Topography of Glycosylation Reactions in the Rough Endoplasmic Reticulum Membrane

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.