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 Tahirovic, Ellie Kuhne, Rodney J. Devenish, Alan L. Munn, Robert C. Piper, Peter Mayinger, Christina A. Mitchell

Research output: Contribution to journalArticle

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Abstract

Phosphoinositides direct membrane trafficking, facilitating the recruitment of effectors to specific membranes. In yeast phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) is proposed 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 Δsac1 Δinp54 and sac1ts Δinp54 mutants. Furthermore, sac1ts Δinp54 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)P 2. 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. Δsac1 Δinp54 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.

Original languageEnglish (US)
Pages (from-to)16295-16307
Number of pages13
JournalJournal of Biological Chemistry
Volume282
Issue number22
DOIs
StatePublished - Jun 1 2007

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Phosphatidylinositol 4,5-Diphosphate
Membrane Fusion
Phosphatidylinositols
Vacuoles
Phosphoric Monoester Hydrolases
Fusion reactions
Membranes
Defects
Cell membranes
Cell Membrane
Yeast
Cathepsin A
Phosphoinositide Phosphatases
Biosensing Techniques
Sorting
Spatial distribution
Saccharomyces cerevisiae
Hydrolysis
Yeasts

ASJC Scopus subject areas

  • Biochemistry

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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. / Wiradjaja, Fenny; Ooms, Lisa M.; Tahirovic, Sabina; Kuhne, Ellie; Devenish, Rodney J.; Munn, Alan L.; Piper, Robert C.; Mayinger, Peter; Mitchell, Christina A.

In: Journal of Biological Chemistry, Vol. 282, No. 22, 01.06.2007, p. 16295-16307.

Research output: Contribution to journalArticle

Wiradjaja, Fenny ; Ooms, Lisa M. ; Tahirovic, Sabina ; Kuhne, Ellie ; Devenish, Rodney J. ; Munn, Alan L. ; Piper, Robert C. ; Mayinger, Peter ; Mitchell, Christina A. / Inactivation of the phosphoinositide phosphatases Sac1p and Inp54p leads to accumulation of phosphatidylinositol 4,5-bisphosphate on vacuole membranes and vacuolar fusion defects. In: Journal of Biological Chemistry. 2007 ; Vol. 282, No. 22. pp. 16295-16307.
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abstract = "Phosphoinositides direct membrane trafficking, facilitating the recruitment of effectors to specific membranes. In yeast phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) is proposed 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 Δsac1 Δinp54 and sac1ts Δinp54 mutants. Furthermore, sac1ts Δinp54 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)P 2. 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. Δsac1 Δinp54 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.",
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AU - Wiradjaja, Fenny

AU - Ooms, Lisa M.

AU - Tahirovic, Sabina

AU - Kuhne, Ellie

AU - Devenish, Rodney J.

AU - Munn, Alan L.

AU - Piper, Robert C.

AU - Mayinger, Peter

AU - Mitchell, Christina A.

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