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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Research Article|
September 08 2000
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
Zlatka KOSTOVA;
Zlatka KOSTOVA
1
*Department of Biochemistry, University of Illinois at Urbana-Champaign, Roger Adams Laboratory, 600 South Mathews Avenue, Urbana, IL 61801, U.S.A.
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David M. RANCOUR;
David M. RANCOUR
1
†Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706, U.S.A.
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Anant K. MENON;
Anant K. MENON
†Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706, U.S.A.
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Peter ORLEAN
Peter ORLEAN
2
*Department of Biochemistry, University of Illinois at Urbana-Champaign, Roger Adams Laboratory, 600 South Mathews Avenue, Urbana, IL 61801, U.S.A.
2To whom correspondence should be addressed (e-mail p-orlean@uiuc.edu).
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Publisher: Portland Press Ltd
Received:
January 10 2000
Revision Received:
May 02 2000
Accepted:
July 06 2000
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 2000
2000
Biochem J (2000) 350 (3): 815–822.
Article history
Received:
January 10 2000
Revision Received:
May 02 2000
Accepted:
July 06 2000
Citation
Zlatka KOSTOVA, David M. RANCOUR, Anant K. MENON, Peter ORLEAN; 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 15 September 2000; 350 (3): 815–822. doi: https://doi.org/10.1042/bj3500815
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