ADP-RIBOSYLATION FACTOR-1 (ARF1) REGULATES RECRUITMENT OF THE AP-3 ADAPTER COMPLEX TO MEMBRANES

Small GTP-binding proteins such as ADP-ribosylation factor 1 (ARF1) an d Sar1p regulate the membrane association of coat proteins involved in intracellular membrane trafficking. ARF1 controls the clathrin coat a daptor AP-1 and the nonclathrin coat COPI, whereas Sar1p controls the nonclathrin coat COPII. In this study, we demonstrate that membrane as sociation of the recently described AP-3 adaptor is regulated by ARF1. Association of AP-3 with membranes in vitro was enhanced by GTP gamma S and inhibited by brefeldin A (BFA), an inhibitor of ARF1 guanine nu cleotide exchange. In addition, recombinant myristoylated ARF1 promote d association of AP-3 with membranes. The role of ARF1 in vivo was exa mined by assessing AP-3 subcellular localization when the intracellula r level of ARF1-GTP was altered through overexpression of dominant ARF 1 mutants or ARF1-GTPase-activating protein (GAP). Lowering ARF1-GTP l evels resulted in redistribution of AP-3 from punctate membrane-bound structures to the cytosol as seen by immunofluorescence microscopy, In contrast, increasing ARF1-GTP levels prevented redistribution of AP-3 to the cytosol induced by BFA or energy depletion. Similar experiment s with mutants of ARF5 and ARF6 showed that these other ARF family mem bers had little or no effect on AP-3. Taken together, our results indi cate that membrane recruitment of AP-3 is promoted by ARF1-GTP. This f inding suggests that ARF1 is not a regulator of specific coat proteins , but rather is a ubiquitous molecular switch that acts as a transduce r of diverse signals influencing coat assembly.