In recent years, a family of membrane translocators that mediate the transport of diverse substrates has been described. Termed the ATP-binding cassette (ABC) superfamily, this group of transporters has representatives in both prokaryotes and eukaryotes. Medically significant representatives include the multi-drug resistance protein (Mdr), which exports chemotherapeutic drugs from mammalian tumor cells, and the cystic fibrosis transmembrane regulator, which is defective in cystic fibrosis patients. The similarities among the ABC transporters suggest a common ancestry and function. The proposed experiments investigate one member of this transport group, an exporter that acts to secrete the peptide antibiotic colicin V from E. coli cells. The long-term goal is to elucidate the mechanism of colicin V transport in order to increase understanding of bacterial ABC export systems and ABC transport in general. The specific goal is to identify through genetic analysis protein interactions among the components of the colicin V pathway. These studies will use the two-hybrid system to detect interactions between the substrate and the individual export proteins and among the export proteins. Specific protein regions that mediate these interactions will also be identified. Mutations will be introduced into interacting domains to determine whether abolished or weakened interaction correlates with loss of function. In other experiments, mutations in the structural gene for the substrate that cause export deficiencies will be characterized in order to identify specific amino acid residues that are recognized by the transporter. Suppressor mutations in the genes encoding the transport proteins will then be selected in order to identify regions of these proteins that interact with the substrate during translocation. Results from these experiments will help to decipher the specific functions of individual components as well as to determine the structure of the overall apparatus, thus shedding light on the mechanism of translocation.
