SAC1-like Domains of Yeast SAC1,INP52, and INP53 and of Human Synaptojanin Encode Polyphosphoinositide Phosphatases

doi:10.1074/jbc.274.19.12990

SAC1-like Domains of Yeast SAC1,INP52, and INP53 and of Human Synaptojanin Encode Polyphosphoinositide Phosphatases*

From the Departments of Pharmacology and Cancer Biology and of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

Abstract

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 recombinantSAC1-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.

Footnotes

↵* This work was supported by a Burroughs Wellcome Fund Career Award in the Biomedical Sciences, a Whitehead Scholar Award, and National Institutes of Health R01-HL 55672.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ To whom correspondence should be addressed: Dept. of Pharmacology and Cancer Biology, Duke University Medical Center, DUMC 3813, Durham, NC 27710. Tel.: 919-681-6414; Fax: 919-684-8922; E-mail:yorkj{at}acpub.duke.edu.
↵2 S. Guo and J. D. York, unpublished observation.
Abbreviations:

PI

phosphatidylinositol

PI(4

5)P₂, phosphatidylinositol (4,5)-bisphosphate

PI(3)P

phosphatidylinositol 3-phosphate

PI(4)P

phosphatidylinositol 4-phosphate

PI(3

5)P₂, phosphatidylinositol (3,5)-bisphosphate

PI(3

4,5)P₃, phosphatidylinositol 3,4,5-triphosphate

M[IP]₂C

mannosyl di-inositol diphosphorylceramide

5-ptase

inositol polyphosphate 5-phosphatase

PPIPase

polyphosphoinositide phosphatase

AP

ammonium phosphate

PCR

polymerase chain reaction

HPLC

high pressure liquid chromatography

groPI

glycerophosphoinositols

PIP

phosphatidylinositol monophosphate

Ins(1

4,5)P₃, inositol 1,4,5-trisphosphate

GST

glutathione S-transferase

DTT

dithiothreitol

MVB

multi-vesicular bodies
- Received February 17, 1999.
- Revision received March 11, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.