The long term goal of the research proposed in this application is to understand the mechanism of ATP-dependent active transport. An extensive biochemical and genetic analysis of the maltose transport system of E. coli is being pursued. This transport system is composed of a periplasmic maltose-binding protein, MBP; and three cytoplasmic membrane components, MalF, MalG, and MalK. These form a heterotetramer with a stoichiometry of 1F:1G:2K[FGK2], which exhibits substrate of ATP- dependent ATPase activity. The MalK subunit shares extensive sequence homology with a variety example, CFTR, which is the altered protein in cystic fibrosis and may play a role in colonization y Pseudomonas; the hylB subunit of the hemolysin exporter of E. coli; and the P-glycoprotein responsible for the multiple-drug resistance phenotype of many cancer cells.
The specific aims are to: (1) Identify regions of contact between MBP and the FGK2 complex through the use of site-directed crosslinking and the analysis of mutants in which the interaction no longer occurs. (2) Characterize the consequences of MalF and malG mutations that result in MBP-independent transport and altered transmembrane signalling. Substrate binding and ATPase activity in the presence and absence of MBP will be measured. (3) Identify substrate recognition sites in the FGK complex by photocrosslinking with a photoactive analog of maltose. In addition the nucleotide sequence alterations in altered specificity mutants in which the FGK complex has acquired the ability to transport a novel substrate, lactose will be determined. (4) Determine the relationship between the ATPase activity of the MalK subunit and its interaction with the MalF and MalG subunits by studying the effects of malK mutations that eliminate ATP binding on a duplicated malK-malK gene. In addition malK mutations that correct defects in uncoupled malF and malG mutants strains will be isolated. (5) Genetic methods will be developed for studying the physical arrangement of the FGK complex. These include the use of an artificial transposon, Tnsnip which introduces translation termination and re-start signals within a gene. The ability of N-terminal and C-terminal fragments of the MalF and MalG proteins to interfere with the activity or assembly of the FGK complex will be tested. (6) Determine the molecular basis for the negative transcriptional regulation exhibited by the MalK subunit. The C-terminal region of the MalK subunit will be expressed separately and tested for regulatory activity.
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