TRANSFER OF FREE POLYMANNOSE-TYPE OLIGOSACCHARIDES FROM THE CYTOSOL TO LYSOSOMES IN CULTURED HUMAN HEPATOCELLULAR-CARCINOMA HEPG2 CELLS

Large, free polymannose oligosaccharides generated during glycoprotein biosynthesis rapidly appear in the cytosol of HepG2 cells where they undergo processing by a cytosolic endo H-like enzyme and a mannosidase to yield the linear isomer of Man(5)GlcNAc (Man[alpha 1-2]Man[alpha 1 -2]Man[alpha 1-3] [Man alpha 1-6]Man[beta 1-4]GlcNAc). Here we have ex amined the fate of these partially trimmed oligosaccharides in intact HepG2 cells. Subsequent to pulse-chase incubations with D-[2-H-3]manno se followed by permeabilization of cells with streptolysin O free olig osaccharides were isolated from the resulting cytosolic and membrane-b ound compartments. Control pulse-chase experiments revealed that total cellular free oligosaccharides are lost from HepG2 cells with a half- life of 3-4 h. In contrast use of the vacuolar H+/ATPase inhibitor, co ncanamycin A, stabilized total cellular free oligosaccharides and enab led us to demonstrate a translocation of partially trimmed oligosaccha rides from the cytosol into a membrane-bound compartment. This translo cation process was unaffected by inhibitors of autophagy but inhibited if cells were treated with either 100 mu M swainsonine, which provoke s a cytosolic accumulation of large free oligosaccharides bearing 8-9 residues of mannose, or agents known to reduce cellular ATP levels whi ch lead to the accumulation of the linear isomer of Man(5)GlcNAc in th e cytosol. Subcellular fractionation studies on Percoll density gradie nts revealed that the cytosol-generated linear isomer of Man(5)GlcNAc is degraded in a membrane-bound compartment that cosediments with lyso somes.