bifunctional reagents, followed by immunoprecipitation, and partial solubilization of membrane will be used to characterize the accessory protein-specified export pathway. Membrane vesicles will be used to test whether Colicin V export system is capable of carrying out energy-dependent drug export. These studies should elucidate the mechanism of CvaB-mediated Colicin V transport, and may be adapted to explore the mechanism of multi-drug resistance.
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