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

Biochem J. 2000 Sep 15;350 Pt 3(Pt 3):815-22.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acetylglucosamine / analogs & derivatives*
  • Acetylglucosamine / biosynthesis*
  • Azides / chemistry*
  • Base Sequence
  • Binding Sites
  • Catalysis
  • DNA Primers
  • Escherichia coli / genetics
  • Glycosylphosphatidylinositols / biosynthesis*
  • Phosphatidylinositols / biosynthesis*
  • Photoaffinity Labels / chemistry*
  • Uracil Nucleotides / chemistry*

Substances

  • 4-(azidoanilido)uridine 5'-triphosphate
  • Azides
  • DNA Primers
  • Glycosylphosphatidylinositols
  • N-acetylglucosaminylphosphatidylinositol
  • Phosphatidylinositols
  • Photoaffinity Labels
  • Uracil Nucleotides
  • Acetylglucosamine