Characterization of a temperature-sensitive yeast vacuolar ATPase mutant with defects in actin distribution and bud morphology

Abstract

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 (vma4-1(ts)) 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 vma4-1(ts) allele. The unassembled 27kDa subunit made in the vma4-1(ts) 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 vma4-1(ts) 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.