GWT1 Gene Is Required for Inositol Acylation of Glycosylphosphatidylinositol Anchors in Yeast

• Published in: The Journal of biological chemistry 2003-06, Vol.278 (26), p.23639-23647
• Main Authors: Umemura, Mariko, Okamoto, Michiyo, Nakayama, Ken-ichi, Sagane, Koji, Tsukahara, Kappei, Hata, Katsura, Jigami, Yoshifumi
• Format: Article
• Language: English
• Subjects: Acyl Coenzyme A - metabolism; Acylation; Amino Acid Sequence; Glycosylphosphatidylinositols - biosynthesis; Glycosylphosphatidylinositols - metabolism; Inositol - metabolism; Molecular Sequence Data; Mutation; Protein Transport; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - physiology; Sequence Alignment; Substrate Specificity; Temperature; Yeasts - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M301044200

Glycosylphosphatidylinositol (GPI) is a conserved post-translational modification to anchor cell surface proteins to plasma membrane in all eukaryotes. In yeast, GPI mediates cross-linking of cell wall mannoproteins to β1,6-glucan. We reported previously that the GWT1 gene product is a target of the novel anti-fungal compound, 1-[4-butylbenzyl]isoquinoline, that inhibits cell wall localization of GPI-anchored mannoproteins in Saccharomyces cerevisiae (Tsukahara, K., Hata, K., Sagane, K., Watanabe, N., Kuromitsu, J., Kai, J., Tsuchiya, M., Ohba, F., Jigami, Y., Yoshimatsu, K., and Nagasu, T. (2003) Mol. Microbiol. 48, 1029–1042). In the present study, to analyze the function of the Gwt1 protein, we isolated temperature-sensitive gwt1 mutants. The gwt1 cells were normal in transport of invertase and carboxypeptidase Y but were delayed in transport of GPI-anchored protein, Gas1p, and were defective in its maturation from the endoplasmic reticulum to the Golgi. The incorporation of inositol into GPI-anchored proteins was reduced in gwt1 mutant, indicating involvement of GWT1 in GPI biosynthesis. We analyzed the early steps of GPI biosynthesis in vitro by using membranes prepared from gwt1 and Δ gwt1 cells. The synthetic activity of GlcN-(acyl)PI from GlcN-PI was defective in these cells, whereas Δ gwt1 cells harboring GWT1 gene restored the activity, indicating that GWT1 is required for acylation of inositol during the GPI synthetic pathway. We further cloned GWT1 homologues in other yeasts, Cryptococcus neoformans and Schizosaccharomyces pombe , and confirmed that the specificity of acyl-CoA in inositol acylation, as reported in studies of endogenous membranes (Franzot, S. P., and Doering, T. L. (1999) Biochem. J. 340, 25–32), is due to the properties of Gwt1p itself and not to other membrane components.