Evidence for coupling between membrane and cytoplasmic domains of the yeast plasma membrane H+-ATPase: An analysis of intragenic revertants of pma1-105

S. L. Harris, D. S. Perlin, D. Seto-Young, J. E. Haber

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Abstract

A genetic approach was used to identify interacting portions of the plasma membrane H+-ATPase from Saccharomyces cerevisiae. The cellular sensitivity of the pma1-105 strain (S368F) to low external pH and to NH4+ was used to select intragenic revertants of two classes: phenotypically wild-type full revertants and partial revertants that were low pH-resistant but retained resistance to hygromycin B. All 10 full revertants had S368 restored. Among five partial revertants mapping to the original site within the phosphorylation domain, S368L and S368V were each found twice. One revertant contained an E367V substitution adjacent to the original S368F alteration. Four of 13 independently isolated second-site revertants mapped to one site, V289F, in the proposed phosphatase domain. Mutations within the proposed phosphatase and phosphorylation domains resulted in enzymes with increased vanadate sensitivity relative to the vanadate-insensitive S368F enzyme. These results suggest that sites S368, E367, and V289 contribute to a vanadate (P(i)) binding domain or are able to interact with such a site within the catalytic domain. The remaining nine partial second-site revertants mapped to six sites within the putative transmembrane regions. Mutations within the transmembrane region had less of an effect on vanadate sensitivity. Most revertant enzymes showed small but significant increases in the rate of ATP hydrolysis relative to the S368F enzyme. Several enzymes no longer displayed the acid-sensitive pH-dependence seen in the S368F enzyme. These data provide novel evidence for an interaction between putative transmembrane helices 1-3 and 7 and the ATP hydrolytic portion of the enzyme.

Original languageEnglish
Pages (from-to)24439-24445
Number of pages7
JournalJournal of Biological Chemistry
Volume266
Issue number36
StatePublished - 1991
Externally publishedYes

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