ADP-Ribosylation factor 1 (ARF1) regulates recruitment of the AP-3 adaptor complex to membranes.
Small GTP-binding proteins such as ADP- ribosylation factor 1 (ARF1) and Sar1p regulate the membrane association of coat proteins involved in intracellular membrane trafficking. ARF1 controls the clathrin coat adaptor AP-1 and the nonclathrin coat COPI, whereas Sar1p controls the nonclathrin coat COPII. In this study, we demonstrate that membrane association of the recently described AP-3 adaptor is regulated by ARF1. Association of AP-3 with membranes in vitro was enhanced by GTPgammaS and inhibited by brefeldin A (BFA), an inhibitor of ARF1 guanine nucleotide exchange. In addition, recombinant myristoylated ARF1 promoted association of AP-3 with membranes. The role of ARF1 in vivo was examined by assessing AP-3 subcellular localization when the intracellular level of ARF1-GTP was altered through overexpression of dominant ARF1 mutants or ARF1- GTPase-activating protein (GAP). Lowering ARF1-GTP levels 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 experiments with mutants of ARF5 and ARF6 showed that these other ARF family members had little or no effect on AP-3. Taken together, our results indicate that membrane recruitment of AP-3 is promoted by ARF1-GTP. This finding suggests that ARF1 is not a regulator of specific coat proteins, but rather is a ubiquitous molecular switch that acts as a transducer of diverse signals influencing coat assembly.
Ooi
C E
CE
Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health (NIH), Bethesda, Maryland 20892, USA.
Dell'Angelica
E C
EC
Bonifacino
J S
JS
D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001921:Brain / Q000378:metabolism; D020126:Brefeldin A; D002417:Cattle; D002478:Cells, Cultured; D003517:Cyclopentanes / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004285:Dogs; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000378:metabolism*; D018942:Macrolides; D008856:Microscopy, Fluorescence; D034041:Monomeric Clathrin Assembly Proteins; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
D033942:Adaptor Proteins, Vesicular Transport; D000900:Anti-Bacterial Agents; D003517:Cyclopentanes; D018942:Macrolides; D034041:Monomeric Clathrin Assembly Proteins; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; C439023:clathrin assembly protein AP180; D020126:Brefeldin A; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors