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J. Biol. Chem., Vol. 282, Issue 22, 16295-16307, June 1, 2007

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Inactivation of the Phosphoinositide Phosphatases Sac1p and Inp54p Leads to Accumulation of Phosphatidylinositol 4,5-Bisphosphate on Vacuole Membranes and Vacuolar Fusion Defects^*

Fenny Wiradjaja, Lisa M. Ooms, Sabina Tahirovic¹, Ellie Kuhne, Rodney J. Devenish, Alan L. Munn^¶, Robert C. Piper^||, Peter Mayinger^**, and Christina A. Mitchell²

From the Department of Biochemistry and Molecular Biology, Monash University, Clayton 3800, Victoria, Australia, ZMBH, University of Heidelberg, INF 282, 69120 Heidelberg, Germany, the ^¶Institute for Molecular Bioscience, Queensland Bioscience Precinct, University of Queensland, St. Lucia, Queensland 4072, Australia, the ^||Department of Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242, and the ^**Division of Nephrology and Hypertension, Oregon Health and Science University, Portland, Oregon 97239

Phosphoinositides direct membrane trafficking, facilitating the recruitment of effectors to specific membranes. In yeast phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) isproposed to regulate vacuolar fusion; however, in intact cells this phosphoinositide can only be detected at the plasma membrane. In Saccharomyces cerevisiae the 5-phosphatase, Inp54p, dephosphorylates PtdIns(4,5)P₂ forming PtdIns(4)P, a substrate for the phosphatase Sac1p, which hydrolyzes (PtdIns(4)P). We investigated the role these phosphatases in regulating PtdIns(4,5)P₂ subcellular distribution. PtdIns(4,5)P₂ bioprobes exhibited loss of plasma membrane localization and instead labeled a subset of fragmented vacuoles in sac1 inp54 and sac1^ts inp54 mutants. Furthermore, sac1^ts inp54 mutants exhibited vacuolar fusion defects, which were rescued by latrunculin A treatment, or by inactivation of Mss4p, a PtdIns(4)P 5-kinase that synthesizes plasma membrane PtdIns(4,5)P₂. Under these conditions PtdIns(4,5)P₂ was not detected on vacuole membranes, and vacuole morphology was normal, indicating vacuolar PtdIns(4,5)P₂ derives from Mss4p-generated plasma membrane PtdIns(4,5)P₂. sac1 inp54 mutants exhibited delayed carboxypeptidase Y sorting, cargo-selective secretion defects, and defects in vacuole function. These studies reveal PtdIns(4,5)P₂ hydrolysis by lipid phosphatases governs its spatial distribution, and loss of phosphatase activity may result in PtdIns(4,5)P₂ accumulation on vacuole membranes leading to vacuolar fragmentation/fusion defects.

Received for publication, February 2, 2007 , and in revised form, March 28, 2007.

^* This work was supported by the Australian Research Council Centre of Excellence-Structural and Functional Microbial Genomics Grant CE0562063, Australian Research Council Grant DP0345120 (to C. A. M.), National Health and Medical Research Council Project Grant 298921 (to A. L. M.), and National Institutes of Health Grant GM071569 (to P. M.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1–5.

¹ Present address: Max Planck Institute for Neurobiology, 82152 Martinsried, Germany.

² To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Monash University, Wellington Rd., Victoria 3800, Australia. Tel.: 613-9905-3790; Fax: 613-9905-3726; E-mail: Christina.Mitchell{at}med.monash.edu.au.

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