TRANSPORT OF FREE POLYMANNOSE-TYPE OLIGOSACCHARIDES FROM THE ENDOPLASMIC-RETICULUM INTO THE CYTOSOL IS INHIBITED BY MANNOSIDES AND REQUIRESA THAPSIGARGIN-SENSITIVE CALCIUM STORE

The transport of free polymannose-type oligosaccharides from the lumen of the endoplasmic reticulum into the cytosol has been recently demon strated (Moore, S.E.H., et al., 1995, EMBO J., 14, 6034-6042), but at present little is known of the characteristics of this process, Here, it is shown that inhibition of the transport of endogenously synthesiz ed metabolically radiolabeled free oligosaccharides out of the endopla smic reticulum into the cytosol of permeabilized HepG2 cells occurs wh en assays are conducted in the presence of mannose (IC50, 4.9 mM), or its derivatives modified at the first carbon (C1) of the sugar ring; a lpha-methyl mannoside (IC50, 2.0 mM), mannoheptulose (IC50, 1.6 mM), a nd alpha-benzyl mannoside (IC50, 0.8 mM), whereas other monosaccharide s (50 mM), differing from mannose at position; C2 (glucose), C3 (altro se), C4 (talose), C5 (L-rhamnose), and C6 (mannoheptose), have little effect, N-Acetylglucosamine does not inhibit oligosaccharide transport and, furthermore, although mannobioses and a mannotriose inhibit free oligosaccharide transport, di-N-acetylchitobiose is without effect, I t is also shown that if the transport assay buffer is either depleted of calcium ions, or supplemented with the Ca2+/Mg2+ ATPase inhibitor, thapsigargin, or with calcium ionophores, free oligosaccharide transpo rt out of the endoplasmic reticulum is inhibited, These results demons trate that the terminal nonreducing mannosyl residues of free polymann ose-type oligosaccharides and not their N-acetylglucosamine-containing reducing termini, play an important role in the interaction of the fr ee oligosaccharide with the transport machinery, and that this transpo rt process requires the presence of calcium sequestered in the lumen o f the endoplasmic reticulum.