Targeting of beta-glucuronidase to lysosomes in mannose 6-phosphate receptor-deficient MOPC 315 cells.
Targeting of beta-glucuronidase to lysosomes in mannose 6-phosphate receptor-deficient MOPC 315 cells.
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DOI：
10.1083/jcb.99.1.296
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发表时间：
1984-07
期刊：
影响因子：
--
通讯作者：
Kornfeld S
中科院分区：
文献类型：
--
作者：
Gabel CA;Kornfeld S
鼠浆细胞系MOPC 315有效地将新合成的酸水解酶靶向溶酶体，尽管甘露糖6-磷酸受体的水平显著不足（Gabel，C.，D. Goldberg和S. Kornfeld，1983，Proc. Natl. Acad. Sci. USA，80：775- 779）。为了更好地理解该细胞系中溶酶体酶的途径，使用[2- 3 H]甘露糖和[35 S]甲硫氨酸进行脉冲追踪实验，然后进行β-葡萄糖醛酸苷酶和伊加的免疫沉淀。经过3小时的追踪，基本上所有新合成的β-葡萄糖醛酸酶都经历了蛋白水解加工，表明分子已经到达溶酶体。此时30%的脉冲标记的伊加仍在细胞内。低聚糖的细胞内伊加的高甘露糖型，而分泌的伊加含有加工，复杂型低聚糖。这表明当所有的β-葡萄糖醛酸酶到达溶酶体时，细胞内伊加仍在内质网或高尔基复合体的早期区域。因此，β-葡萄糖醛酸酶和伊加必须退出内质网或早期高尔基复合体以不同的速度，这一发现是不一致的，这些蛋白质的体相运动从内质网的反高尔基复合体。添加离子载体莫能菌素大大减慢了MOPC 315细胞分泌伊加的速率，并且分泌的分子具有不完全加工的寡糖。相比之下，莫能菌素仅略微延迟新合成的β-葡萄糖醛酸苷酶转运至溶酶体，并且不引起寡糖磷酸化程度的显著改变，该过程似乎发生在早期（顺式）高尔基复合体中。然而，标记的β-葡萄糖醛酸酶缺乏唾液酸化、磷酸化的杂合寡糖，其生物合成需要后期寡糖加工酶的作用，该酶被认为位于反式高尔基体复合体中。
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.