Mayorga L S, Diaz R, Stahl P D
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110.
J Biol Chem. 1989 Apr 5;264(10):5392-9.
The presence of acid proteases in the endosomal compartment of macrophages has been recently demonstrated (Diment, S., Leech, M. S., and Stahl, P. D. (1988) J. Biol. Chem. 263, 6901-6907). This proteolytic activity allows the early degradation of ligands internalized by receptor-mediated endocytosis. To study the early steps that initiate the proteolytic processing of ligands, immune complexes formed with anti-dinitrophenol monoclonal IgG and radiolabeled dinitrophenol-derivatized bovine serum albumin were bound at 4 degrees C to Fc receptors of J774 macrophages. Cells were allowed to internalize immune complexes bound to the plasma membrane for different periods of time at 37 degrees C. Vesicle preparations generated from these cells were incubated in vitro at acidic pH to allow the hydrolysis of ligands located in protease-positive compartments. Ligand hydrolysis was observed after about 5 min of internalization, suggesting that at earlier times immune complexes were located in protease-free vesicles. Upon incubation of cell lysates under conditions that support in vitro endosome-endosome fusion, early protease-free endosomes containing ligand acquire proteolytic activity. Reconstitution of fusion-dependent proteolysis required energy, ions, membrane-associated factors, and cytosol. Cytosol was inactivated by incubation with N-ethylmaleimide. The proteolytic compartment formed upon in vitro incubation colocalized with endosomes in the light region of a Percoll gradient. Reconstitution was also achieved using an endosomal preparation separated from lysosomes in a Percoll gradient. Our results indicate that a fusion step between newly formed endocytic vesicles and a light density, protease-positive compartment triggers the proteolytic processing of ligands internalized by receptor-mediated endocytosis.
最近已证实巨噬细胞内体区室中存在酸性蛋白酶（迪门特，S.，利奇，M. S.，和斯塔尔，P. D.（1988年）《生物化学杂志》263卷，6901 - 6907页）。这种蛋白水解活性使得通过受体介导的内吞作用内化的配体能够早期降解。为了研究启动配体蛋白水解加工的早期步骤，将用抗二硝基苯酚单克隆IgG和放射性标记的二硝基苯酚衍生化牛血清白蛋白形成的免疫复合物在4℃下与J774巨噬细胞的Fc受体结合。使细胞在37℃下将结合到质膜上的免疫复合物内化不同时间段。从这些细胞产生的囊泡制剂在酸性pH下进行体外孵育，以使位于蛋白酶阳性区室中的配体发生水解。内化约5分钟后观察到配体水解，这表明在较早时间免疫复合物位于无蛋白酶的囊泡中。在支持体外内体 - 内体融合的条件下孵育细胞裂解物时，含有配体的早期无蛋白酶内体获得蛋白水解活性。融合依赖性蛋白水解的重建需要能量、离子、膜相关因子和胞质溶胶。胞质溶胶通过与N - 乙基马来酰亚胺孵育而失活。体外孵育时形成的蛋白水解区室与Percoll梯度轻区的内体重叠。使用在Percoll梯度中与溶酶体分离的内体制剂也实现了重建。我们的结果表明，新形成的内吞囊泡与低密度、蛋白酶阳性区室之间的融合步骤触发了通过受体介导的内吞作用内化的配体的蛋白水解加工。
