Targeting of ß-Glucuronidase to Lysosomes in Mannose 6- Phosphate Receptor-deficient MOPC 315 Cells

The murine plasma cell line MOPC 315 efficiently targets newly synthesized acid hydrolases to lysosomes in spite of a marked deficiency in the level of the mannose 6phosphate receptor (Gabel, C ., D . Goldberg, and S . Kornfeld, 1983, Proc . Nad. Acad . Sci. USA, 80:775-779) . To better understand the routing of lysosomal enzymes in this cell line, pulsechase experiments were performed with [23H]mannose and [ 35S]methionine followed by immunoprecipitation of ß-glucuronidase and IgA . By 3 h of chase, essentially all of the newly synthesized ß-glucuronidase had undergone proteolytic processing, suggesting that the molecules had reached lysosomes . At this time 30% of the pulse-labeled IgA was still intracellular . The oligosaccharides on the intracellular IgA were of the high mannose-type, while the secreted IgA contained processed, complex-type oligosaccharides . This indicates that the intracellular IgA was still in the endoplasmic reticulum or an early region of the Golgi complex when all of the ß-glucuronidase had reached lysosomes . Therefore, ß-glucuronidase and IgA must exit from the endoplasmic reticulum or the early Golgi complex at different rates, a finding that is inconsistent with bulk phase movement of these proteins from the endoplasmic reticulum to the trans Golgi complex . The addition of the ionophore monensin greatly slows the rate of IgA secretion from MOPC 315 cells and the molecules secreted have incompletely processed oligosaccharides . In contrast, monensin only slightly delays the transport of newly synthesized ß-glucuronidase to lysosomes and causes no significant alteration in the extent of oligosaccharide phosphorylation, a process that appears to occur in the early (cis) Golgi complex . However, the labeled #glucuronidase was deficient in sialylated, phosphorylated hybrid oligosaccharides whose biosynthesis requires the action of late stage oligosaccharide processing enzymes assumed to be localized in the trans Golgi complex . Lysosomal hydrolases, like membrane and secretory proteins, are synthesized in the rough endoplasmic reticulum (ER) as preproteins (1-4) . Following their biosynthesis the acid hydrolases must be sorted from other proteins present in the lumen of the endoplasmic reticulum and delivered to lysosomes. The best understood mechanism for lysosomal enzyme sorting involves the mannose 6-phosphate (Man-6-P)` recognition system . Newly synthesized acid hydrolases acquire Man-6-P residues by the following two step reaction. First, Nacetylglucosamine-l-phosphate is transferred to the C-6 position of mannose residues present on asparagine-linked high mannose oligosaccharides (5, 6) . The enzyme catalyzing this Abbreviations used in this paper: Man-6-P, mannose 6-phosphate . 296 reaction, UDP-N-acetylglucosamine :lysosomal enzyme Nacetylglucosaminylphosphotransferase, is membrane-bound and is associatedwith the Golgi apparatus (7, 8) . In the second step, the N-acetylglucosamine residue is removed by another Golgi-associated enzyme, N-acetylglucosaminylphosphoglycosidase, to generate the Man-6-P monoester (9, 10) . These phosphomonoesters mediate binding of the acid hydrolase to a specific receptor (the Man-6-P receptor) (11-14), and the resulting receptor-ligand complex is translocated to lysosomes or a prelysosomal compartment where the low pH stimulates dissociation and completes the delivery process (15) . Although the Man-6-P receptor-mediated pathway has been shown to be a general mechanism by which acid hydrolases reach lysosomes, alternative pathways must also exist (16, THE JOURNAL OF CELL BIOLOGY " VOLUME 99 JULY 1984 296-305 0 The Rockefeller University Press 0021-9525/84/01/0296/10 $1 .00 on M arch 1, 2017 D ow nladed fom Published July 1, 1984