Temperature-sensitive yeast GPI anchoring mutants gpi2 and gpi3 are defective in the synthesis of N-acetylglucosaminyl phosphatidylinositol. Cloning of the GPI2 gene

S D Leidich; Z Kostova; R R Latek; L C Costello; D A Drapp; W Gray; J S Fassler; P Orlean

doi:10.1074/jbc.270.22.13029

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Temperature-sensitive yeast GPI anchoring mutants gpi2 and gpi3 are defective in the synthesis of N-acetylglucosaminyl phosphatidylinositol. Cloning of the GPI2 gene

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Temperature-sensitive yeast GPI anchoring mutants gpi2 and gpi3 are defective in the synthesis of N-acetylglucosaminyl phosphatidylinositol. Cloning of the GPI2 gene

S D Leidich, Z Kostova, R R Latek, L C Costello, D A Drapp, W Gray, J S Fassler and P Orlean

The Journal of biological chemistry, Vol.270(22), pp.13029-13035

06/02/1995

DOI: 10.1074/jbc.270.22.13029

PMID: 7768896

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Abstract

To identify genes required for the synthesis of glycosyl phosphatidylinositol (GPI) membrane anchors in yeast, we devised a way to isolate GPI anchoring mutants in which colonies are screened for defects in [3H]-inositol incorporation into protein. The gpi1 mutant, identified in this way, is temperature sensitive for growth and defective in vitro in the synthesis of GlcNAc-phosphatidylinositol, the first intermediate in GPI biosynthesis (Leidich, S. D., Drapp, D. A., and Orlean, P. (1994) J. Biol. Chem. 269, 10193-10196). We report the isolation of two more conditionally lethal mutants, gpi2 and gpi3, which, like gpi1, have a temperature-sensitive defect in the incorporation of [3H]inositol into protein and which lack in vitro GlcNAc-phosphatidylinositol synthetic activity. Haploid Saccharomyces cerevisiae strains containing any pairwise combination of the gpi1, gpi2, and gpi3 mutations are inviable. The GPI2 gene, cloned by complementation of the gpi2 mutant's temperature sensitivity, encodes a hydrophobic 269-amino acid protein that resembles no other gene product known to participate in GPI assembly. Gene disruption experiments show that GPI2 is required for vegetative growth. Overexpression of the GPI2 gene causes partial suppression of the gpi1 mutant's temperature sensitivity, a result that suggests that the Gpi1 and Gpi2 proteins interact with one another in vivo. The gpi3 mutant is defective in the SPT14 gene, which encodes a yeast protein similar to the product of the mammalian PIG-A gene, which complements a GlcNAc-phosphatidylinositol synthesis-defective human cell line. In yeast, at least three gene products are required for the first step in GPI synthesis, as is the case in mammalian cells, and utilization of several different proteins at this stage is therefore likely to be a general characteristic of the GPI synthetic pathway.

Amino Acid Sequence

Base Sequence

Cell Line

Cloning, Molecular

DNA, Fungal

Glycosylphosphatidylinositols - genetics

Humans

Molecular Sequence Data

Mutation

Phosphatidylinositols - biosynthesis

Saccharomyces cerevisiae - genetics

Saccharomyces cerevisiae - metabolism

Temperature

Details

Title: Subtitle: Temperature-sensitive yeast GPI anchoring mutants gpi2 and gpi3 are defective in the synthesis of N-acetylglucosaminyl phosphatidylinositol. Cloning of the GPI2 gene
Creators: S D Leidich - Department of Biochemistry, University of Illinois at Urbana-Champaign 61801, USA
Z Kostova
R R Latek
L C Costello
D A Drapp
W Gray
J S Fassler
P Orlean
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.270(22), pp.13029-13035
DOI: 10.1074/jbc.270.22.13029
PMID: 7768896
NLM abbreviation: J Biol Chem
ISSN: 0021-9258
eISSN: 1083-351X
Grant note: GM46220 / NIGMS NIH HHS GM40306 / NIGMS NIH HHS
Language: English
Date published: 06/02/1995
Academic Unit: Biology
Record Identifier: 9984217425202771

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