Functional assay of effectors of ADP ribosylation factor 6 during clathrin/AP-2 coat recruitment to membranes.
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.
Krauss
Michael
M
Department of Biochemistry II, Center for Biochemistry and Molecular Cell Biology, University of Göttingen, Germany.
Haucke
Volker
V
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*
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