Diaz R, Colombo M I, Koval M, Mayorga L, Stahl P
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis 63110.
Eur J Cell Biol. 1991 Dec;56(2):223-32.
Dinitrophenol (DNP)-beta-glucuronidase and mannosylated anti-DNP IgG, which are endocytosed by the mannose receptor and delivered to lysosomes, were previously developed as probes for examination of fusion between early endosomes in a cell-free system. In this study, these probes were found to be transported by intact cells to endocytic vesicles with heavy buoyant density at different rates, as determined by Percoll gradient fractionation of cell homogenates. There was a concomitant loss of in vitro fusion activity as the ligands moved to dense compartments. In monensin-treated cells, DNP-beta-glucuronidase was retained in a light compartment corresponding to intracellular vesicles capable of fusion in vitro. Pulse-chase studies using a DNP-derivatized transferrin-alkaline phosphatase conjugate showed that a recycling ligand was always found in light intracellular vesicles that were capable of fusion to early endosomes in vitro. In contrast to cell-free systems, intact cells sequentially labeled with DNP-beta-glucuronidase and then mannosylated anti-DNP IgG showed ligand mixing in both early and late endocytic compartments. Treatment with nocodazole or colchicine did not affect the rate of DNP-beta-glucuronidase transport to heavy vesicles in intact cells, however, the extent of ligand mixing in late endosomes was decreased by microtubule disruption. Using sequentially labeled cells split into two groups, we directly compared ligand mixing in vitro to mixing by intact cells. Fusion alone does not mediate increases in vesicle density, since DNP-beta-glucuronidase/anti-DNP IgG complexes formed in vitro were found in light vesicles, while intact cells showed immune complexes predominantly in heavy vesicles. These results suggest that the density shift is an initial step in targeting to lysosomes.
二硝基苯酚（DNP）-β-葡萄糖醛酸酶和甘露糖基化抗DNP IgG可被甘露糖受体内化并转运至溶酶体，此前已被开发为在无细胞系统中检测早期内体融合的探针。在本研究中，通过对细胞匀浆进行Percoll梯度分级分离发现，这些探针在完整细胞中以不同速率被转运至具有高浮力密度的内吞小泡。随着配体向致密区室移动，体外融合活性随之丧失。在莫能菌素处理的细胞中，DNP-β-葡萄糖醛酸酶保留在一个轻区室中，该轻区室对应于能够在体外融合的细胞内小泡。使用DNP衍生化转铁蛋白-碱性磷酸酶偶联物进行脉冲追踪研究表明，回收配体总是存在于能够在体外与早期内体融合的轻细胞内小泡中。与无细胞系统不同，先用DNP-β-葡萄糖醛酸酶然后用甘露糖基化抗DNP IgG顺序标记的完整细胞在早期和晚期内吞区室中均显示出配体混合。用诺考达唑或秋水仙碱处理不影响完整细胞中DNP-β-葡萄糖醛酸酶向重小泡的转运速率，然而，微管破坏会降低晚期内体中配体混合的程度。通过将顺序标记的细胞分成两组，我们直接比较了体外配体混合与完整细胞的配体混合。单独的融合不会介导小泡密度增加，因为在体外形成的DNP-β-葡萄糖醛酸酶/抗DNP IgG复合物存在于轻小泡中，而完整细胞中的免疫复合物主要存在于重大泡中。这些结果表明，密度变化是靶向溶酶体的初始步骤。
