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Title:Isolation and molecular genetic analysis of conditionally lethal yeast mutants in glycosylphosphatidylinositol anchoring
Author(s):Leidich, Steven Donald
Doctoral Committee Chair(s):Orlean, Peter A.
Department / Program:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Biology, Genetics
Biology, Cell
Biology, Microbiology
Abstract:Glycosylphosphatidylinositol (GPI) anchors are a recently described class of glycolipids attached through covalent linkage to protein. To explore the functions of GPIs, I investigated the effects of GPI anchoring deficiencies in the unicellular eukaryote Saccharomyces cerevisiae.
I developed a way to isolate yeast GPI anchoring mutants. Yeast colonies are screened for defects in ($\sp3$H) inositol incorporation into protein, hence GPI anchoring. I report the isolation of three mutants, gpi1, gpi2, and gpi3. These mutants are all temperature-sensitive for growth, indicating that GPI synthesis fulfills an essential function in S. cerevisiae. The biochemical defects in the three mutants have been identified. All three are defective in vitro in the first step of GPI assembly, the synthesis of N-acetylglucosaminyl phosphatidylinositol. The wild-type counterparts of the defective genes in each of the mutants were isolated. The Gpi1 and Gpi2 proteins so far resemble no known proteins. The gpi3 mutant is defective in the yeast counterpart of the human PIG-A gene, which complements a mammalian GlcNAc-PI synthesis mutant.
The finding that at least three gene products are required for GlcNAc-PI synthesis raises the possibility that this step in GPI synthesis is carried out by a complex of proteins. Supportive of this notion are the findings that haploids containing any pairwise combination of gpi mutations exhibit double mutant lethality, and that overexpression of the GPI2 gene gives partial multicopy suppression of the gpi mutants temperature-sensitivity.
GPI anchoring deficient mutants show aberrations in cell surface growth and are defective in the developmental process leading to ascospore formation. In addition, the gpi1, gpi2, and gpi3 mutants have an intriguing transcriptional phenotype in that they suppress the effects of insertion of the Ty917 trans-poson into the regulatory region of the HIS4 gene.
The S. cerevisiae mutants I have isolated have provided the first demonstration that GPI synthesis is an essential process in a unicellular eukaryote.
Issue Date:1995
Rights Information:Copyright 1995 Leidich, Steven Donald
Date Available in IDEALS:2011-05-07
Identifier in Online Catalog:AAI9624415
OCLC Identifier:(UMI)AAI9624415

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