Garred Ø, Rodal S K, van Deurs B, Sandvig K
Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway.
Traffic. 2001 Jan;2(1):26-36. doi: 10.1034/j.1600-0854.2001.020105.x.
This paper studies the endocytosis of ricin at the apical pole of polarized MDCK II cells after permeabilization of the cells basolaterally with streptolysin O. Ricin endocytosis after the addition of cytosol with an ATP-regenerating system was 2-3-fold higher than after the addition of a transport medium. A similar increase in ricin endocytosis was obtained by reconstitution of dialyzed cytosol with the nonhydrolyzable GTP analog, GTP gamma S, in the presence of an ATP-regenerating system. The nonhydrolyzable GDP analog, GDP beta S, did not increase ricin uptake. In contrast to the data obtained with ricin, GTP gamma S was found to inhibit apical transferrin uptake in MDCK II cells transfected with the human transferrin receptor, and the data thus imply that GTP gamma S supports clathrin-independent endocytosis. Electron microscopy (EM) demonstrated that free endocytic vesicles were formed from the apical pole of permeabilized MDCK II cells in the presence of GTP gamma S and that both a ricin-HRP conjugate, HRP, and cationized gold were endocytosed. Ricin endocytosis in the presence of intact cytosol, as well as GTP gamma S-stimulated ricin uptake, was inhibited by Clostridium botulinum C3 transferase, an enzyme found to inactivate Rho proteins. The data demonstrate that apical clathrin-independent endocytosis functions in the presence of GTP gamma S, and suggest that one or more of the small GTP binding proteins of the Rho family is involved in regulation of the apical clathrin-independent endocytosis in MDCK II cells.
本文研究了在用链球菌溶血素O从基底外侧通透极化的MDCK II细胞后，蓖麻毒素在其顶端极的内吞作用。加入含有ATP再生系统的胞质溶胶后，蓖麻毒素的内吞作用比加入转运培养基后高2 - 3倍。在存在ATP再生系统的情况下，用不可水解的GTP类似物GTPγS重构透析后的胞质溶胶，可使蓖麻毒素内吞作用有类似的增加。不可水解的GDP类似物GDPβS则不会增加蓖麻毒素的摄取。与用蓖麻毒素获得的数据相反，发现GTPγS抑制转染了人转铁蛋白受体的MDCK II细胞中顶端转铁蛋白的摄取，因此这些数据表明GTPγS支持不依赖网格蛋白的内吞作用。电子显微镜（EM）显示，在存在GTPγS的情况下，从通透的MDCK II细胞的顶端极形成了游离的内吞小泡，并且蓖麻毒素 - HRP偶联物、HRP以及阳离子化金都被内吞。肉毒杆菌C3转移酶（一种能使Rho蛋白失活的酶）抑制了在完整胞质溶胶存在下的蓖麻毒素内吞作用以及GTPγS刺激的蓖麻毒素摄取。数据表明，在存在GTPγS的情况下顶端不依赖网格蛋白的内吞作用发挥功能，并且提示Rho家族的一种或多种小GTP结合蛋白参与了MDCK II细胞中顶端不依赖网格蛋白的内吞作用的调节。
