title: Functional characterization of a mammalian Sac1 and mutants exhibiting substrate-specific defects in phosphoinositide phosphatase activity.
abstract: The Saccharomyces cerevisiae SAC1 gene was identified via independent analyses of mutations that modulate yeast actin function and alleviate the essential requirement for phosphatidylinositol transfer protein (Sec14p) activity in Golgi 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 membrane phosphoinositide phosphatases, the synaptojanins, and these Sac1 domains define novel phosphoinositide phosphatase modules. We now report the characterization of a rat counterpart of Sac1p. Rat Sac1 is a ubiquitously expressed 65-kDa integral membrane protein of the endoplasmic reticulum that is found at particularly high levels in cerebellar Purkinje cells. Like Sac1p, rat Sac1 exhibits intrinsic phosphoinositide phosphatase activity directed toward phosphatidylinositol 3-phosphate, phosphatidylinositol 4-phosphate, and phosphatidylinositol 3,5-bisphosphate substrates, and we identify mutant rat sac1 alleles that evoke substrate-specific defects in this enzymatic activity. Finally, rat Sac1 expression in Deltasac1 yeast strains complements a wide phenotypes associated with Sac1p insufficiency. Biochemical and in vivo data indicate that rat Sac1 phosphatidylinositol-4-phosphate phosphatase activity, but not its phosphatidylinositol-3-phosphate or phosphatidylinositol-3, 5-bisphosphate phosphatase activities, is essential for the heterologous complementation of Sac1p defects in vivo. Thus, yeast Sac1p and rat Sac1 are integral membrane lipid phosphatases that play evolutionary conserved roles in eukaryotic cell physiology.
authors.affiliation: Brain Science Institute, The Institute of Physical and Chemical Research, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Nemoto
authors.forename: B G
authors.initials: BG
authors.lastname: Kearns
authors.forename: M R
authors.initials: MR
authors.lastname: Wenk
authors.forename: H
authors.initials: H
authors.lastname: Chen
authors.forename: K
authors.initials: K
authors.lastname: Mori
authors.forename: J G
authors.initials: JG
authors.lastname: Alb
authors.forename: P
authors.initials: P
authors.lastname: De Camilli
authors.forename: V A
authors.initials: VA
authors.lastname: Bankaitis
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D017931:DNA Primers; D004721:Endoplasmic Reticulum / Q000378:metabolism; D007295:Inositol Phosphates / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D007295:Inositol Phosphates; D008565:Membrane Proteins; C416669:Sac1 protein, mammalian; C089504:polyphosphoinositide phosphatase; D010744:Phosphoric Monoester Hydrolases