Photoaffinity labelling with P3-(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 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 P(3)-(4-azidoanilido)-uridine 5'-triphosphate (AAUTP). We report that Gpi3p bearing the FLAG epitope at its C-terminus becomes cross-linked to AAUTP[alpha-(32)P], 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.
Kostova
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Department of Biochemistry, University of Illinois at Urbana-Champaign, Roger Adams Laboratory, 600 South Mathews Avenue, Urbana, IL 61801, USA.
Rancour
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DM
Menon
A K
AK
Orlean
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P
D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000096:biosynthesis*; D001386:Azides / Q000737:chemistry*; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D017261:Glycosylphosphatidylinositols / Q000096:biosynthesis*; D010716:Phosphatidylinositols / Q000096:biosynthesis*; D019920:Photoaffinity Labels / Q000737:chemistry*; D014500:Uracil Nucleotides / Q000737:chemistry*
C114327:4-(azidoanilido)uridine 5'-triphosphate; D001386:Azides; D017931:DNA Primers; D017261:Glycosylphosphatidylinositols; C081027:N-acetylglucosaminylphosphatidylinositol; D010716:Phosphatidylinositols; D019920:Photoaffinity Labels; D014500:Uracil Nucleotides; D000117:Acetylglucosamine