Purification and properties of a phospholipid acyl hydrolase from plasma membranes ofSaccharomyces cerevisiae

Characterisation of glucose transport in with plasma membrane vesicles (countertransport) and intact cells (initial uptake) with single Hxt1, Hxt2, Hxt3, Hxt4, Hxt6, Hxt7 or Gal2 transporters

“…As shown in other wild-type strains [8,12,13,15^17] MC996A also demonstrated the phenomenon of countertransport. As shown in other wild-type strains [8,12,13,15^17] MC996A also demonstrated the phenomenon of countertransport.…”

“…The method of vesicle preparation described by Fuhrmann et al [8] was modi¢ed slightly. Mitochondrial vesicles were aggregated at pH 4.5 instead of pH 4 and glycine/KCl solution (0.3 M KCl and 0.1 M glycine/HCl, pH 7.0) was used as osmotic stabiliser throughout the whole preparation [12] with addition of 150 WM phenylmethylsulfonyl £uoride (PMSF, prepared in methanol), 0.9 WM pepstatin (prepared in ethanol) and 1.8 mM EDTA for proteolysis protection. Before aggregation of mitochondrial vesicles at pH 4.5, PMSF, pepstatin and EDTA were added again to the glycine/KCl solution.…”

Production and reutilization of an extracellular phosphatidylinositol catabolite, glycerophosphoinositol, by Saccharomyces cerevisiae

“…GroPIns, GroPIns 4-phosphate, and GroPIns 4,5-bisphosphate are produced from PI, PI 4-phosphate, and PI 4,5-bisphosphate, respectively, by a phospholipase A and a lysophospholipase acting sequentially or by a phospholipase B. Phospholipase B activities have been found in the plasma membrane, the periplasmic space, and in the medium of S. cerevisiae (11,(21)(22)(23). Recently, a gene (PLB1) encoding a protein predicted to have a 45% amino acid sequence identity with phospholipase B from Penicillium notatum has been isolated from S. cerevisiae (13).…”

“…Thus, the extracellular accumulation of GroPIns was shown to be specific, regulated, and a major route of PI turnover in a growing yeast culture. Recently, Hawkins et al (9) confirmed that the addition of glucose to stationary-phase cultures results in the extracellular production of not only GroPIns but also low levels of GroPIns 4-phosphate and GroPIns 4,5-bisphosphate.GroPIns, GroPIns 4-phosphate, and GroPIns 4,5-bisphosphate are produced from PI, PI 4-phosphate, and PI 4,5-bisphosphate, respectively, by a phospholipase A and a lysophospholipase acting sequentially or by a phospholipase B. Phospholipase B activities have been found in the plasma membrane, the periplasmic space, and in the medium of S. cerevisiae (11,(21)(22)(23). Recently, a gene (PLB1) encoding a protein predicted to have a 45% amino acid sequence identity with phospholipase B from Penicillium notatum has been isolated from S. cerevisiae (13).…”

Characterization and Function in Vivo of Two Novel Phospholipases B/Lysophospholipases fromSaccharomyces cerevisiae

“…The highly glycosylated enzyme of about 220 kDa (73 kDa for the protein part, predicted from the sequence) is enriched in the yeast plasma membrane but was also found in the periplasmic space and in the culture supernatant. The lysophospholipase activity of Plb1p greatly exceeds the activity catalyzing the first step of hydrolysis; thus, lyso-phospholipids do not accumulate as intermediate products of Plb1p activity (2)(3)(4). In addition, this enzyme has transacylase activity, catalyzing the synthesis of phosphatidylcholine (PtdCho) 1 from two molecules of lyso-phosphatidylcholine.…”