Distribution of ARF6 between membrane and cytosol is regulated by its GTPase cycle.

The ADP-ribosylation factor (ARF) subfamily of small GTPases regulates intracellular transport. Although much is known about how ARF1 regulates transport in the secretory pathways, regulation of the endocytic pathways by ARF6 remains less understood. In particular, whereas cycling of ARF1 between membrane and cytosol represents a major mechanism of regulating its function, this regulation has been questioned for ARF6. In this study, we found that ARF6 is distributed both on membranes and in the cytosol. Cytosolic ARF6 is recruited to membranes in a GTP-dependent manner that is fundamentally similar to ARF1. However, unlike ARF1, release of membrane-bound ARF6 to the cytosol requires hydrolysis of GTP that is sensitive to the level of magnesium. These findings suggest that the GTPase cycle of ARF6 also regulates its distribution between membrane and cytosol and that this form of regulation will also likely be important for the function of ARF6. Moreover, as ARF6 has little intrinsic ability to hydrolyze GTP, magnesium concentration most likely affects the release of membrane-bound ARF6 by altering the activity of its GTPase-activating protein.