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Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892
ADP-ribosylation factor (ARF) 6 localizes
to the plasma membrane (PM) in its GTP state and to a
tubulovesicular compartment in its GDP state in HeLa
cells that express wild-type or mutant forms of this
GTPase. Aluminum fluoride (AlF) treatment of ARF6-transfected cells redistributes ARF6 to the PM and stimulates the formation of actin-rich surface protrusions.
Here we show that cytochalasin D (CD) treatment inhibited formation of the AlF-induced protrusions and
shifted the distribution of ARF6 to a tubular membrane
compartment emanating from the juxtanuclear region
of cells, which resembled the compartment where the
GTP-binding defective mutant of ARF6 localized. This
membrane compartment was distinct from transferrin-positive endosomes, could be detected in the absence of
ARF6 overexpression or CD treatment, and was accessible to loading by PM proteins lacking clathrin/AP-2
cytoplasmic targeting sequences, such as the IL-2 receptor 
subunit Tac. ARF6 and surface Tac moved into this
compartment and back out to the PM in the absence of
pharmacologic treatment. Whereas AlF treatment
blocked internalization, CD treatment blocked the recycling of wild-type ARF6 and Tac back to the PM; these
blocks were mimicked by expression of ARF6 mutants
Q67L and T27N, which were predicted to be in either the
GTP- or GDP-bound state, respectively. Thus, the ARF6
GTP cycle regulates this membrane traffic pathway. The
delivery of ARF6 and membrane to defined sites along
the PM may provide components necessary for remodeling the cell surface and the underlying actin cytoskeleton.
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