SAC1-like domains of yeast SAC1, INP52, and INP53 and of human synaptojanin encode polyphosphoinositide phosphatases

The SAC1 gene product has been implicated in the regulation of actin cytosk eleton, secretion from the Golgi, and microsomal ATP transport; yet its fun ction is unknown. Within SAC1 is an evolutionarily conserved 300-amino acid region, designated a SAC1-like domain, that is also present at the amino t ermini of the inositol polyphosphate 5-phosphatases, mammalian synaptojanin , and certain yeast INP5 gene products. Here we report that SAC1-like domai ns have intrinsic enzymatic activity that defines a new class of polyphosph oinositide phosphatase (PPIPase), Purified recombinant SAC1-like domains co nvert yeast lipids phosphatidylinositol (PI) S-phosphate, PI 4-phosphate, a nd PI 3,5-bisphosphate to PI, whereas PI 4,5-bisphosphate is not a substrat e. Yeast lacking Sac1p exhibit 10-, 2.5-, and a-fold increases in the cellu lar levels of PI 4-phosphate, PI 3,5-bisphosphate, and PI 3-phosphate, resp ectively. The 5-phosphatase domains of synaptojanin, Inp52p, and Inp53p are also catalytic, thus representing the first examples of an inositol signal ing protein with two distinct lipid phosphatase active sites within a singl e polypeptide chain. Together, our data provide a long sought mechanism as to how defects in Sac1p overcome certain actin mutants and bypass the requi rement for yeast phosphatidylinositol/phosphatidylcholine transfer protein, Sec14p. We demonstrate that PPIPase activity is a key regulator of membran e trafficking and actin cytoskeleton organization and suggest signaling rol es for phosphoinositides other than PI 4,5-bisphosphate in these processes. Additionally, the tethering of PPIPase and B-phosphatase activities indica te a novel mechanism by which concerted phosphoinositide hydrolysis partici pates in membrane trafficking.