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

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 degreesC). Sequencing revealed that a single point mutation, D145G, was responsible for the phenotypes of the vma4-1(ts) allele. The unassembled 27-kDa subunit made in the vma4-1(ts) cells is rapidly degraded, particularly at 37 degreesC, 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 degrees 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.