GENETIC AND BIOCHEMICAL-CHARACTERIZATION OF A PHOSPHATIDYLINOSITOL-SPECIFIC PHOSPHOLIPASE-C IN SACCHAROMYCES-CEREVISIAE

Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphat idylinositol-specific phospholipase C (PI-PLC) generates two second me ssengers, inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. The pol ymerase chain reaction was used to isolate a Saccharomyces cerevisiae gene (PLC1) that encodes a protein of 869 amino acids (designated Plc1 p) that bears greatest resemblance to the delta isoforms of mammalian PI-PLC in terms of overall sequence similarity and domain arrangement. Plc1p contains the conserved X and Y domains found in all higher euka ryotic PI-PLCs (51 and 29% identity, respectively, to the correspondin g domains of rat delta1 PI-PLC) and also contains a presumptive Ca2+-b inding site (an E-F hand motif). Plc1p, modified by in-frame insertion of a His, tract and a c-myc epitope near its amino terminus, was over expressed from the GAL1 promoter, partially purified by nickel chelate affinity chromatography, and shown to be an active PLC enzyme in vitr o with properties similar to those of its mammalian counterparts. Plc1 p activity was strictly Ca2+ dependent: at a high Ca2+ concentration ( 0.1 mM), the enzyme hydrolyzed PIP2 at a faster rate than phosphatidyl inositol, and at a low Ca2+ concentration (0.5 muM), it hydrolyzed PIP , exclusively. Cells carrying either of two different deletion-inserti on mutations (plc1DELTA1::HIS3 and plc1DELTA2::LEU2) were viable but d isplayed several distinctive phenotypes, including temperature-sensiti ve growth (inviable above 35-degrees-C), osmotic sensitivity, and defe cts in the utilization of galactose, raffinose, and glycerol at permis sive temperatures (23 to 30-degrees-C). The findings reported here sug gest that hydrolysis of PIP2 in S. cerevisiae is required for a number of nutritional and stress-related responses.