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The SAC1 gene product has been implicated in the regulation of actin cytoskeleton, secretion from the Golgi, and microsomal ATP transport; yet its function is unknown. Within SAC1 is an evolutionarily conserved 300-amino acid region, designated a SAC1-like domain, that is also present at the amino termini of the inositol polyphosphate 5-phosphatases, mammalian synaptojanin, and certain yeast INP5 gene products. Here we report that SAC1-like domains have intrinsic enzymatic activity that defines a new class of polyphosphoinositide phosphatase (PPIPase). Purified recombinant SAC1-like domains convert yeast lipids phosphatidylinositol (PI) 3-phosphate, PI 4-phosphate, and PI 3,5-bisphosphate to PI, whereas PI 4,5-bisphosphate is not a substrate. Yeast lacking Sac1p exhibit 10-, 2.5-, and 2-fold increases in the cellular levels of PI 4-phosphate, PI 3,5-bisphosphate, and PI 3-phosphate, respectively. The 5-phosphatase domains of synaptojanin, Inp52p, and Inp53p are also catalytic, thus representing the first examples of an inositol signaling protein with two distinct lipid phosphatase active sites within a single polypeptide chain. Together, our data provide a long sought mechanism as to how defects in Sac1p overcome certain actin mutants and bypass the requirement for yeast phosphatidylinositol/ phosphatidylcholine transfer protein, Sec14p. We demonstrate that PPIPase activity is a key regulator of membrane trafficking and actin cytoskeleton organization and suggest signaling roles for phosphoinositides other than PI 4,5-bisphosphate in these processes. Additionally, the tethering of PPIPase and 5-phosphatase activities indicate a novel mechanism by which concerted phosphoinositide hydrolysis participates in membrane trafficking.Phosphoinositides are essential components of eukaryotic membranes and are key regulators of membrane trafficking and actin cytoskeleton (1-3). Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) 1 serves as a precursor to second messengers and as a signaling molecule itself by regulating protein activities and through interactions with protein modules (3-5). Additionally, roles for PI(3)P, PI(3,5)P 2 and PI(3,4,5)P 3 in membrane movement have been defined (1, 6 -10). Homeostasis of phosphoinositides occurs both spatially and temporally via a plethora of lipase, kinase, and phosphatase activities, thereby providing several unique points of regulation (for reviews see .A role for inositol lipid phosphatases in membrane trafficking has come from the characterization and cloning of synaptojanin, a mammalian neuronal inositol polyphosphate 5-phosphatase (5-ptase) involved in synaptic vesicle recycling (18). Additionally, studies of three yeast INP5 gene products (also known as SJLs) demonstrate that although they are collectively essential, the individual proteins also have nonredundant roles in regulating membrane trafficking, cell wall synthesis, osmo-sensitivity, and actin cytoskeleton structure (19 -22). Synaptojanin and the Inp5ps are members of a large 5-ptase gene family, conserved from ...

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Sequence analysis of Saccharomyces cerevisiae chromosome IX identified a 946 amino acid open reading frame (YIL002C), designated here as INP51, that has carboxyl-and amino-terminal regions similar to mammalian inositol polyphosphate 5-phosphatases and to yeast SAC1. This two-domain primary structure resembles the mammalian 5-phosphatase, synaptojanin. We report that Inp51p is associated with a particulate fraction and that recombinant Inp51p exhibits intrinsic phosphatidylinositol 4,5-bisphosphate 5-phosphatase activity. Deletion of INP51 (inp51) results in a "coldtolerant" phenotype, enabling significantly faster growth at temperatures below 15°C as compared with a parental strain. Complementation analysis of an inp51 mutant strain demonstrates that the cold tolerance is strictly due to loss of 5-phosphatase catalytic activity. Furthermore, deletion of PLC1 in an inp51 mutant does not abrogate cold tolerance, indicating that Plc1p-mediated production of soluble inositol phosphates is not required. Cells lacking INP51 have a 2-4-fold increase in levels of phosphatidylinositol 4,5-bisphosphate and inositol 1,4,5-trisphosphate, whereas cells overexpressing Inp51p exhibit a 35% decrease in levels of phosphatidylinositol 4,5-bisphosphate. We conclude that INP51 function is critical for proper phosphatidylinositol 4,5-bisphosphate homeostasis. In addition, we define a novel role for a 5-phosphatase loss of function mutant that improves the growth of cells at colder temperatures without alteration of growth at normal temperatures, which may have useful commercial applications.Of central importance to the inositol signaling pathway is phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ), 1 which serves as a precursor to second messengers and is a signaling molecule itself (1-3). PI(4,5)P 2 has a direct functional role in signaling via interactions with actin-binding proteins, phospholipase D, and pleckstrin homology domains (3-7). Levels of PI(4,5)P 2 are tightly maintained by a balance of kinase, lipase, transferase, and phosphatase activities. The importance of this regulation is manifested by disease states that arise as a result of mutation in these enzymes. For example, in Drosophila deletion of the norpA phospholipase C ␤ gene results in blindness (8); ablation of an eye-specific CDP-diacylglycerol synthetase results in retinal degeneration that can be pharmacologically rescued by re-addition of PI(4,5)P 2 (9); and recent studies by Milligan et al. (10) demonstrate that the phosphatidylinositol transfer protein domain of the rdgB gene is necessary for proper photoreceptor function in response to prolonged light stimuli and serves to protect photoreceptor degeneration. In addition, the human disease oculocerebrorenal syndrome (Lowe's syndrome) arises from mutations in the OCRL-1 gene (11), which encodes an inositol polyphosphate 5-phosphatase (5-ptase) (12).5-Ptase enzymes comprise a family of critical proteins that remove the 5-position phosphate from either soluble or lipid inositol polyphosphates, or both (revi...