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1998; Claude et al., 1999). In contrast, ARF6, the least-conserved ARF-
family member (67% amino-acid identity with ARF1), is localized to the
plasma membrane and early endosomal compartments where it regulates
endocytic tra
c at the cell periphery (Chavrier and Goud, 1999). Here, we
review recent observations that implicate ARF6 as a major regulator of
plasma membrane organization, coordinating endocytic trac with cortical
actin cytoskeleton remodelling. In agreement with its proposed function,
ARF6 has been found to be essential during cell motility and phagocytosis,
two processes requiring membrane movement toward the cell surface and
actin cytoskeleton organization.
Intracellular localization of ARF6
In ARFs the classical GDP/GTP nucleotide switch is coupled to a cytosol/
membrane transition. The N-terminal myristoylated helix is retracted in
ARF:GDP and is released during the GDP-to-GTP conformational switch to
insert into the lipid bilayer (Pasqualato et al., 2001). However, despite highly
conserved structural features, ARF proteins differ in respect to their steady-
state association with membranes: ARF1, ARF3 and ARF5 are found
predominantly in the cytosol (GDP-bound), while a major fraction of ARF6
(50% to 100% depending on the cell lines) is membrane-bound (Cavenagh et
al., 1996; Prigent M. and Chavrier P., unpublished). These observations could
suggest that ARF6 is predominantly in the GTP-bound active conformation.
However, using a pull-down assay selectively to precipitate GTP-bound
ARF6, we found that ARF6:GTP is low in cultured cells (few percent of total
both in Hela cells and mouse RAW 264.7 macrophages). Therefore, we should
also consider the possibility that ARF6:GDP is able to associate stably with
membranes by interacting with some unknown lipid(s) or protein(s). More-
over, this is not the only example ARF6's special properties. Indeed, a mutant
of ARF6 (Q67L) predicted to be defective for GTP hydrolysis (thus mainly
GTP-bound) accumulates at the plasma membrane where it induces the
formation of actin-based protrusions, while another mutant, ARF6T27N,
thought to be in the GDP-bound inactive state, is mostly localized to internal
tubulovesicular endosomes (Peters et al., 1995; D'Souza-Schorey et al., 1998).
These observations, which suggest differential localization for the active and
inactive ARF6 conformations, have been taken as evidence for a role of ARF6
activation for targeted delivery of recycling endosomal vesicles to the plasma
membrane (see below and in D'Souza-Schorey et al., 1998; Brown et al.,
2001). The mechanism of ARF6 activation has,
therefore, become an
important question.
