Glycosylphosphatidylinositols (GPIs) are made by all eukaryotes. The first step in their synthesis is the transfer of GlcNAc from UDP-GlcNAc to phosphatidylinositol (PI). Four proteins in mammals and at least three in yeast make up a complex that carries out this reaction. Three of the proteins are highly conserved between yeast and mammals: the Gpi1 protein, the Pig-C/Gpi2 protein and the Pig-A/Gpi3 protein. The function of the individual subunits is not known, but of the three, the Pig-A/Gpi3 proteins resemble members of a large family of nucleotide-sugar-utilizing glycosyltransferases. To establish whether Gpi3p is the UDP-GlcNAc-binding subunit of the yeast GlcNAc-PI synthetic complex, we tested its ability to become cross-linked to the photoactivatable substrate analogue P3-(4-azidoanilido)-uridine 5´-triphosphate (AAUTP). We report that Gpi3p bearing the FLAG epitope at its C-terminus becomes cross-linked to AAUTP[α-32P], but that Gpi2p-FLAG does not. Furthermore, Gpi3p-FLAG expressed in Escherichiacoli is also cross-linked. These results indicate that Gpi3p is the UDP-GlcNAc-binding and probable catalytic subunit of the GlcNAc-PI synthetic complex.

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Author notes


These authors contributed equally to this work.