ISOLATION OF TEMPERATURE-SENSITIVE YEAST GPI-ANCHORING MUTANTS

We are using a genetic approach to explore the synthesis and function of glycosylphosphatidylinositol (GPI). We have developed a novel strategy to isolate Saccharomyces cerevisiae mutants blocked in GPI anchoring by screening colonies of mutagenized yeast cells for those that fail to incorporate [H-3]inositol into protein. Among our isolates are strains blocked in mannosylation of the GPI-anchor precursor, and strains defective in the synthesis of N-acetylglucosaminyl phosphatidyl inositol (GlcNAc-PI). We have characterized one mutant, gpil, further. This strain is defective in GlcNAc-PI synthesis and is temperature-sensitive for growth. Completion of the first step in GPI assembly is therefore required for the growth of the unicellular eukaryote S. cerevisiae. We have isolated plasmids that complement the gpil mutation from S. cerevisiae genomic DNA- and fission yeast cDNA libraries.