Establishment of a real-time analytical method for free oligosaccharide transport from the ER to the cytosol.

During N-glycosylation of proteins, significant amounts of free unconjugated glycans are also generated in the lumen of the endoplasmic reticulum (ER). These ER-derived free glycans are translocated into the cytosol by a putative transporter on the ER membrane for further processing. However, the molecular nature of the transporter remains to be determined. Here, we report the establishment of a novel assay method for free oligosaccharide transport from the ER lumen using chemically synthesized fluorescence-labeled N-glycan derivatives. In this method, fluorescence-labeled glycan substrates were encapsulated inside mouse liver microsomes, followed by incubation with the cytosol and a fluorescence-quenching agent (anti-fluorophore antibody). The rate of substrate efflux was then monitored in real time by the decrease in the fluorescence intensity. The present data clearly demonstrated that the oligosaccharide transport activity under the current assay conditions was both ATP and cytosol dependent. The transporter activity was also found to be glycan structure specific because free glucosylated glycans were unable to be transported out of the microsomes. This new assay method will be a useful tool for identifying the transporter protein on the ER membrane.