TY - JOUR
T1 - Glycopeptide export from mammalian microsomes is independent of calcium and is distinct from oligosaccharide export
AU - Ali, Bassam R.S.
AU - Field, Mark C.
PY - 2000/4
Y1 - 2000/4
N2 - Glycopeptides are exported from the endoplasmic reticulum to the cytosol of eukaryotic membranes in an ATP- and cytosol-requiring process. Oligosaccharides of the polymannose-type are also exported from the endoplasmic reticulum of mammalian cells to the cytosol in an ATP-dependent fashion. These findings raise 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 investigated the mechanism by which a glycopeptide is exported from rat liver microsomes, and compare this to the export of free polymannose oligosaccharides. Using EGTA and the endoplasmic reticulum calcium mobilizing agents thapsigargicin and calcium ionophores A23187 and ionomycin, we show that glycopeptides, in contrast to oligosaccharides, are exported by a calcium-independent mechanism. On the other hand, Mg2+ is required in the assay for the transport of glycopeptide from mammalian microsomes which is in common with oligosaccharide export. Deoxynojirimycin and castanospermine, inhibitors of ER glucosidases, when added to rat liver microsomes prior to loading with peptide that bears an N-glycosylation sequon, had no effect on the release of glucosylated glycopeptides from membranes, indicating that removal of the α-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 inhibitors of glycopeptide export. Taken together, these data suggest that glycopeptides are exported by a distinct mechanism from oligosaccharides of the polymannose-type and that the peptide moiety is an important structural determinant for glycopeptide export and capable of directing translocation of substrates to a specific transport pathway.
AB - Glycopeptides are exported from the endoplasmic reticulum to the cytosol of eukaryotic membranes in an ATP- and cytosol-requiring process. Oligosaccharides of the polymannose-type are also exported from the endoplasmic reticulum of mammalian cells to the cytosol in an ATP-dependent fashion. These findings raise 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 investigated the mechanism by which a glycopeptide is exported from rat liver microsomes, and compare this to the export of free polymannose oligosaccharides. Using EGTA and the endoplasmic reticulum calcium mobilizing agents thapsigargicin and calcium ionophores A23187 and ionomycin, we show that glycopeptides, in contrast to oligosaccharides, are exported by a calcium-independent mechanism. On the other hand, Mg2+ is required in the assay for the transport of glycopeptide from mammalian microsomes which is in common with oligosaccharide export. Deoxynojirimycin and castanospermine, inhibitors of ER glucosidases, when added to rat liver microsomes prior to loading with peptide that bears an N-glycosylation sequon, had no effect on the release of glucosylated glycopeptides from membranes, indicating that removal of the α-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 inhibitors of glycopeptide export. Taken together, these data suggest that glycopeptides are exported by a distinct mechanism from oligosaccharides of the polymannose-type and that the peptide moiety is an important structural determinant for glycopeptide export and capable of directing translocation of substrates to a specific transport pathway.
KW - ERAD
KW - Endoplasmic reticulum
KW - Glycopeptide
KW - Oligosaccharide
KW - Transport
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U2 - 10.1093/glycob/10.4.383
DO - 10.1093/glycob/10.4.383
M3 - Article
C2 - 10764826
AN - SCOPUS:0034026440
SN - 0959-6658
VL - 10
SP - 383
EP - 391
JO - Glycobiology
JF - Glycobiology
IS - 4
ER -