Osmotic stress–induced increase of phosphatidylinositol 3,5-bisphosphate requires Vac14p, an activator of the lipid kinase Fab1p

Abstract

Phosphatidylinositol 3,5-bisphosphate (PtdIns[3,5]P2) was first identified as a nonabundant phospholipid whose levels increase in response to osmotic stress. In yeast, Fab1p catalyzes formation of PtdIns(3,5)P2 via phosphorylation of PtdIns(3)P. We have identified Vac14p, a novel vacuolar protein that regulates PtdIns(3,5)P2 synthesis by modulating Fab1p activity in both the absence and presence of osmotic stress. We find that PtdIns(3)P levels are also elevated in response to osmotic stress, yet, only the elevation of PtdIns(3,5)P2 levels are regulated by Vac14p. Under basal conditions the levels of PtdIns(3,5)P2 are 18–28-fold lower than the levels of PtdIns(3)P, PtdIns(4)P, and PtdIns(4,5)P2. After a 10 min exposure to hyperosmotic stress the levels of PtdIns(3,5)P2 rise 20-fold, bringing it to a cellular concentration that is similar to the other phosphoinositides. This suggests that PtdIns(3,5)P2 plays a major role in osmotic stress, perhaps via regulation of vacuolar volume. In fact, during hyperosmotic stress the vacuole morphology of wild-type cells changes dramatically, to smaller, more highly fragmented vacuoles, whereas mutants unable to synthesize PtdIns(3,5)P2 continue to maintain a single large vacuole. These findings demonstrate that Vac14p regulates the levels of PtdIns(3,5)P2 and provide insight into why PtdIns(3,5)P2 levels rise in response to osmotic stress.

Assembly of a Fab1 Phosphoinositide Kinase Signaling Complex Requires the Fig4 Phosphoinositide Phosphatase

“…In contrast, fig4-1 stabilizes the Fab1-Vac14 interaction (positive signal) in the absence of PtdIns(3,5)P 2 turnover, resulting in a significant increase in PtdIns(3,5)P 2 levels. In fact, the level of PtdIns(3,5)P 2 in the absence of Vac14 and Fig4 is only ϳ0.1 times that of wild-type cells but in the presence of Vac14 and Fig4-1 it is 3 times that of wild-type (thus, ϳ30-fold difference; Bonangelino et al, 2002;Dove et al, 2002;Gary et al, 2002;Rudge et al, 2004). Because elimination of phosphatase activity cannot solely explain this increase, it suggests that binding of Vac14-Fig4 to Fab1 stimulates its intrinsic kinase activity.…”

Ent5p Is Required with Ent3p and Vps27p for Ubiquitin-dependent Protein Sorting into the Multivesicular Body

“…Additionally, the vacuoles of Fab1p-depleted cells display a defect in acidification, whereas the fragmented vacuoles in ent3⌬ent5⌬ cells are properly acidified. In fab1⌬ cells the enlarged vacuole is the result of the lack of PtdIns(3,5)P 2 synthesis (Bonangelino et al, 2002), whereas it is likely that ent3⌬ent5⌬ cells are not affected in PtdIns(3,5)P 2 synthesis, but rather in PtdIns(3,5)P 2 binding. The ent3⌬ent5⌬ cells do not display classical class E phenotype, which might be due to their implication in different membrane trafficking pathways to the vacuole (Duncan et al, 2002).…”

The transient receptor potential channel on the yeast vacuole is mechanosensitive

“…On an osmotic upshock, the vacuoles shrink and crenellate (23,24,29,38 (29), and the enzymes for its production, as well as other gene products, are required for the final crenellation. Mutants defective in these proteins have defective vacuolar inheritance and enlarged vacuoles with or without osmotic stress (39,29). Other mutants have protein-sorting defects and are sensitive to osmotic stress (40)(41)(42).…”

“…The procedure entails an overnight culturing of the spheroplasts, in which the multivesicular looking vacuole(s) in each logarithmic-phase cell (ref. 29; Fig. 5) became a single spherical vacuole.…”