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  • Title: Functional assay of effectors of ADP ribosylation factor 6 during clathrin/AP-2 coat recruitment to membranes.
    Author: Krauss M, Haucke V.
    Journal: Methods Enzymol; 2005; 404():388-98. PubMed ID: 16413285.
    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.
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