Inactivation of the phosphoinositide phosphatases Sac1p and Inp54p leads to accumulation of phosphatidylinositol 4,5-bisphosphate on vacuole membranes and vacuolar fusion defects

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 sac1^ts Δinp54 mutants. Furthermore, sac1^ts 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.