ENERGY COUPLING BY THE YEAST PLASMA MEMBRANE-ATPASE

Project Details

Description

The long term objective of this project is to understand the
physical mechanism for energy coupling by ion-translocating
ATPases. For the immediate proposal, the proton-ATPase of
yeast plasma membranes will be probed biochemically and
genetically to determine which protein domains are involved with
coupling proton transport to ATP hydrolysis. The yeast plasma
membrane-ATPase closely resembles in structure and function the
Na+, K+-ATPase and Ca2+-ATPase of higher eukaryotes and
shares some sequence homology with these enzymes. A working hypothesis will be examined in which a local
conformational change acts as the linkage between ATP
hydrolysis and proton transport. This project will examine 1) the topography of the enzyme in the
bilayer by correlating hydropathy profiles determined from the
primary amino acid sequence with data from mapping studies
using hydrophilic and hydrophobic probes; 2) localized
conformational states of enzyme stopped at different stages of
turnover by evaluating peptide profiles following limited
proteolysis and protein cross-linking; 3) expression of the plasma
membrane-ATPase gene in yeast and E. coli; 4) uncoupled ATPase
mutants produced by localized random mutagenesis and 5)
uncoupled enzymes produced by protein cross-linking and limited
proteolysis. Biochemical methods will include protein labelling with
photoactivatable probes, gel filtration, preparative gel
electrophoresis, CNBr-cleavage, HPLC separation, amino acid
analysis and micro-sequencing. Genetic methods will include large-scale- and mini-plasmid
preparations, DNA fragment isolation, agarose gel
electrophoresis, plasmid subcloning, yeast and bacterial
transformation and DNA sequencing.
StatusFinished
Effective start/end date4/1/8711/30/04

Funding

  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $386,356.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $409,611.00
  • National Institutes of Health: $397,813.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $421,762.00
  • National Institutes of Health
  • National Institutes of Health

ASJC

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)

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