THE STRESS-ACTIVATED PHOSPHATIDYLINOSITOL 3-PHOSPHATE 5-KINASE FAB1P IS ESSENTIAL FOR VACUOLE FUNCTION IN SACCHAROMYCES-CEREVISIAE

Polyphosphoinositides have many roles in cell signalling and vesicle t rafficking [1-3]. Phosphatidylinositol 3,5-bisphosphate (PI(3,5)P-2) i s recently discovered PIP2 isomer, is ubiquitous in eukaryotic cells a nd rapidly accumulates in hyperosmotically stressed yeast PI(3,5)P-2 i s synthesised from PI(3)P in both yeast and mammalian cells [4,5]. A s earch of the Saccharomyces cerevisiae genome database identified FAB1, a gene encoding a PIP kinase homologue and potential PI(3)P 5-kinase. Fab1p shows PI(3)P B-kinase activity both in vivo and in vitro. A yea st strain in which FAB1 had been deleted was unable to synthesise PI(3 ,5)P-2, either in the presence or absence of osmotic shock. A loss of PI(3,5)P-2 was observed also in a temperature-sensitive FAB1 strain at the non-permissive temperature. A recombinant glutathione-S-transfera se (GST)-Fab1p fusion protein was shown to have selective PI(3)P 5-kin ase activity in vitro. Thus, we have demonstrated that Fab1p is a Pr(3 )P specific 5-kinase and represents a third class of PIP kinase activi ty, which we have termed type Ill. Deletion of the FAB1 gene produces a loss of vacuolar morphology [6]; it is therefore concluded that PI(3 ,5)P-2, the lipid product of Fab1p, is required for normal vacuolar fu nction.