In support of such a scenario, we find in MDCK cells that two ARF1-regulated coats, COPI and AP-3, display differential sensitivity to BFA. 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 Imidapril (Tanatril) specific coat proteins, but rather is a ubiquitous molecular switch that acts as a transducer of diverse signals influencing coat assembly. (Madison, WI). GTPS, ATP, creatine phosphate, and creatine kinase were from (St. Louis, MO). Recombinant Proteins GST Fusion with -Adaptin Fragment (GG1). DNA encoding residues 752C 839 of -adaptin was generated Imidapril (Tanatril) by PCR and then cloned into pGEX-5X-1 (to yield the postnuclear supernatant. The postnuclear supernatant was adjusted to 40.6% sucrose, then overlaid sequentially with 35% sucrose, 25% sucrose, and HB (all sucrose solutions contained 3 mM imidazole, pH 7.4). The gradient was centrifuged in a SW41 rotor (Beckman Instrs.) at 40,000 rpm for 90 min. Membrane fractions enriched for endosomal or Golgi membranes were collected from the 35/25% and the 40.6/35% sucrose interface, respectively (Aniento et al., 1996). Membranes were washed with salt by incubation on ice for 20 min after addition of an equal volume of 2M KCl. The stripped membranes were collected by centrifugation at 100,000 for 90 min, resuspended in coat-binding (CB) buffer (see for 40 min followed by a second Imidapril (Tanatril) centrifugation step at 105,000 for 90 min, at 4C. This fraction (30 mg/ml protein) was designated bovine brain cytosol and used in the in vitro membrane-binding assays. To generate a high molecular weight (HMW) fraction of the cytosol that contained AP-3 but not ARF, the cytosol was subjected to gel filtration chromatography on a Superose 6 column (= 5 for AP-3; = Imidapril (Tanatril) 2 for ARF). The amounts of protein bound in the presence of cytosol and GTPS are considered 100% binding. (and and and and and and and and and and and and and and and and and and and and and and and and and and and and and and and mutant, which indicate that the orthologue of ARF1 in yeast is required for maintenance of both Golgi and endosome structure and function (Gaynor et al., 1998). The finding that ARF1 regulates three different coats, COPI, AP-1, and now AP-3, reinforces the idea that ARF1 is not a regulator of specific coats, but rather a common molecular switch that transduces signals from upstream modulators of coat assembly. A candidate for such a modulator is the ARF1 GEF that activates ARF1 by guanine nucleotide exchange. Four different cytosolic and/ or Golgi ARF1 GEFs have recently been identified (Chardin et al., 1996; Rosa et al., 1996; Meacci et al., 1997; Morinaga et al., 1997), and a Golgi membrane-associated ARF1 GEF has been described but not yet isolated (Donaldson et al., 1992; Helms and Rothman, 1992; Randazzo et al., 1993). It is possible that activation of ARF1 by these distinct GEF proteins leads to recruitment of different coat proteins. In support of such a scenario, we find in MDCK cells that two ARF1-regulated coats, COPI and AP-3, display differential sensitivity to BFA. Since BFA appears to be an inactivator of ARF1 GEF, this implies the existence of distinct ARF1 GEFs that control different coats via ARF1. In addition, two Golgi-localized ARF1-regulated coats, COPI and AP-1, also display differential sensitivity to BFA (Wagner et al., 1994; this study). Thus, different subdomains of an organelle can exhibit independent ARF1-regulated events. The GTPase-activating protein Rabbit Polyclonal to RFA2 ARF1-GAP is another ARF1 regulatory protein that may represent a modulator of ARF1 activity that is specific for a particular coat. We cannot distinguish at present whether the effect of the Golgi-localized ARF1-GAP on AP-3 in our experiments is due to a direct or indirect effect of ARF1-GAP. Conceivably, the latter case could arise from sequestration of the pool of available intracellular ARF1 by cytosolic overexpressed ARF1-GAP. Finally, the finding that the KDEL receptor ERD2 regulates ARF1-mediated events by recruiting ARF1-GAP (Aoe et al., 1997) raises the possibility that other as yet unidentified transmembrane receptors modulate different coat proteins via ARF1-GAP. The molecular mechanism by which ARF1 recruits coat proteins is not known. Two prevailing views are (mutants (Stearns et al., 1990; Kahn et al., 1991), and have similar in vitro activities (Liang and Kornfeld, 1997), they do not appear to be strictly redundant.