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J. Biol. Chem., Vol. 282, Issue 33, 23878-23891, August 17, 2007

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Core Protein Machinery for Mammalian Phosphatidylinositol 3,5-Bisphosphate Synthesis and Turnover That Regulates the Progression of Endosomal Transport

NOVEL SAC PHOSPHATASE JOINS THE ArPIKfyve-PIKfyve COMPLEX^*

Diego Sbrissa¹, Ognian C. Ikonomov¹, Zhiyao Fu, Takeshi Ijuin², Jean Gruenberg^¶, Tadaomi Takenawa², and Assia Shisheva³

From the Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201, the ^¶Department of Biochemistry, Sciences II, University of Geneva, Geneva 4CH-1211, Switzerland, and the Department of Biochemistry, Institute of Molecular Science, University of Tokyo, Tokyo 113-0032, Japan

Perturbations in phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P₂)-synthesizing enzymes result in enlarged endocytic organelles from yeast to humans, indicating evolutionarily conserved function of PtdIns(3,5)P₂ in endosome-related events. This is reinforced by the structural and functional homology of yeast Vac14 and human Vac14 (ArPIKfyve), which activate yeast and mammalian PtdIns(3,5)P₂-producing enzymes, Fab1 and PIKfyve, respectively. In yeast, PtdIns(3,5)P₂-specific phosphatase, Fig4, in association with Vac14, turns over PtdIns(3,5)P₂, but whether such a mechanism operates in mammalian cells and what the identity of mammalian Fig4 may be are unknown. Here we have identified and characterized Sac3, a Sac domain phosphatase, as the Fig4 mammalian counterpart. Endogenous Sac3, a widespread 97-kDa protein, formed a stable ternary complex with ArPIKfyve and PIKfyve. Concordantly, Sac3 cofractionated and colocalized with ArPIKfyve and PIKfyve. The intrinsic Sac3^WT phosphatase activity preferably hydrolyzed PtdIns(3,5)P₂ in vitro, although the other D5-phosphorylated polyphosphoinositides were also substrates. Ablation of endogenous Sac3 by short interfering RNAs elevated PtdIns(3,5)P₂ in ³²P-labeled HEK293 cells. Ectopically expressed Sac3^WT in COS cells colocalized with and dilated EEA1-positive endosomes, consistent with the PtdIns(3,5)P₂ requirement in early endosome dynamics. In vitro reconstitution of carrier vesicle formation from donor early endosomes revealed a gain of function upon Sac3 loss, whereas PIKfyve or ArPIKfyve protein depletion produced a loss of function. These data demonstrate a coupling between the machinery for PtdIns(3,5)P₂ synthesis and turnover achieved through a physical assembly of PIKfyve, ArPIKfyve, and Sac3. We suggest that the tight regulation in PtdIns(3,5)P₂ homeostasis is mechanistically linked to early endosome dynamics in the course of cargo transport.

Received for publication, December 20, 2006 , and in revised form, May 22, 2007.

^* This work was supported by National Institutes of Health Grant DK58058 and American Diabetes Association Research grants (to A. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² Present address: Dept. of Lipid Biochemistry, Kobe-University Graduate School of Medicine, 650-0017, Kobe, Japan.

³ To whom correspondence should be addressed: Dept. of Physiology, Wayne State University School of Medicine, 540 E. Canfield, Detroit, MI 48201. Tel.: 313-577-5674; Fax: 313-577-5494; E-mail: ashishev{at}med.wayne.edu.

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