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 Michelle Ooms, Sabina Tahirovic, Ellersley Shae Kuhne, Rodney James Devenish, Alan L Munn, Robert C Piper, Peter Mayinger, Christina Anne Mitchell

Research output: Contribution to journalArticleResearchpeer-review

13 Citations (Scopus)

Abstract

Phosphoinositides direct membrane trafficking, facilitating the recruitment of effectors to specific membranes. In yeast PtdIns(4,5)P(2) is proposed to regulate vacuolar fusion, however, in intact cells this phosphoinositide can only be detected at the plasma membrane. In Saccharomyces cerevisiae the inositol polyphosphate 5-phosphatase, Inp54p, dephosphorylates PtdIns(4,5)P(2) forming PtdIns(4)P, a substrate for the phosphoinositide phosphatase Sac1p, which hydrolyzes PtdIns(4)P forming PtdIns. We investigated the role these lipid phosphatases co-ordinately play in regulating PtdIns(4,5)P(2) subcellular distribution. The PtdIns(4,5)P(2) bioprobe PH-PLCdelta1 exhibited loss of plasma membrane localization and instead labeled a subset of fragmented vacuoles in delta sac1 deltainp54 and sac1(ts) deltainp54 mutants. Furthermore sac1(ts) deltainp54 mutants exhibited vacuolar fusion defects, which were rescued by Latrunculin A treatment, which blocks endocytosis, or by inactivation of Mss4p, a PtdIns(4)P 5-kinase which synthesizes the bulk of PtdIns(4,5)P(2) at the plasma membrane. Under these conditions PtdIns(4,5)P(2) was not detected on vacuole membranes and vacuole morphology was normal, indicating vacuolar PtdIns(4,5)P(2) derives from Mss4p-generated PtdIns(4,5)P(2) at the plasma membrane. deltasac1 deltainp54 mutants also exhibited delayed CPY sorting, cargo-selective secretion defects and defects in vacuole function. These studies reveal PtdIns(4,5)P(2) hydrolysis by lipid phosphatases governs its spatial distribution and loss of phosphatase activity may result in PtdIns(4,5)P(2) accumulation on vacuole membranes leading to vacuolar fragmentation/fusion defects.
Original languageEnglish
Pages (from-to)16295 - 16307
Number of pages13
JournalThe Journal of Biological Chemistry
Volume282
Issue number22
Publication statusPublished - 2007

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