FAB1P IS ESSENTIAL FOR PTDINS(3)P 5-KINASE ACTIVITY AND THE MAINTENANCE OF VACUOLAR SIZE AND MEMBRANE HOMEOSTASIS

The Saccharomyces cerevisiae FAB1 gene encodes a 257-kD protein that c ontains a cysteine-rich RING-FYVE domain at its NH2-terminus and a kin ase domain at its COOH terminus. Based on its sequence, Fab1p was init ially proposed to function as a phosphatidylinositol 4-phosphate (PtdI ns(4)P) 5-kinase (Yamamoto et al., 1995). Additional sequence analysis of the Fab1p kinase domain, reveals that Fab1p defines a subfamily of putative PtdInsP kinases that is distinct from the kinases that synth esize PtdIns(4,5)P-2. Consistent with this, we find that unlike wild-t ype cells, fab1 Delta, fab1(tsf) and fab1 kinase domain point mutants lack detectable levels of PtdIns(3,5)P-2, a phosphoinositide recently identified both in yeast and mammalian cells. PtdIns(4,5)P-2 synthesis , on the other hand, is only moderately affected even in fab1 Delta mu tants. The presence of PtdIns(3)P in fab1 mutants, combined with previ ous data, indicate that PtdIns(3,5)P-2 synthesis is a two step process , requiring the production of PtdIns(3)P by the Vps34p PtdIns 3-kinase and the subsequent Fab1p-dependent phosphorylation of PtdIns(3)P yiel ding PtdIns(3,5)P-2. Although Vps34p-mediated synthesis of PtdIns(3)P is required for the proper sorting of hydrolases from the Golgi to the vacuole, the production of PtdIns(3,5)P-2 by Fab1p does not directly affect Golgi to vacuole trafficking, suggesting that PtdIns(3,5)P-2 ha s a distinct function. The major phenotypes resulting from Fab1p kinas e inactivation include temperature-sensitive growth, vacuolar acidific ation defects, and dramatic increases in vacuolar size. Based on our s tudies, we hypothesize that whereas Vps34p is essential for anterograd e trafficking of membrane and protein cargoes to the vacuole, Fab1p ma y play an important compensatory role in the recycling/turnover of mem branes deposited at the vacuole. Interestingly, deletion of VAC7 also results in an enlarged vacuole morphology and has no detectable PtdIns (3,5)P-2, suggesting that Vac7p functions as an upstream regulator, pe rhaps in a complex with Fab1p. We propose that Fab1p and Vac7p are com ponents of a signal transduction pathway which functions to regulate t he efflux or turnover of vacuolar membranes through the regulated prod uction of PtdIns(3,5)P-2.