Abstract
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.
Highlights
The ADP-ribosylation factors (Arfs) are small GTP-binding proteins that regulate intracellular vesicular transport along secretory and endocytic pathways
We have cloned and characterized an Arf6specific exchange factor, EFA6, that is exclusively found at the plasma membrane, implying that Arf6-GDP should be found at the plasma membrane (Franco et al, 1999)
In vivo, a proportion of the Arf6(T27N) mutant is engaged in a high-affinity nucleotidefree complex with the exchange factor, whereas most denatures and accumulates into aggresomes
Summary
The ADP-ribosylation factors (Arfs) are small GTP-binding proteins that regulate intracellular vesicular transport along secretory and endocytic pathways. Two mutants of Arf, Q67L and T27N, are considered to mimic the GTP- and GDP-bound forms, respectively, and have been used extensively to apprehend the localization and the function of the protein Both immunoelectron microscopy and immunofluorescence (IF) analyses have revealed that the Q67L mutant is restricted to the plasma membrane, whereas the T27N mutant is found on internal structures resembling endosomes (D’Souza-Schorey et al, 1998; Peters et al, 1995; Radhakrishna et al, 1996). Based on the localization of these two mutants, it has been proposed that Arf cycles between the plasma membrane (Arf6-GTP) and a recycling endosomal compartment (Arf6GDP) In this model, Arf6-GDP would encounter its exchange factor at the surface of an internal compartment to promote the formation of vesicles destined to the plasma membrane. We have cloned and characterized an Arf6specific exchange factor, EFA6, that is exclusively found at the plasma membrane, implying that Arf6-GDP should be found at the plasma membrane (Franco et al, 1999)
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