Photoaffinity labelling with P-3-(4-azidoanilido)uridine 5 '-triphosphate identifies Gpi3p as the UDP-GlcNAc-binding subunit of the enzyme that catalyses formation of GlcNAc-phosphatidylinositol, the first glycolipid intermediate in glycosylphosphatidylinositol synthesis

Glycosylphosphatidylinositols (GPIs) are made by all eukaryotes. The first step in their synthesis is the transfer of GlcNAc from UDP-GlcNAc to phosph atidylinositol (PI). Four proteins in mammals and at least three in yeast m ake up a complex that carries out this reaction. Three of the proteins are highly conserved between yeast and mammals: the Gpil protein, the Pig-C/Gpi 2 protein and the Pig-A/Gpi3 protein. The function of the individual subuni ts is not known, but of the three, the PigA/Gpi3 proteins resemble members of a large family of nucleotide-sugar-utilizing glycosyltransferases. To es tablish 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 P-3-(4-azidoanilido)uridine 5'-triphosp hate (AAUTP). We report that Gpi3p bearing the FLAG epitope at its C-termin us becomes cross-linked to AAUTP[alpha-P-32], but that Gpi2p-FLAG does not. Furthermore, Gpi3p-FLAG expressed in Escherichia coli is also cross-linked . These results indicate that Gpi3p is the UDP-GlcNAc-binding and probable catalytic subunit of the GlcNAc-PI synthetic complex.