MUTATIONAL ANALYSIS OF VAM4 YPT7P FUNCTION IN THE VACUOLAR BIOGENESISAND MORPHOGENESIS IN THE YEAST, SACCHAROMYCES-CEREVISIAE/

The vacuole is one of the most prominent compartments in yeast cells. The wild-type yeast cells have a large vacuolar compartment which occu pies approximately a quarter of the cell volume, while the vam4 mutant cells exhibit highly fragmented vacuolar morphology. We isolated the VAM4 gene and found that the VAM4 is identical to the YPT7 which encod es a member of small GTP-binding protein superfamily. We introduced mu tations to the VAM4/YPT7 which alter nucleotide binding characteristic s of the gene product specifically, and their activities for the vacuo lar morphogenesis were examined by transforming the mutant genes into yeast cells. The Thr22Asn mutation, which was expected to fix the prot ein in the GDP-bound state, resulted in loss of function in the vacuol ar morphogenesis. Subcellular fractionation analysis indicated that th e mutant molecule did nor associate with intracellular membranes effic iently. In contrast, Vam4/Ypt7p with the Gln68Leu mutation, which was expected to be the GTP-bound form, complemented the fragmented vacuola r morphology of Delta vam4 mutant cells. Vam4/Ypt7p with the Gln68Leu mutation also complemented the defects in the biogenesis of vacuolar a lkaline phosphatase whose maturation requires the proper function of V am4/Ypt7p. Overexpression of the mutant proteins in wild-type cells di d not develop dominant-negative effects on the vacuolar assembly. Thes e results indicated that the GTP-bound form of Vam4/Ypt7p promotes the biogenesis and morphogenesis of the yeast vacuolar compartment.