Open AccessThe GDP-bound form of Arf6 is located at the plasma membrane
Author(s) -
Eric Macia,
Frédéric Luton,
Mariagrazia Partisani,
Jacqueline Cherfils,
Pierre Teilhard de Chardin,
Michel Franco
Publication year - 2004
Publication title -
journal of cell science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.384
H-Index - 278
eISSN - 1477-9137
pISSN - 0021-9533
DOI - 10.1242/jcs.01090
Subject(s) - mutant , biology , gtp' , microbiology and biotechnology , guanosine diphosphate , phosphatidylinositol , in vivo , membrane , biochemistry , guanosine triphosphate , signal transduction , gene , genetics , enzyme
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.