Brown W J, Goodhouse J, Farquhar M G
J Cell Biol. 1986 Oct;103(4):1235-47. doi: 10.1083/jcb.103.4.1235.
We have examined the distribution of mannose-6-phosphate (Man6P) receptors (215 kD) for lysosomal enzymes in cultured Clone 9 hepatocytes at various times after the addition or removal of lysosomotropic weak bases (chloroquine or NH4Cl). Our previous studies demonstrated that after treatment with these agents, Man6P receptors are depleted from their sorting site in the Golgi complex and accumulate in dilated vacuoles that could represent either endosomes or lysosomes (Brown, W. J., E. Constantinescu, and M. G. Farquhar, 1984, J. Cell Biol., 99:320-326). We have now investigated the nature of these vacuoles by labeling NH4Cl-treated cells simultaneously with anti-Man6P receptor IgG and lysosomal or endosomal markers. The structures in which the immunolabeled receptors are found were identified as endosomes based on the presence of endocytic tracers (lucifer yellow and cationized ferritin). The lysosomal membrane marker, lgp120, was associated with a separate population of swollen vacuoles that did not contain detectable Man6P receptors. When cells were allowed to recover from weak base treatment, the receptors reappeared in the Golgi cisternae of most cells (approximately 90%) within approximately 20 min, indicating that as the intra-endosomal pH drops and lysosomal enzymes dissociate, the entire population of receptors rapidly recycles to Golgi cisternae. When NH4Cl-treated cells were allowed to endocytose Man6P, a competitive inhibitor of lysosomal enzyme binding, the receptors also recycled to the Golgi cisternae, suggesting that lysosomal enzymes can dissociate from the receptors under these conditions (high pH + presence of competitive inhibitor). From these results it can be concluded that the intracellular itinerary of the 215-kD Man6P receptor involves its cycling via coated vesicles between the Golgi complex and endosomes, ligand dissociation is both necessary and sufficient to trigger the recycling of Man6P receptors to the Golgi complex, and endosomes rather than secondary lysosomes represent the junction where endocytosed material and primary lysosomes carrying receptor-bound lysosomal enzymes meet.(ABSTRACT TRUNCATED AT 400 WORDS)
我们研究了在培养的克隆9肝细胞中，溶酶体促渗性弱碱（氯喹或氯化铵）添加或去除后不同时间点，溶酶体酶的甘露糖-6-磷酸（Man6P）受体（215 kD）的分布情况。我们之前的研究表明，用这些试剂处理后，Man6P受体从高尔基体复合体中的分拣位点耗尽，并积累在扩张的液泡中，这些液泡可能代表内体或溶酶体（Brown, W. J., E. Constantinescu, and M. G. Farquhar, 1984, J. Cell Biol., 99:320 - 326）。我们现在通过用抗Man6P受体IgG与溶酶体或内体标记物同时标记氯化铵处理的细胞，来研究这些液泡的性质。根据内吞示踪剂（荧光黄和阳离子化铁蛋白）的存在，发现免疫标记受体的结构被鉴定为内体。溶酶体膜标记物lgp120与另一群肿胀的液泡相关，这些液泡中未检测到Man6P受体。当细胞从弱碱处理中恢复时，大多数细胞（约90%）的受体在约20分钟内重新出现在高尔基体潴泡中，这表明随着内体pH值下降且溶酶体酶解离，整个受体群体迅速循环回到高尔基体潴泡。当氯化铵处理的细胞被允许内吞Man6P（一种溶酶体酶结合的竞争性抑制剂）时，受体也循环回到高尔基体潴泡，这表明在这些条件下（高pH值 + 竞争性抑制剂存在）溶酶体酶可从受体上解离。从这些结果可以得出结论，215-kD Man6P受体的细胞内行程涉及通过被膜小泡在高尔基体复合体和内体之间循环，配体解离对于触发Man6P受体循环回到高尔基体复合体既是必要的也是充分的，并且内体而非次级溶酶体代表内吞物质与携带受体结合的溶酶体酶的初级溶酶体相遇的交汇点。（摘要截断于400字）
