Gabel C A, Foster S A
J Cell Biol. 1986 Mar;102(3):943-50. doi: 10.1083/jcb.102.3.943.
During their transport from the endoplasmic reticulum to lysosomes, newly synthesized acid hydrolases are phosphorylated in the Golgi apparatus to generate a common recognition marker, mannose 6-phosphate (Man 6-P). The phosphorylated acid hydrolases then bind to the Man 6-P receptor and are transported by an unknown route to lysosomes. To learn more about the delivery pathway, we examined the fate of the phosphorylated oligosaccharides synthesized by a Man 6-P receptor-positive line of mouse L-cells. In contrast to the rapid degradation of the recognition marker previously observed in mouse lymphoma cells (Gabel, C. A., D. E. Goldberg, and S. Kornfield. 1982. J. Cell Biol., 95:536-542), the number of high mannose oligosaccharides phosphorylated by the L-cells after a 30-min pulse labeling with [2-3H]mannose increased continuously during a subsequent 4-h chase period to a maximum of 9.3% of the total cell-associated structures. After 19 h of chase the absolute number of phosphorylated oligosaccharides declined, but the loss was accompanied by a general loss of cellular oligosaccharides such that 7.4% of the cell-associated high mannose oligosaccharides remained phosphorylated. The longevity of the Man 6-P recognition marker in the L-cells was verified by analyzing the ability of an individual acid hydrolase, beta-glucuronidase, to serve as a ligand for the Man 6-P receptor. At least 60% of the steady state beta-glucuronidase molecules isolated from the L-cells could undergo receptor-mediated endocytosis into enzyme-deficient human fibroblasts. Dense lysosomal granules isolated by metrizamide gradient centrifugation from [3H]mannose-labeled L-cells were found to be highly enriched in their content of phosphomonoester-containing oligosaccharides. The data indicate that acid hydrolases may retain their Man 6-P recognition markers within lysosomes, and suggest the possibility that dephosphorylation occurs at a nonlysosomal location through which the newly synthesized enzymes pass en route to lysosomes.
在从内质网运输到溶酶体的过程中，新合成的酸性水解酶在高尔基体中被磷酸化，以生成一个共同的识别标记——甘露糖6-磷酸（Man 6-P）。然后，磷酸化的酸性水解酶与Man 6-P受体结合，并通过一条未知途径被运输到溶酶体。为了更多地了解递送途径，我们研究了由小鼠L细胞的Man 6-P受体阳性细胞系合成的磷酸化寡糖的命运。与先前在小鼠淋巴瘤细胞中观察到的识别标记的快速降解情况（Gabel, C. A., D. E. Goldberg, and S. Kornfield. 1982. J. Cell Biol., 95:536 - 542）不同，在用[2-³H]甘露糖进行30分钟脉冲标记后，L细胞磷酸化的高甘露糖寡糖数量在随后的4小时追踪期内持续增加，最高达到细胞相关结构总量的9.3%。追踪19小时后，磷酸化寡糖的绝对数量下降，但这种损失伴随着细胞寡糖的普遍损失，以至于细胞相关的高甘露糖寡糖中有7.4%仍保持磷酸化状态。通过分析单个酸性水解酶β-葡萄糖醛酸酶作为Man 6-P受体配体的能力，证实了L细胞中Man 6-P识别标记的持久性。从L细胞中分离出的至少60%的稳态β-葡萄糖醛酸酶分子能够通过受体介导的内吞作用进入缺乏该酶的人成纤维细胞。通过甲泛葡胺梯度离心从[³H]甘露糖标记的L细胞中分离出的致密溶酶体颗粒，其含磷酸单酯的寡糖含量高度富集。数据表明酸性水解酶可能在溶酶体内保留其Man 6-P识别标记，并提示在新合成的酶通往溶酶体的途中，去磷酸化可能发生在一个非溶酶体位置。
