Membrane Voltage Effects on Proton Transport by a Yeast H+ -ATPase.

摘要

The objective of this two year project was to identify protein structure domains participating in proton transport and membrane voltage interactions by the plasma membrane H(+)-ATPase from Saccharomyces cerevisiae. H+-ATPase mutants (pmal) were generated by random and site-directed mutagensis techniques that cause a deploarization of the cellular membrane potential. All pmal mutant enzymes were active in proton transport although one mutant Gly158--Asp, appeared to be partially uncoupled from ATP hydrolysis. Three loci, one within a putative transmembrane domain (GLY158) and the other two (Ser368, Pro640) within putative membrane/cytoplasmic interface domains, were found to cause the most prominent effect on cellular membrane potential. To better understand how membrane voltage effects the H(+)-ATPase, a new procedure was developed to produce large and sustained membrane potentials in reconstituted proteoliposomes. The results of this project will serve as a foundation for probing electrogenic proton transport by the H(+)-ATPase with the eventual goal of developing a structural model for ion translocation. Keywords: Cations, Phosphorus Hydrolases. (AW)