title: Vacuole size control: regulation of PtdIns(3,5)P2 levels by the vacuole-associated Vac14-Fig4 complex, a PtdIns(3,5)P2-specific phosphatase.
abstract: In the budding yeast Saccharomyces cerevisiae, phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) is synthesized by a single phosphatidylinositol 3-phosphate 5-kinase, Fab1. Cells deficient in PtdIns(3,5)P2 synthesis exhibit a grossly enlarged vacuole morphology, whereas increased levels of PtdIns(3,5)P2 provokes the formation of multiple small vacuoles, suggesting a specific role for PtdIns(3,5)P2 in vacuole size control. Genetic studies have indicated that Fab1 kinase is positively regulated by Vac7 and Vac14; deletion of either gene results in ablation of PtdIns(3,5)P2 synthesis and the formation of a grossly enlarged vacuole. More recently, a suppressor of vac7Delta mutants was identified and shown to encode a putative phosphoinositide phosphatase, Fig4. We demonstrate that Fig4 is a magnesium-activated PtdIns(3,5)P2-selective phosphoinositide phosphatase in vitro. Analysis of a Fig4-GFP fusion protein revealed that the Fig4 phosphatase is localized to the limiting membrane of the vacuole. Surprisingly, in the absence of Vac14, Fig4-GFP no longer localizes to the vacuole. However, Fig4-GFP remains localized to the grossly enlarged vacuoles of vac7 deletion mutants. Consistent with these observations, we found that Fig4 physically associates with Vac14 in a common membrane-associated complex. Our studies indicate that Vac14 both positively regulates Fab1 kinase activity and directs the localization/activation of the Fig4 PtdIns(3,5)P2 phosphatase.
authors.affiliation: Department of Cellular and Molecular Medicine, and the Howard Hughes Medical Institute, University of California at San Diego, School of Medicine, La Jolla, California 92093-0668, USA.
authors.forename: Simon A
authors.initials: SA
authors.lastname: Rudge
authors.forename: Deborah M
authors.initials: DM
authors.lastname: Anderson
authors.forename: Scott D
authors.initials: SD
authors.lastname: Emr
mesh_terms: D004789:Enzyme Activation / Q000502:physiology; D005420:Flavoproteins / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D049452:Green Fluorescent Proteins; D007425:Intracellular Membranes / Q000378:metabolism; D008164:Luminescent Proteins; D008274:Magnesium / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D009154:Mutation; D018129:Phosphatidylinositol Phosphates / Q000096:biosynthesis*; D010744:Phosphoric Monoester Hydrolases; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014617:Vacuoles / Q000201:enzymology
chemical_list: D005420:Flavoproteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D018129:Phosphatidylinositol Phosphates; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C452435:VAC14 protein, S cerevisiae; C452436:VAC7 protein, S cerevisiae; C106336:phosphatidylinositol 3,5-diphosphate; D049452:Green Fluorescent Proteins; C110669:FIG4 protein, S cerevisiae; D010744:Phosphoric Monoester Hydrolases; D008274:Magnesium