Temperature-sensitive Yeast GPI Anchoring Mutants gpi2 and gpi3 Are Defective in the Synthesis of N-Acetylglucosaminyl Phosphatidylinositol.

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.

SETH1andSETH2, Two Components of the Glycosylphosphatidylinositol Anchor Biosynthetic Pathway, Are Required for Pollen Germination and Tube Growth in Arabidopsis [W]

“…Saccharomyces cerevisiae GPI-deficient haploid ascospores are able to germinate and complete up to four cell divisions (Leidich et al, 1995). Moreover, GAPs added to animal cell cultures can be incorporated into surface membranes and exert native functions (Premkumar et al, 2001); therefore, it is possible that pollen tubes may acquire GPI from surrounding pistil tissues.…”

“…In human, an X-linked mutation of PIG-A in hematopoietic stem cells leads to paroxysmal nocturnal hemoglobinuria, an acquired clonal disease (Rosti, 2000). In haploid Saccharomyces cerevisiae strains, PIG-C mutations affect vegetative growth such that ascospores carrying the mutation germinate but complete no more than four cell divisions (Leidich et al, 1995). Partial deficiency of DPM biosynthesis in human causes the congenital disorder of glycosylation, resulting in seizures, hypotonia, and dysmorphic features (Imbach et al, 2000).…”

Regulation of the PIS1-encoded Phosphatidylinositol Synthase in Saccharomyces cerevisiae by Zinc

“…In addition to being a major structural component of the membrane, PI serves as the precursor for sphingolipids (7,8), the D-3, D-4, and D-5 phosphoinositides (3, 9 -12), and glycosylphosphatidylinositol anchors (13,14) (Fig. 1).…”

The Candida albicans homologue of PIG-P, CaGpi19p: gene dosage and role in growth and filamentation

“…This step is catalysed in humans by a complex of seven proteins: PIG-A, PIG-P, PIG-C, PIG-H, PIG-Q, PIG-Y and DPM2 (Watanabe et al, 1996(Watanabe et al, , 1998(Watanabe et al, , 2000Murakami et al, 2005), collectively referred to as the GPIGnT complex. Homologues of all proteins, except DPM2, have also been identified in members of the genus Saccharomyces (Vossen et al, 1995;Leidich et al, 1995;Leidich & Orlean, 1996;Yan et al, 2001). In C. albicans, very few studies have been carried out on the GPI anchor biosynthetic pathway itself and these have been restricted to genes further downstream in the pathway, for example CaSMP3 and Candida GPI7/LAS2 (Grimme et al, 2004;Richard et al, 2002).…”