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© The Rockefeller University Press, 0021-9525/1999//7 $5.00
The Journal of Cell Biology, Volume 147, Number 1, , 1999 7-12

Brief Report

Adp-Ribosylation Factor 6 and Endocytosis at the Apical Surface of Madin-Darby Canine Kidney Cells



Y. Altschulera, S.-H. Liub, L. Katza, K. Tanga, S. Hardyd, F. Brodskyb, G. Apodacac, and K. Mostova

a Department of Anatomy, University of California, San Francisco, California 94143-0452
b G.W. Hooper Foundation, Department of Microbiology and Immunology, Department of Biopharmaceutical Sciences, and Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0552
c Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
d Cell Genesis, Foster City, California 94404
Department of Anatomy, Box 0452, University of California, San Francisco, California 94143-0452.(415) 476-4845(415) 502-4450

yoram{at}itsa.ucsf.edu

We report that the small GTPase, ADP-ribosylation factor 6 (ARF6), is present only on the apical surface of polarized MDCK epithelial cells. Overexpression of a mutant of ARF6, ARF6–Q67L, which is predicted to be in the GTP-bound form, stimulates endocytosis exclusively at this surface. Surprisingly, overexpression of the mutant ARF6–T27N, which is predicted to be in the GDP-bound form, also stimulated apical endocytosis, though to a lesser extent. ARF6-stimulated endocytosis is inhibited by a dominant-negative form of dynamin, or a dominant-negative hub fragment of clathrin heavy chain, indicating that it is mediated by clathrin. Correspondingly, overexpression of either mutant of ARF6 leads to an increase in the number of clathrin-coated pits at the apical plasma membrane. When ARF6–Q67L is overexpressed in the presence of the dominant-negative dynamin, the ARF6–Q67L colocalizes with clathrin and with IgA bound to its receptor. We conclude that ARF6 is an important modulator of clathrin-mediated endocytosis at the apical surface of epithelial cells.

Key Words: receptor-mediated endocytosis • polarized Madin-Darby canine kidney cells • ADP-ribosylation factor 6 • apical • clathrin

© 1999 The Rockefeller University Press

1.used in this paper: AP, apical; ARF6, ADP-ribosylation factor 6; ARF6–T27N, mutant ARF6 predicted to be in the GDP-bound form; ARF6–Q67L, mutant ARF6 predicted to be in the GTP-bound form; ARF6–WT, wild-type ARF6; BL, basolateral; DX, doxycycline; gal, galactosidase; pIgR, polymeric immunoglobulin receptor; PM, plasma membrane; WT, wild-type


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