ADP-RIBOSYLATION FACTOR-6 REGULATES A NOVEL PLASMA-MEMBRANE RECYCLINGPATHWAY

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 redistribu tes 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 juxtanuc lear region of cells, which resembled the compartment where the GTP-bi nding defective mutant of ARF6 localized. This membrane compartment wa s distinct from transferrin-positive endosomes, could be detected in t he 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 alpha subunit Tac. ARF6 and surfa ce Tac moved into this compartment and back out to the PM in the absen ce of pharmacologic treatment. Whereas AlF treatment blocked internali zation, CD treatment blocked the recycling of wild-type ARF6 and Tac b ack to the PM; these blocks were mimicked by expression of ARF6 mutant s Q67L and T27N, which were predicted to be in either the GTP- or GDP- bound state, respectively. Thus, the ARF6 GTP cycle regulates this mem brane traffic pathway. The delivery of ARF6 and membrane to defined si tes along the PM may provide components necessary for remodeling the c ell surface and the underlying actin cytoskeleton.