Genetic and Biochemical Characterization of a Phosphatidylinositol-Specific Phospholipase C in Saccharomyces cerevisiae

Abstract

Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) by phosphatidylinositol-specific phospholipase C (PI-PLC) generates two second messengers, inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. The polymerase chain reaction was used to isolate a Saccharomyces cerevisiae gene (PLC1) that encodes a protein of 869 amino acids (designated Plc1p) that bears greatest resemblance to the δ 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 eukaryotic PI-PLCs (51 and 29% identity, respectively, to the corresponding domains of rat δ 1 PI-PLC) and also contains a presumptive Ca²⁺-binding site (an E-F hand motif). Plc1p, modified by in-frame insertion of a His₆ tract and a c-myc epitope near its amino terminus, was overexpressed from the GAL1 promoter, partially purified by nickel chelate affinity chromatography, and shown to be an active PLC enzyme in vitro with properties similar to those of its mammalian counterparts. Plc1p activity was strictly Ca²⁺ dependent: at a high Ca²⁺ concentration (0.1 mM), the enzyme hydrolyzed PIP₂ at a faster rate than phosphatidylinositol, and at a low Ca²⁺ concentration (0.5 microM), it hydrolyzed PIP₂ exclusively. Cells carrying either of two different deletion-insertion mutations (plc1 δ 1::HIS3 and plc1 δ 2::LEU2) were viable but displayed several distinctive phenotypes, including temperature-sensitive growth (inviable above 35 degrees C), osmotic sensitivity, and defects in the utilization of galactose, raffinose, and glycerol at permissive temperatures (23 to 30 degrees C). The findings reported here suggest that hydrolysis of PIP₂ in S. cerevisiae is required for a number of nutritional and stress-related responses.