PMID: 2117613Aug 25, 1990Paper

Topography of initiation of N-glycosylation reactions.

The Journal of Biological Chemistry
C Abeijon, C B Hirschberg

Abstract

Previous studies on the topography of the reactions leading to the formation of dolichol-P-P-Glc-NAc2Man9Glc3 have shown that these occur on both sides of the endoplasmic reticulum membrane (Hirschberg, C. B., and Snider, M. D. (1987) Annu. Rev. Biochem. 56, 63-87). Dolichol-P-P-GlcNAc2Man5 has been detected on the cytoplasmic side of the endoplasmic reticulum membrane while the subsequent dolichol-oligosaccharide intermediates face the lumen. Less clear is the side of the membrane where dolichol-P-P-GlcNAc2 is assembled. We now present evidence strongly suggesting that the active sites of the enzymes catalyzing the synthesis of this latter intermediate are on the cytoplasmic side of the endoplasmic reticulum membrane. In addition, dolichol-P-P-GlcNAc2 has also been detected on this side. Incubations of sealed, "right side out" rat liver endoplasmic reticulum-derived vesicles with [beta-32P] UDP-GlcNAc in the presence of 5-Br-UMP resulted in the formation of radiolabeled dolichol-P-P-GlcNAc and dolichol-P-P-GlcNAc2 under conditions where there was complete inhibition of transport of the nucleotide sugar. In other experiments with the above radiolabeled nucleotide sugar and sealed vesicles, it was demonstrated that EDTA (a membr...Continue Reading

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