title: ADP-ribosylation factor 1 transiently activates high-affinity adaptor protein complex AP-1 binding sites on Golgi membranes.
abstract: Association of the Golgi-specific adaptor protein complex 1 (AP-1) with the membrane is a prerequisite for clathrin coat assembly on the trans-Golgi network (TGN). The AP-1 adaptor is efficiently recruited from cytosol onto the TGN by myristoylated ADP-ribosylation factor 1 (ARF1) in the presence of the poorly hydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). Substituting GTP for GTPgammaS, however, results in only poor AP-1 binding. Here we show that both AP-1 and clathrin can be recruited efficiently onto the TGN in the presence of GTP when cytosol is supplemented with ARF1. Optimal recruitment occurs at 4 microM ARF1 and with 1 mM GTP. The AP-1 recruited by ARF1.GTP is released from the Golgi membrane by treatment with 1 M Tris-HCl (pH 7) or upon reincubation at 37 degreesC, whereas AP-1 recruited with GTPgammaS or by a constitutively active point mutant, ARF1(Q71L), remains membrane bound after either treatment. An incubation performed with added ARF1, GTP, and AlFn, used to block ARF GTPase-activating protein activity, results in membrane-associated AP-1, which is largely insensitive to Tris extraction. Thus, ARF1. GTP hydrolysis results in lower-affinity binding of AP-1 to the TGN. Using two-stage assays in which ARF1.GTP first primes the Golgi membrane at 37 degreesC, followed by AP-1 binding on ice, we find that the high-affinity nucleating sites generated in the priming stage are rapidly lost. In addition, the AP-1 bound to primed Golgi membranes during a second-stage incubation on ice is fully sensitive to Tris extraction, indicating that the priming stage has passed the ARF1.GTP hydrolysis point. Thus, hydrolysis of ARF1.GTP at the priming sites can occur even before AP-1 binding. Our finding that purified clathrin-coated vesicles contain little ARF1 supports the concept that ARF1 functions in the coat assembly process rather than during the vesicle-uncoating step. We conclude that ARF1 is a limiting factor in the GTP-stimulated recruitment of AP-1 in vitro and that it appears to function in a stoichiometric manner to generate high-affinity AP-1 binding sites that have a relatively short half-life.
authors.affiliation: Division of Hematology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Zhu
authors.forename: L M
authors.initials: LM
authors.lastname: Traub
authors.forename: S
authors.initials: S
authors.lastname: Kornfeld
mesh_terms: D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D001665:Binding Sites; D002457:Cell Extracts; D002966:Clathrin / Q000378:metabolism; D018699:Coated Vesicles / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006868:Hydrolysis; D007425:Intracellular Membranes / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013696:Temperature; D014325:Tromethamine
chemical_list: D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D002457:Cell Extracts; D002966:Clathrin; D008565:Membrane Proteins; D014325:Tromethamine; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors