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Targeting of beta-glucuronidase to lysosomes in mannose 6-phosphate receptor-deficient MOPC 315 cells
Abstract:
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 6-phosphate receptor (Gabel, C., D. Goldberg, and S. Kornfeld, 1983, Proc. Natl. Acad. Sci. USA, 80:775-779). To better understand the routing of lysosomal enzymes in this cell line, pulse-chase experiments were performed with [2-3H]mannose and [35S]methionine followed by immunoprecipitation of beta-glucuronidase and IgA. By 3 h of chase, essentially all of the newly synthesized beta-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 beta-glucuronidase had reached lysosomes. Therefore, beta-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 beta-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 beta-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.
Insights
Lysosomal enzymes like beta-glucuronidase are efficiently routed to lysosomes in MOPC 315 cells, even with low mannose 6-phosphate receptor levels. This suggests distinct protein transport pathways separate from IgA secretion.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The MOPC 315 murine plasma cell line efficiently delivers newly synthesized acid hydrolases to lysosomes.
- This occurs despite a significant deficiency in the mannose 6-phosphate receptor, a key protein in lysosomal targeting.
- Understanding lysosomal enzyme routing in this cell line provides insights into alternative protein trafficking pathways.
Purpose of the Study:
- To investigate the mechanism of lysosomal enzyme targeting in MOPC 315 cells.
- To compare the transport kinetics of beta-glucuronidase and IgA within the secretory pathway.
- To elucidate the role of the Golgi complex in differential protein processing and sorting.
Main Methods:
- Pulse-chase experiments using radiolabeled mannose and methionine.
- Immunoprecipitation of beta-glucuronidase and IgA.
- Analysis of oligosaccharide processing and modification.
- Treatment with the ionophore monensin to assess transport rates.
Main Results:
- Newly synthesized beta-glucuronidase rapidly reached lysosomes, undergoing proteolytic processing within 3 hours.
- Intracellular IgA retained high mannose-type oligosaccharides, indicating it was in the endoplasmic reticulum or early Golgi.
- Monensin treatment significantly slowed IgA secretion but only slightly delayed beta-glucuronidase transport to lysosomes.
- Beta-glucuronidase showed deficiencies in late Golgi-processed oligosaccharides, suggesting limited transit through the trans Golgi network.
Conclusions:
- Beta-glucuronidase and IgA exit the endoplasmic reticulum or early Golgi at different rates, contradicting bulk flow transport models.
- The MOPC 315 cell line utilizes distinct pathways for lysosomal enzyme and secretory protein trafficking.
- Oligosaccharide processing patterns indicate that beta-glucuronidase may not extensively traverse the trans Golgi network for lysosomal targeting in this cell line.