title: Site-specific cross-linking reveals a differential direct interaction of class 1, 2, and 3 ADP-ribosylation factors with adaptor protein complexes 1 and 3.
abstract: We have used a site-specific photo-cross-linking approach to identify direct interactions between clathrin adaptor protein (AP)1 complexes and small GTPases of the ADP-ribosylation factor (ARF) family and to explore the specificity of this interaction on immature secretory granule (ISG) membranes. ISG membranes are a well-characterized, highly enriched preparation of membranes that has previously been shown to have the membrane-associated factors for ARF1 recruitment that are not present on artificial liposomes. All three classes of ARF proteins could be recruited to ISG membranes, displaying differential requirements for GTPgammaS. We found that ARF1, ARF5, and ARF6 interacted directly with the beta1-adaptin subunit of AP-1 in the presence of GTPgammaS. Furthermore, we observed a direct interaction between the switch 1 region of ARF1 and the N-terminal trunk domains of gamma- and beta1-adaptin. In addition, both ARF1 and ARF6 but not ARF5 interacted directly with the beta3- and delta-adaptin subunits of AP-3. No interaction was observed between AP-2 and any of the ARF proteins. Our results delineate the specificity and provide evidence of a direct interaction between different ARF proteins and the AP complexes AP-1 and AP-3 on natural ISG membranes and show that residues in the switch 1 region of ARF proteins can selectively bind to the trunk domains of these complexes.
authors.affiliation: Secretory Pathways Laboratory, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, England.
authors.forename: Carol
authors.initials: C
authors.lastname: Austin
authors.forename: Markus
authors.initials: M
authors.lastname: Boehm
authors.forename: Sharon A
authors.initials: SA
authors.lastname: Tooze
mesh_terms: D020823:ADP-Ribosylation Factor 1 / Q000737:chemistry / Q000378:metabolism*; D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000737:chemistry / Q000378:metabolism*; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D000906:Antibodies; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D003432:Cross-Linking Reagents; D020558:GTP Phosphohydrolases / Q000378:metabolism; D008081:Liposomes; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D034041:Monomeric Clathrin Assembly Proteins; D016297:Mutagenesis, Site-Directed; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins
chemical_list: D000089982:ADP-Ribosylation Factor 6; D033942:Adaptor Proteins, Vesicular Transport; D000906:Antibodies; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D008081:Liposomes; D008565:Membrane Proteins; D034041:Monomeric Clathrin Assembly Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C439023:clathrin assembly protein AP180; C116686:ARF2 protein, S cerevisiae; D020558:GTP Phosphohydrolases; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; C116691:ARF5 protein, human; C000715348:ARF6 protein, human