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 ⌬sac1 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.Phosphoinositides regulate diverse cellular processes such as cell proliferation, signal transduction, organization of the cytoskeleton, and membrane trafficking. They do so by regulating the intracellular localization and biological properties of a variety of proteins involved in these processes. Such effector proteins recognize specific phosphoinositides via lipid binding modules such as C2 domains, pleckstrin homology domains, FYVE domains, and SH2 1 domains (reviewed in Refs.

The Sac1 Phosphoinositide Phosphatase Regulates Golgi Membrane Morphology and Mitotic Spindle Organization in Mammals

“…Under those conditions, mSac1-GFP colocalized with an ER marker (calnexin), a cis-Golgi marker (GM130), and a TGN marker (TGN46; Figure 3A). This pattern is in agreement with the distribution of endogenous Sac1 in mammalian cells (Nemoto et al, 2000;Rohde et al, 2003). mSac1-GFP showed a higher degree of colocalization with the cis-Golgi marker GM130 than with the TGN marker TGN46 ( Figure 3A).…”

“…Site-directed mutagenesis of murine SAC1 was performed as described previously (Nemoto et al, 2000). Mutagenic primers were as follows: for msac1 D391N , 5Ј-CGCAGCA ACTGCATGAATTGTCTAGACAG-3Ј and 5Ј-CT-GTCTAGACAATTCATGCAGTTGCTGCG-3Ј; for msac1 A442V , 5Ј-CCTGGGC-CGATAATGTTAATGCTTGTGCC-3Ј and 5Ј-GGCACAAGCATTAACA TTATCGGCCCAGG-3Ј; and for msac1 R480H , 5Ј-GGCTTCAACTCATTATTA-CACTATTACAAGAACA AC-3Ј and 5Ј-GTTGTTCTTGTAATAGTGTAATA-ATGAGTTGAAGCC-3Ј.…”

“…First, ySac1 degrades a PtdIns-4-P pool produced by the Stt4 PtdIns 4-OH kinase, one of three PtdIns 4-OH kinases in this organism (Nemoto et al, 2000;Foti et al, 2001). Second, ySac1 is an integral membrane protein of endoplasmic reticulum (ER) and Golgi membranes (Cleves et al, 1989;Whitters et al, 1993;Nemoto et al, 2000). This disposition is determined by two C-terminal membrane anchor sequences with the large catalytic domain Abbreviations used: BrdU, bromodeoxyuridine; BSA, bovine serum albumin; ER, endoplasmic reticulum; ES, embryonic stem; FACS, fluorescence-activated cell sorting; GAG, glycosaminoglycan; GFP, green fluorescent protein; Ins, inositol; LOF, loss-of-function; MT, microtubule; PBS, phosphate-buffered saline; PIP, phosphoinositide; PITP, phosphatidylinositol transfer protein; PtdIns, phosphatidylinositol; RFP, red fluorescent protein; RT-PCR, reverse transcriptase-polymerase chain reaction; TGN, trans-Golgi network; VSV-G, vesicular stomatitis virus glycoprotein.…”

“…ySac1 homologues are conserved throughout the Eukaryota (Supplemental Figure S1B). Mammalian Sac1 is an integral membrane protein with an anchor-sequence arrangement similar to that of ySac1p (Supplemental Figure S1, A and B), and it primarily resides in ER membranes (Nemoto et al, 2000). ER retention of the mammalian enzyme is controlled by a COP1-Sac1 binding interaction that captures enzyme in the Golgi system and recycles it back to the ER in COP1 vesicles (Rohde et al, 2003).…”

“…ySac1 is an unusual PIP phosphatase in several respects. First, ySac1 degrades a PtdIns-4-P pool produced by the Stt4 PtdIns 4-OH kinase, one of three PtdIns 4-OH kinases in this organism (Nemoto et al, 2000;Foti et al, 2001). Second, ySac1 is an integral membrane protein of endoplasmic reticulum (ER) and Golgi membranes (Cleves et al, 1989;Whitters et al, 1993;Nemoto et al, 2000).…”