Regulation of Fab1 Phosphatidylinositol 3-Phosphate 5-Kinase Pathway by Vac7 Protein and Fig4, a Polyphosphoinositide Phosphatase Family Member

Abstract

The Saccharomyces cerevisiae FAB1 gene encodes the sole phosphatidylinositol 3-phosphate [PtdIns(3)P] 5-kinase responsible for synthesis of the polyphosphoinositide PtdIns(3,5)P₂. VAC7 encodes a 128-kDa transmembrane protein that localizes to vacuolar membranes. Bothvac7 and fab1 null mutants have dramatically enlarged vacuoles and cannot grow at elevated temperatures. Additionally, vac7Δ mutants have nearly undetectable levels of PtdIns(3,5)P₂, suggesting that Vac7 functions to regulate Fab1 kinase activity. To test this hypothesis, we isolated a fab1 mutant allele that bypasses the requirement for Vac7 in PtdIns(3,5)P₂ production. Expression of this fab1 allele in vac7Δmutant cells suppresses the temperature sensitivity, vacuolar morphology, and PtdIns(3,5)P₂ defects normally exhibited byvac7Δ mutants. We also identified a mutant allele ofFIG4, whose gene product contains a Sac1polyphosphoinositide phosphatase domain, which suppressesvac7Δ mutant phenotypes. Deletion ofFIG4 in vac7Δ mutant cells suppresses the temperature sensitivity and vacuolar morphology defects, and dramatically restores PtdIns(3,5)P₂ levels. These results suggest that generation of PtdIns(3,5)P₂ by the Fab1 lipid kinase is regulated by Vac7, whereas turnover of PtdIns(3,5)P₂ is mediated in part by theSac1 polyphosphoinositide phosphatase family member Fig4.