SAC1p is an integral membrane protein that influences the cellular requirement for phospholipid transfer protein function and inositol in yeast

Abstract

Abstract. Mutations in the SAC1 gene exhibit allelespecific genetic interactions with yeast actin structural gene defects and effect a bypass of the cellular requirement for the yeast phosphatidylinositol/phosphatidylcholine transfer protein (SEC14p), a protein whose function is essential for sustained Golgi secretory function. We report that SAClp is an integral membrane protein that localizes to the yeast Golgi complex and to the yeast ER, but does not exhibit a detectable association with the bulk of the yeast F-actin cytoskeleton. The data also indicate that the profound in vivo effects on Golgi secretory function and the organization of the actin cytoskeleton observed in sad mutants result from loss of SAClp function. This cosuppression of actin and SEC14p defects is a unique feature of sad alleles as mutations in other SAC genes that result in a suppression of actin defects do not result in phenotypic suppression of SEC14p defects. Finally, we report that sac1 mutants also exhibit a specific inositol auxotrophy that is not exhibited by the other sac mutant strains. This sad-associated inositol auxotrophy is not manifested by measurable defects in de novo inositol biosynthesis, nor is it the result of some obvious defect in the ability of sad mutants to utilize inositol for phosphatidylinositol biosynthesis. Thus, sac1 mutants represent a novel class of inositol auxotroph in that these mutants appear to require elevated levels of inositol for growth. On the basis of the collective data, we suggest that SAClp dysfunction exerts its pleiotropic effects on yeast Golgi function, the organization of the actin cytoskeleton, and the cellular requirement for inositol, through altered metabolism of inositol glycerophospholipids.

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

“…However, scanning electron microscopy revealed no significant reductions in dorsal surface filopodial density in hSac1-insufficient cells (0.34 Ϯ 0.14 filopodia/ m 2 ) compared with controls (0.46 Ϯ 0.09 filopodia/ m 2 ). Functional ablation of ySac1 alters yeast actin organization , an effect interpreted as an indirect consequence derived from deregulated interactions between actin binding proteins and PIPs (Cleves et al, 1991;Whitters et al, 1993). Visualization of F-actin by phalloidin staining showed no dramatic disorganization of actin stress fibers in hSac1-depleted cells, although stress fibers seemed more pronounced relative to those in controls (Supplemental Figure S5C).…”

“…Ablation of ySac1 PIP phosphatase function evokes pleiotropic phenotypes that include a cold sensitivity for growth, Ins auxotrophy, allele-specific genetic interactions with mutations in the single yeast actin structural gene, and bypass for the normally essential cellular requirement for Sec14, the major yeast PtdIns/phosphatidylcholine transfer protein (Cleves et al, 1989;Novick et al, 1989;Whitters et al, 1993). Mutations that compromise ySac1 enzymatic activity phenocopy sac1 null mutations, whereas substitutions outside the ySac1 catalytic motif evoke defects that are likely regulatory in nature (Nemoto et al, 2000;Li et al, 2002).…”

“…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.…”

“…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 This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E07-12-1290) on May 14, 2008. oriented toward the cytosolic face (Konrad et al, 2002;Supplemental Figure S1A).…”

Phosphoinositides in Constitutive Membrane Traffic

“…One group of these enzymes contains only a NH 2 -terminal Sac domain, and the second class contains an additional, 5-phosphatase domain in the center of the protein followed by various other domains (155). Sac1p was identified in a screen for mutations that relieved the block in secretion caused by loss of activity of the S. cerevisiae PITP, Sec14p (51,385), and was subsequently discovered to have PIP phosphatase activity (47,124). In S. cerevisiae there are two such proteins, Sac1p and Fig4p, and in humans three, KIAA0274, KIAA0966, and KIAA0851.…”