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J. Biol. Chem., Vol. 266, Issue 36, 24439-24445, Dec, 1991
SL Harris, DS Perlin, D Seto-Young and JE Haber
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 (Pi) 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.
Evidence for coupling between membrane and cytoplasmic domains of the yeast plasma membrane H(+)-ATPase. An analysis of intragenic revertants of pma1-105
Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02254.
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