Topography of initiation of N-glycosylation reactions

Journal Article · Sat Aug 25 04:00:00 EDT 1990 · Journal of Biological Chemistry; (USA)

OSTI ID:6759807

Abeijon, C; Hirschberg, C B ^[1]

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1. Univ. of Massachusetts Medical Center, Worcester (USA)

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. 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.

• OSTI ID: 6759807
• Journal Information: Journal of Biological Chemistry; (USA), Journal Name: Journal of Biological Chemistry; (USA) Vol. 265:24; ISSN JBCHA; ISSN 0021-9258
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
ANIMALS
ANTIMETABOLITES
AZINES
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOSYNTHESIS
BROMOURACILS
BUDR
CARBOHYDRATES
CARBOXYLIC ACIDS
CELL CONSTITUENTS
CELL MEMBRANES
CHELATING AGENTS
DAYS LIVING RADIOISOTOPES
DRUGS
EDTA
ENDOPLASMIC RETICULUM
ENZYMES
GLYCOSYL TRANSFERASES
HETEROCYCLIC COMPOUNDS
HYDROXY COMPOUNDS
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
MAMMALS
MEMBRANE TRANSPORT
MEMBRANES
NUCLEI
NUCLEOSIDES
NUCLEOTIDES
ODD-ODD NUCLEI
OLIGOSACCHARIDES
ORGANIC ACIDS
ORGANIC BROMINE COMPOUNDS
ORGANIC COMPOUNDS
ORGANIC HALOGEN COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANOIDS
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
POST-TRANSLATION MODIFICATION
PYRIMIDINES
RADIOISOTOPES
RATS
RIBOSIDES
RODENTS
SACCHARIDES
SYNTHESIS
TRACER TECHNIQUES
TRANSFERASES
URACILS
VERTEBRATES