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First published online May 4, 2004
doi: 10.1242/10.1242/jcs.01090
Research Article |
1 Institut de Pharmacologie Moléculaire et Cellulaire, CNRS-UMR 6097, 660 route des Lucioles, 06560 Valbonne Sophia-Antipolis, France
2 Laboratoire d'Enzymologie et de Biochimie Structurales, CNRS, 1, avenue de la Terrasse, 91198 Gif sur Yvette, France
3 Institut de Pharmacologie Moléculaire et Cellulaire, CNRS-UMR 6097, 660 route des Lucioles, 06560 Valbonne-Sophia-Antipolis, France
* Author for correspondence (e-mail: franco{at}ipmc.cnrs.fr)
Accepted 8 January 2004
The function of Arf6 has been investigated largely by using the T27N and the Q67L mutants, which are thought to be blocked in GDP- and GTP-bound states, respectively. However, these mutants have been poorly characterized biochemically. Here, we found that Arf6(T27N) is not an appropriate marker of the inactive GDP-bound form because it has a high tendency to lose its nucleotide in vitro and to denature. As a consequence, most of the protein is aggregated in vivo and localizes to detergent-insoluble structures. However, a small proportion of Arf6(T27N) is able to form a stable complex with its exchange factor EFA6 at the plasma membrane, accounting for its dominant-negative phenotype. To define the cellular localization of Arf6-GDP, we designed a new mutant, Arf6(T44N). In vitro, this mutant has a 30-fold decreased affinity for GTP. In vivo, it is mostly GDP bound and, in contrast to the wild type, does not switch to the active conformation when expressed with EFA6. This GDP-locked mutant is found at the plasma membrane, where it localizes with EFA6 and Ezrin in actin- and phosphatidylinositol (4,5)-bisphosphate-enriched domains. From these results, we conclude that the Arf6 GDP-GTP cycle takes place at the plasma membrane.
Key words: Aggresomes, Arf6, Dominant-negative mutant, Endocytosis, Guanine-nucleotide-exchange factor
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