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 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.
Guo
S
S
Departments of Pharmacology and Cancer Biology and of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Stolz
L E
LE
Lemrow
S M
SM
York
J D
JD
D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D000072181:Inositol Polyphosphate 5-Phosphatases; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*
D005656:Fungal Proteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; C097702:synaptojanin; D010744:Phosphoric Monoester Hydrolases; D000072181:Inositol Polyphosphate 5-Phosphatases