Title

Topography of initiation of N-glycosylation reactions

UMMS Affiliation

Department of Biochemistry and Molecular Biology

Publication Date

8-25-1990

Document Type

Article

Subjects

Animals; Bromodeoxyuridine; Edetic Acid; Endoplasmic Reticulum; Galactosyltransferases; Glycosylation; Liver; Models, Biological; Phosphorus Radioisotopes; Polyisoprenyl Phosphate Sugars; Protein Processing, Post-Translational; Rats; Tritium; Tunicamycin; Uridine Diphosphate N-Acetylglucosamine; Uridine Diphosphate Sugars

Disciplines

Life Sciences | Medicine and Health Sciences

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 membrane-impermeable reagent) inhibited the N-acetylglucosamine-1-phosphate transferase under conditions where transport of the nucleotide sugar into the lumen was unaffected. Finally, sealed vesicles were first incubated with [32P]UDP-GlcNAc and subsequently with UDP-Gal and soluble galactosyltransferase. This resulted in galactosylation of dolichol-P-P-GlcNAc2. The above results, together with the previous observations, strongly suggest that all reactions leading to this latter dolichol intermediate occur on the cytosolic side of the endoplasmic reticulum membrane.

Rights and Permissions

Citation: J Biol Chem. 1990 Aug 25;265(24):14691-5.

Related Resources

Link to Article in PubMed

Journal/Book/Conference Title

The Journal of biological chemistry

PubMed ID

2117613