Reconstitution of clathrin-independent endocytosis at the apical domain of permeabilized MDCK II cells: Requirement for a Rho-family GTPase

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 gammaS, in the presence of an ATP-regenerating system. The nonhydrolyzable GDP analog, GDP betaS, did not increase ricin uptake. In contrast to the data obtained with ricin, GTP IS was found to inhibit apical transferrin uptake in MDCK II cells transfected with the human transferrin receptor, and the data thus imply that GTP gammaS 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 IS 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 gammaS-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 gammaS, and suggest that one or more of the small GTP binding proteins of the Rho family is involved in regulation of the epical clathrin-independent endocytosis in MDCK II cells.