Complexity of the Late Stages of GPI Anchor Biosynthesis in Saccharomyces Cerevisiae

148 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1999.I identified a yeast homolog of the human PIG-F protein, a protein suspected of adding phosphoethanolamine to the third mannose of the mammalian GPI anchor. Genetic disruption of the yeast PIG-F gene (termed GPI11), demonstrated that it is essential for vegetative growth. Yeast gpi11 mutants in which the Gpi11 protein (Gpi11p) could be conditionally depleted were constructed and tested for GPI anchoring defects. Cells depleted of Gpi11p were unable to incorporate [3H]inositol into proteins, were unable to make yeast complete GPI precursors, and accumulated two base-labile PI-PLC-resistant GPI intermediates. Structural characterization of these two lipids revealed that they both contained four mannoses and differed in the position and probably the number of polar P-EthN substituents. The less polar of the two lipids bears a P-EthN on its second mannose demonstrating that the yeast GPI, like the mammalian anchor, becomes modified at this position. The more polar lipid has P-EthN on its third mannose, indicating that Gpi11p is likely not the P-EthN transferase responsible for adding P-EthN to Man-3 of the anchor. The most likely model for the structures of the lipids that accumulate upon Gpi11p deficiency implies that these lipids are generated independently, in turn suggesting branching or sub-compartmentalization in the yeast GPI synthetic pathway.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD