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J Biol Chem, Vol. 273, Issue 29, 18470-18480, July 17, 1998
Characterization of a Temperature-sensitive Yeast Vacuolar ATPase
Mutant with Defects in Actin Distribution and Bud Morphology
Jing Wei
Zhang,
Karlett J.
Parra,
Jianzhong
Liu, and
Patricia M.
Kane
From the Department of Biochemistry & Molecular Biology, State
University of New York, Health Science Center at Syracuse,
Syracuse, New York 13210
The 27-kDa E subunit, encoded by the
VMA4 gene, is a peripheral membrane subunit of the yeast
vacuolar H+-ATPase. We have randomly mutagenized the
VMA4 gene in order to examine the structure and function of
the 27-kDa subunit. Cells lacking a functional VMA4 gene
are unable to grow at pH > 7 or in elevated concentrations of
CaCl2. Plasmid-borne, mutagenized vma4 genes
were screened for failure to complement these phenotypes. Mutants
producing Vma4 proteins detectable by immunoblot were selected; one
(vma4-1ts) is temperature conditional, exhibiting
the Vma phenotype only at elevated temperature
(37 °C). Sequencing revealed that a single point mutation, D145G,
was responsible for the phenotypes of the
vma4-1ts allele. The unassembled
27-kDa subunit made in the vma4-1ts
cells is rapidly degraded, particularly at 37 °C, but can be protected from degradation by prior assembly into the V-ATPase complex.
In purified vacuolar vesicles from the mutant cells, the peripheral
subunits are localized to the vacuolar membrane at decreased levels and
a comparably decreased level of ATPase activity (14% of the activity
in wild-type vesicles) is observed. When vma4-1ts
mutant cells are shifted to pH 7.5 medium at 37 °C, the cells become
enlarged and exhibit multiple large buds, elongated buds, and other
abnormal morphologies, together with delocalization of actin and
chitin, within 4 h. These phenotypes suggest connections between
the vacuolar ATPase, bud morphology, and cytokinesis that had not been
recognized previously.
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.

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Copyright © 1998 by the American Society for Biochemistry and Molecular Biology.
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