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Characterization of yeast V-ATPase mutants lacking Vph1p or Stv1p and the effect on endocytosis
Department of Biochemistry, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel
* Author for correspondence (e-mail: nelson{at}post.tau.ac.il )
Accepted 11 February 2002
Subunit a of V-ATPase in the yeast Saccharomyces cerevisiae, in
contrast to its other subunits, is encoded by two genes VPH1 and
STV1. While disruption of any other gene encoding the V-ATPase
subunits results in growth arrest at pH 7.5, null mutants of Vph1p or Stv1p
can grow at this pH. We used a polyclonal antibody to yeast Stv1p and a
commercially available monoclonal antibody to Vph1p for analysis of yeast
membranes by sucrose gradient fractionation, and two different vital dyes to
characterize the phenotype of vph1 
and stv1
mutants as compared to the double mutant and the wild-type cells.
Immunological assays of sucrose gradient fractions revealed that the amount of
Stv1p was elevated in the vph1 strain, and that vacuoles
purified by this method with no detectable endosomal contamination contain an
assembled V-ATPase complex, but with much lower activity than the wild type.
These results suggest that Stv1p compensates for the loss of Vph1p in the
vph1 strain. LysoSensor Green DND-189 was used as a pH sensor
to demonstrate unexpected changes in vacuolar acidification in stv1
as the Vph1p-containing V-ATPase complex is commonly considered to
acidify the vacuoles. In the vph1 strain, the dye revealed
slight but definite acidification of the vacuole as well. The lipophilic dye
FM4-64 was used as an endocytic marker. We show that the null V-ATPase
mutants, as well as the vph1 one, markedly slow down
endocytosis of the dye.
Key words: V-ATPase, subunit a, yeast, Saccharomyces cerevisiae, biogenesis, endocytosis, proton pumping
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