Glycopeptide export from mammalian microsomes is independent of calcium and is distinct from oligosaccharide export

Glycopeptides are exported from the endoplasmic reticulum to the cytosol of eukaryotic membranes in an ATP-and cytosol-requiring process (Romisch and All, 1997, Proc, Natl Acad, Sci, USA, 94, 6730-6734). Oligosaccharides of t he polymannose-type are also exported from the endoplasmic reticulum of mam malian cells to the cytosol in an ATP-dependent fashion. These findings rai se the strong possibility that the two substrate classes are transported by the same mechanism but the precise identity of the translocation machinery for each substrate class has not been fully defined, Here we have investig ated the mechanism by which a glycopeptide is exported from rat liver micro somes, and compare this to the export of free polymannose oligosaccharides. Using EGTA and the endoplasmic reticulum calcium mobilizing agents thapsig argicin and calcium ionophores A23187 and ionomycin, we show that glycopept ides, in contrast to oligosaccharides, are exported by a calcium-independen t mechanism. On the other hand, Mg2+ is required in the assay for the trans port of glycopeptide from mammalian microsomes which is in common with olig osaccharide export. Deoxynojirimycin and castanospermine, inhibitors of ER glucosidases, when added to rat liver microsomes prior to loading with pept ide that bears an N-glycosylation sequon, had no effect on the release of g lucosylated glycopeptides from membranes, indicating that removal of the a- glucose units from the oligomannose glycan structure of the glycopeptide is not required for export. In contrast to oligosaccharides, where transport is efficiently inhibited, mannosides were without effect or only weak inhib itors of glycopeptide export. Taken together, these data suggest that glyco peptides are exported by a distinct mechanism from oligosaccharides of the polymannose-type and that the peptide moiety is an important structural det erminant for glycopeptide export and capable of directing translocation of substrates to a specific transport pathway.