Nakajima Y, Pfeffer S R
Department of Biochemistry, Stanford University School of Medicine, California 94305-5307, USA.
Mol Biol Cell. 1997 Apr;8(4):577-82. doi: 10.1091/mbc.8.4.577.
Mannose 6-phosphate receptors carry newly synthesized lysosomal hydrolases from the trans-Golgi network to endosomes, then return to the trans-Golgi network for another round of enzyme delivery. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, interferes with the delivery of newly synthesized lysosomal enzymes to lysosomes. We used two independent assays of mannose 6-phosphate receptor trafficking to determine the precise step that is blocked by wortmannin. Using an assay that monitors resialylation of desialylated cell surface 300-kDa mannose 6-phosphate receptors, we found that receptor endocytosis and transport to the trans-Golgi network were not inhibited by 2 microM wortmannin. In addition, this concentration of drug had no effect on the transport of the mannose 6-phosphate receptor from late endosomes to the trans-Golgi network using a system that reconstitutes this transport process in cell extracts. Under the same conditions, wortmannin significantly inhibited the generation of mature cathepsin D. In addition, the structurally unrelated phosphatidylinositol 3-kinase inhibitor, LY294002, was also without effect when added to in vitro endosome-trans-Golgi network transport reactions. These experiments demonstrate that the interruption in lysosomal enzyme targeting is most likely due to a wortmannin-sensitive process required for the export of these receptors from the trans-Golgi network, consistent with the established role of phosphatidylinositol 3-kinase in the equivalent transport process in Saccharomyces cerevisiae.
甘露糖 6 - 磷酸受体将新合成的溶酶体水解酶从反式高尔基体网络转运至内体，然后返回反式高尔基体网络进行新一轮的酶递送。渥曼青霉素是一种磷脂酰肌醇 3 - 激酶抑制剂，它会干扰新合成的溶酶体酶向溶酶体的递送。我们使用了两种独立的甘露糖 6 - 磷酸受体转运检测方法来确定被渥曼青霉素阻断的精确步骤。通过监测去唾液酸化的细胞表面 300 kDa 甘露糖 6 - 磷酸受体的再唾液酸化的检测方法，我们发现 2 μM 渥曼青霉素不会抑制受体内吞作用以及向反式高尔基体网络的转运。此外，在细胞提取物中重建该转运过程的系统中，此药物浓度对甘露糖 6 - 磷酸受体从晚期内体转运至反式高尔基体网络没有影响。在相同条件下，渥曼青霉素显著抑制了成熟组织蛋白酶 D 的生成。此外，在体外晚期内体 - 反式高尔基体网络转运反应中添加结构不相关的磷脂酰肌醇 激酶抑制剂 LY294002 也没有作用。这些实验表明，溶酶体酶靶向作用的中断很可能是由于这些受体从反式高尔基体网络输出所需的一个对渥曼青霉素敏感的过程，这与磷脂酰肌醇 3 - 激酶在酿酒酵母等效转运过程中已确立的作用一致。
