| Glycosylphosphatidylinositol (GPI) anchors are glycophospholipids that serve to attach certain soluble proteins to the external leaflet of the plasma membrane in all eukaryotes. GPI biosynthesis begins with the synthesis of GlcNAc-PI. The enzyme that catalyzes this first step in yeast has been shown to be composed of three subunits: Gpi1p, Gpi2p, and Gpi3p. However, the mammalian version of this enzyme appeared to have more subunits, including two---Pig-Hp and Pig-Pp---that had not yet been identified in yeast. I identified the S. cerevisiae homologues of these proteins: Gpi15p/Ynl038wp and Ydr437wp. In addition, I found that the putative catalytic subunit of this enzyme, Gpi3p, which belongs to the superfamily of EX 7E-retaining glycosyltransferases, has a more stringent requirement for the second glutamate in its EX7E motif than the first in vivo, in contrast to findings in bacterial members of the EX7E glycosyltransferase family. I also investigated possible regulatory mechanisms of the GlcNAc-PI synthetic complex. The SWI1 gene had been previously identified in our laboratory by complementation of a mutant isolated in a screen designed to identify mutations that are synthetically lethal with gpi1. I found that membranes from a strain carrying a temperature-sensitive allele of SWI1 is unable to synthesize [14C]GlcNAc-PI in vitro at its restrictive temperature. Therefore, the SWI1 gene is required for efficient synthesis of GPI anchors in our strain background.; I also found that hyperactive Ras2 protein inhibits [14C]GlcNAc-PI synthesis in vitro, whereas Ras2p-deficient yeast membranes synthesize greater quantities of this glycolipid, and therefore that Ras2p may serve as a regulator of GPI synthesis.; Together, these results provide an enhanced view of the GlcNAc-PI synthase complex that initiates GPI assembly in yeast, as well as a basis for comparison to the human enzyme. |
