Functional characterization of a mammalian Sac1 and mutants exhibiting substrate-specific defects in phosphoinositide phosphatase activity

The Saccharomyces cerevisiae SAC1 gene was identified via independent analy ses of mutations that modulate yeast actin function and alleviate the essen tial requirement for phosphatidylinositol transfer protein (Sec14p) activit y in Gels secretory function. The SAC1 gene product (Sac1p) is an integral membrane protein of the endoplasmic reticulum and the Golgi complex, Sac1p shares primary sequence homology with a subfamily of cytosolic/peripheral m embrane phosphoinositide phosphatases, the synaptojanins, and these Sad dom ains define novel phosphoinositide phosphatase modules. We now report the c haracterization of a rat counterpart of Sac1p, Rat Sad is a ubiquitously ex pressed 65-kDa integral membrane protein of the endoplasmic reticulum that is found at particularly high levels in cerebellar Purkinje cells. Like Sac 1p, rat Sad exhibits intrinsic phosphoinositide phosphatase activity direct ed toward phosphatidylinositol 3-phosphate, phosphatidylinositol 4-phosphat e, and phosphatidylinositol 3,5-bisphosphate substrates, and we identify mu tant rat sad alleles that evoke substrate-specific defects in this enzymati c activity. Finally, rat Sad expression in Delta sac1 yeast strains complem ents a wide phenotypes associated with Sac1p insufficiency, Biochemical and in vivo data indicate that rat Sad phosphatidylinositol-4-phosphate phosph atase activity, but not its phosphatidylinositol-3-phosphate or pho sphatid ylinositol-3,5 bisphosphate phosphatase activities, is essential for the he terologous complementation of Sac1p defects in vivo. Thus, yeast Sac1p and rat Sad are integral membrane lipid phosphatases that play evolutionary con served roles in eukaryotic cell physiology.