Cytosol-to-lysosome transport of free polymannose-type oligosaccharides - Kinetic and specificity studies using rat liver lysosomes

In hepatocellular carcinoma HepG2 cells, free polymannose-type oligosacchar ides appearing in the cytosol during the biosynthesis and quality control o f glycoproteins are rapidly translocated into lysosomes by an as yet pearly defined process (Saint-Pol, A., Bauvy, C., Codogno, P., and Moore, S. E. H . (1997) J. Cell Biol. 136, 45-59), Here, we demonstrate an ATP-dependent a ssociation of [2-H-3]mannose-labeled Man(5)GlcNAc with isolated rat liver l ysosomes, This association was only observed in the presence of swainsonine , a mannosidase inhibitor, which was required for the protection of sedimen table, but not nonsedimentable, Man(5)GlcNAc from degradation, indicating t hat oligosaccharides were transported into lysosomes, Saturable high affini ty transport (K-uptake, 22.3 mu M, V-max, 7.1 fmol/min/unit of P hexosamini dase) was dependent upon the hydrolysis of ATP but independent of vacuolar H+/ATPase activity. Transport. was inhibited strongly by NEM and weakly by vanadate but not by sodium azide, and, in addition, the sugar transport inh ibitors phloretin, phloridzin, and cytochalasin B were without effect on tr ansport. Oligosaccharide import did not show absolute specificity but was s elective toward partially demannosylated and dephosphorylated oligosacchari des, and, furthermore, inhibition studies revealed that the free reducing G lcNAc residue of the oligosaccharide was of critical importance for its int eraction with the transporter. These results demonstrate the presence of a novel lysosomal free oligosaccharide transporter that must work in concert with cytosolic hydrolases in order to clear the cytosol of endoplasmic reti culum-generated free oligosaccharides.