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Reconstitution of Clathrin‐Independent Endocytosis at the Apical Domain of Permeabilized MDCK II cells: Requirement for a Rho‐Family GTPase
Author(s) -
Garred Øystein,
Kjersti Rodal Siv,
Van Deurs Bo,
Sandvig Kirsten
Publication year - 2001
Publication title -
traffic
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.677
H-Index - 130
eISSN - 1600-0854
pISSN - 1398-9219
DOI - 10.1034/j.1600-0854.2001.020105.x
Subject(s) - endocytosis , ricin , gtp' , clathrin , biology , microbiology and biotechnology , receptor mediated endocytosis , endocytic cycle , cytosol , gtpase , bulk endocytosis , transferrin receptor , biochemistry , transferrin , receptor , toxin , enzyme
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, GTPγS, in the presence of an ATP‐regenerating system. The nonhydrolyzable GDP analog, GDPβS, did not increase ricin uptake. In contrast to the data obtained with ricin, GTPγS was found to inhibit apical transferrin uptake in MDCK II cells transfected with the human transferrin receptor, and the data thus imply that GTPγS 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 GTPγS and that both a ricin–HRP conjugate, HRP, and cationized gold were endocytosed. Ricin endocytosis in the presence of intact cytosol, as well as GTPγS‐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 GTPγS, 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.