Genetic characterization of genes encoding enzymes catalyzing addition of phospho-ethanolamine to the glycosylphosphatidylinositol anchor in Saccharomyces cerevisiae

摘要

MPC1/GPI13/YLL031C, one of the genes involved in the addition of phosphoethanolamine to the glycosylphosphatidylinositol (GPI) anchor core, is an essential gene. Three available temperature-sensitive mutant alleles, mpc1-3, mpc1-4, and mpc1-5, displayed different phenotypes to each other and, correspondingly, these mutants were found to have different mutations in the MPC1 ORF. Temperature-sensitivity of mpc1-5 mutants was suppressed by 5 mM ZnSO4 and by 5 mM MnCl2. Multicopy suppressors were isolated from mpc1-5 mutant. Suppressors commonly effective to mpcl-4 and mpcl-5 mutations are PSD1, encoding phosphatidylserine decarboxylase, and ECM33, which were found to suppress the temperature-sensitive phenotype shown by the fsr2-1 and las21Delta mutants, those of which have defects in the GPI anchor synthesis. PSD2, encoding another phosphatidylserine decarboxylase that is localized in Golgi/vacuole, was found to be able to serve as a multicopy suppressor of mpc1 and fsr2-1 mutants but not of the 1as21Delta mutant. In contrast to psd1Delta, psd2Delta showed a synthetic growth defect with mpc1 mutants but not with fsr2-1 or las21Delta. Furthermore, psd1Delta psd2Delta mpc1 triple mutants did not form colonies on nutrient medium unless ethanolamine was supplied to the medium, whereas psd1Delta psd2Delta fsr2-1 or psd1Delta psd2Delta las21Delta triple mutants grew on nutrient medium without supplementation of ethanolamine. These observations suggest that Mpc1 preferentially utilizes phosphatidylethanolamine produced by Psd2 that is localized in Golgi/vacuole. fsr2-1 dpl1Delta psd1Delta strains showed slower growth than fsr2-1 dpl1Delta psd2Delta, suggesting that Fsr2 enzyme depends more on Dpl1 and Psd1 for production of phosphatidylethanolamine. Las21 did not show preference for the metabolic pathway to produce phosphatidylethanolamine.