title: Functional assay of effectors of ADP ribosylation factor 6 during clathrin/AP-2 coat recruitment to membranes.
abstract: Arf proteins play pivotal roles in membrane traffic, cell signaling, and actin cytoskeletal rearrangements. We describe here methods to functionally analyze interacting partner proteins of recombinantly produced N-myristoylated Arf6. Combined evidence from affinity purification and chemical crosslinking experiments, in vitro recruitment assays, and the analysis of lipid kinase activities indicates that Arf6-GTP facilitates clathrin/AP-2 recruitment to synaptic membranes by direct binding and activation of the brain-specific phosphatidylinositol 4-phosphate 5-kinase type Igamma (PIPK Igamma). These methods shall help to mechanistically dissect the role of Arf6 in regulating exo-endocytic vesicle cycling at synapses and in related membrane trafficking events.
authors.affiliation: Department of Biochemistry II, Center for Biochemistry and Molecular Cell Biology, University of Göttingen, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Krauss
authors.forename: Volker
authors.initials: V
authors.lastname: Haucke
mesh_terms: D020823:ADP-Ribosylation Factor 1 / Q000235:genetics; D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000378:metabolism*; D033962:Adaptor Protein Complex 2 / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000166:cytology; D022181:COP-Coated Vesicles / Q000502:physiology; D002458:Cell Fractionation / Q000379:methods; D002966:Clathrin / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D019814:Myristic Acid / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods; D051381:Rats; D013570:Synaptic Membranes / Q000378:metabolism*
chemical_list: D000089982:ADP-Ribosylation Factor 6; D033962:Adaptor Protein Complex 2; D002966:Clathrin; D019814:Myristic Acid; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human; C000715349:Arf6 protein, rat