Diaz R, Mayorga L, Stahl P
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110.
J Biol Chem. 1988 May 5;263(13):6093-100.
Receptor-mediated endocytosis and receptor recycling involve a series of intracellular membrane fusion events that appear to play an important role in the regulation of the overall rate and efficiency of the process. An endosome-endosome fusion assay is described using two ligands that (i) rapidly and efficiently enter the endosomal compartment via the macrophage mannose receptor and (ii) that mutually recognize each other. Dinitrophenol-derivatized beta-glucuronidase (DNP-beta-glucuronidase), a ligand for the mannose receptor, was endocytosed by one population of J774 E clone cells, and mannose-derivatized monoclonal anti-DNP IgG (Man-IgG) was internalized by a second set of cells. Both ligands were localized in endosomes as determined by fractionation on Percoll gradients. Incubation of vesicles prepared from the two set of cells resulted in vesicle fusion as indicated by the formation of DNP-beta-glucuronidase-Man-IgG complexes. Under standard conditions, fusion was time-, ATP-, and temperature-dependent. KCl was required for fusion. Fusion required both cytosolic- and membrane-associated proteins. N-Ethylmaleimide treatment of cytosol inhibited fusion. Proton ionophores and amines had no effect on the fusion reaction. ATP-dependent fusion was only observed between early endocytic compartments. While in the presence of a Ca2+ chelator fusion was ATP-dependent, in its absence fusion was also observed in an ATP-independent fashion.
受体介导的内吞作用和受体循环涉及一系列细胞内膜融合事件，这些事件似乎在调节该过程的整体速率和效率中发挥重要作用。本文描述了一种内体-内体融合测定法，使用两种配体，即（i）通过巨噬细胞甘露糖受体快速有效地进入内体区室的配体，以及（ii）相互识别的配体。甘露糖受体的配体二硝基苯酚衍生的β-葡萄糖醛酸酶（DNP-β-葡萄糖醛酸酶）被一群J774 E克隆细胞内吞，而甘露糖衍生的抗DNP单克隆IgG（Man-IgG）被另一组细胞内化。通过在Percoll梯度上分级分离确定，两种配体均定位于内体中。将从两组细胞制备的囊泡一起孵育，会形成DNP-β-葡萄糖醛酸酶-Man-IgG复合物，这表明囊泡发生了融合。在标准条件下，融合是时间、ATP和温度依赖性的。融合需要KCl。融合需要胞质和膜相关蛋白。用N-乙基马来酰亚胺处理胞质溶胶会抑制融合。质子载体和胺对融合反应没有影响。仅在早期内吞区室之间观察到ATP依赖性融合。虽然在存在Ca2+螯合剂的情况下融合是ATP依赖性的，但在不存在Ca2+螯合剂的情况下，也能观察到非ATP依赖性的融合。
