Characterization and function in vivo of two novel phospholipases B/lysophospholipases from Saccharomyces cerevisiae

The yeast genome contains two genes, designated as PLB2 and PLB3, that are 67% and 62% identical, respectively, to PLB1, which codes for a phospholipa se B/lysophospholipase in yeast (Lee, S. K., Patton, J. L., Fido, M., Dines , L. K., Kohlwein, S. D., Paltauf, F,, Henry, S. A., and Levin, D. E. (1994 ) J. Biol, Chem. 269, 19725-19730). Deletion and overexpression studies and in vivo and in vitro activity measurements suggest that both genes indeed code for phospholipases B/lysophospholipases. In cell free extracts of a pl b1 plb2 plb3 triple mutant, no phospholipase B activity was detectable. Upo n overexpression of PLB2 in a plb1 plb3 mutant background, phospholipase B activity was detectable in the plasma membrane, periplasmic space extracts and the culture supernatant. Similar to Plb1p, Plb2p appears to accept all major phospholipid classes, with a preference for acidic phospholipids incl uding phosphatidylinositol 3',4'-bisphosphate and phosphatidic acid. Consis tent with a function as an extracellular lysophospholipase, PLB2 overexpres sion conferred resistance to lyso-phosphatidylcholine. Deletion of Plb2p fu nction had no effect on glycerophosphoinositol or glycerophosphocholine re. lease in vivo, in contrast to a deletion of Plb3p function, which resulted in a 50% reduction of phosphatidylinositol breakdown and glycerophosphoino sitol release from the cells, lit vitro, Plb3p hydrolyzes only phosphatidyl inositol and phosphatidylserine and, to a lesser extent, their lyso-analogs . Plb3p activity in a plb1 plb2 mutant background was observed in periplasm ic space extracts. Both Plb3p and Plb2p display transacylase activity in vi tro, in the presence or absence, respectively, of detergent.