Abstract
The translocation of UDP-glucose and GDP-mannose from an external to a luminal compartment has been examined in rat liver vesicles derived from the rough endoplasmic reticulum (RER). RER vesicles with the same topographical orientation as in vivo were incubated with a mixture of [3H]UDP-glucose and UDP-[14C]glucose to demonstrate that the intact sugar nucleotide was translocated into the lumen of the vesicles. The translocation of UDP-glucose was dependent on temperature and was saturable at high concentrations of the sugar nucleotide. The transfer of glucose to endogenous acceptors was dependent on the translocation of UDP-glucose into the lumen of the RER since leaky vesicles resulted in both a decrease in transport and transfer of glucose to endogenous acceptors. Preliminary results suggest that the mechanism of UDP-glucose transport into RER-derived vesicles is via a coupled exchange with luminal UMP. Using the same experimental approach to detect translocation of UDP-glucose into the lumen of RER vesicles, we were unable to detect transport of GDP-mannose. Incubation of leaky vesicles with GDP-mannose resulted in stimulation of the amount of mannose transferred to endogenous acceptors, in marked contrast to that observed for UDP-glucose and UDP-N-acetylglucosamine. These results suggest that whereas UDP-glucose is translocated across the RER membrane in vitro, GDP-mannose is not transported. In addition, these results tentatively suggest that there is asymmetric synthesis of the lipid-linked oligosaccharides within the membrane of the RER.
Highlights
The translocation of UDP-glucose was dependent on temperature and was saturabaltehigh concentrations of the sugar nucleotide
These systems function to translocation of UDP-glucose into the lumen of RER translocate GDP-fucose, CMP-sialic acid, UDP-galactose, vesicles, we were unable to detect transportof GDP- and UDP-GlcNAc from the cytoplasmicside of the membrane mannose
Accumulation of Radioactive Solutes within VesicZes-UDP[3H]Glcwas incubated with rat liver RER vesicles which were sealed and of the same membrane topography as found in vim
Summary
The firststepin the synthesis of an asparagine-linked lipid-linked oligosaccharides occurs on the cytoplasmic, as glycoproteinitshteransfer of the oligosaccharide well as theluminal, side of the RER membrane. One symmetrical peak was obtained which co-migrated with standard UDP-[3H]Glc in the following systems: Whatman 3MM paper chromatography in ethanol/ammonium acetate (1M, pH 7.5, 3:2) and ethanol/ammonium acetate (1M, pH 3.8, 52); chromatography on Dowex 2 formate columns; thin-layer chromatography on polyethyleneimine (Brinkman Polygram CEL 300 PEI) in LiCl(1M); high pressure liquid chromatography using a SynChrom, Inc. Extraction and Identification of RadiolabeledProducts in the Vesicle Pellets-To measure the incorporation of radiolabeled glucose and mannose into lipids and oligosaccharide fractions, the procedure of Snider et al[28]was used as previously described by Perez and Hirschberg [7]. The dolichol-associated chloroform/methanol extracts were examined by radioautography following thin layer chromatography on silica gel plateisn chloroform/methanol/water (60:25:4) Both dolichol-['4C]glucosyl phosphate and dolichol-["C] mannosyl phosphate derived from rat liver RER co-migrated with authentic d~lichol-[~~C]glucopsyhlosphate isolated from calf pancreas microsomes, a generous gift from Dr Annette Herscovics (McGill University).
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