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

摘要

The objective of this study examine structural mechanisms for proton transport and membrane voltage interactions by the plasma membrane H+ -ATPase from Saccharomyces cerevisiae. H+ -ATPase mutants (pmal), generated by random and site directed mutagenesis techniques, have been isolated that cause depolarization of the cellular membrane potential. 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. All pmal mutant enzymes were active in proton transport, although one mutant, Gly158-->Asp, appeared to be partially uncoupled from ATP hydrolysis. The locus causing the most severe effect on membrane potential. Ser368, was subjected a detailed revertant and site-directed mutagenesis analysis. Amino acid substitutions of the, Val or Leu resulted in membrane potential depolarizations. Finally, to examine the effects of membrane voltage on mutant enzymes, a new procedure was developed to produce large and sustained membrane potentials in reconstituted proteoloposomes. In the next year, transport and to study the voltage dependence of proton transport and ATP hydrolysis in existing and new mutant enzymes. (KT)