title: Multiple interactions of auxilin 1 with clathrin and the AP-2 adaptor complex.
abstract: The removal of the clathrin coat is essential for vesicle fusion with acceptor membranes. Disassembly of the coat involves hsc70, which is specifically recruited by members of the auxilin protein family to clathrin lattices. In vitro, this function of auxilin does not require the globular amino-terminal domain of the clathrin heavy chain, which is known to play a prominent role in the interaction of clathrin with adaptors and numerous endocytic accessory proteins. Here we report the unexpected finding that the neuron-specific form of auxilin (auxilin 1) can also associate with the clathrin amino-terminal domain. This interaction is mediated through tandemly arranged sites within the auxilin 1 carboxyl-terminal segment 547-910. The overlapping auxilin 1 fragments 547-714 and 619-738 bind the clathrin terminal domain with high affinity, whereas auxilin 1-(715-901) interacts only poorly with it. All three fragments also associate with the clathrin distal domain and the alpha-appendage domain of AP-2. Moreover, they support efficient assembly of clathrin triskelia into regular cages. A novel uncoating assay was developed to demonstrate that auxilin 1-(715-901) functions efficiently as a cofactor for hsc70 in the uncoating of clathrin-coated vesicles. The multiple protein-protein interactions of auxilin 1 suggest that its function in endocytic trafficking may be more complex than previously anticipated.
authors.affiliation: Department of Cell Biology, Center of Anatomy, Hannover Medical School, D-30125 Hannover, Germany.
authors.forename: U
authors.initials: U
authors.lastname: Scheele
authors.forename: C
authors.initials: C
authors.lastname: Kalthoff
authors.forename: E
authors.initials: E
authors.lastname: Ungewickell
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D002966:Clathrin / Q000737:chemistry / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000378:metabolism; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006801:Humans; D015151:Immunoblotting; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D013552:Swine; D014357:Trypsin / Q000494:pharmacology
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D002352:Carrier Proteins; D002966:Clathrin; D018076:DNA, Complementary; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; C494371:HSPA8 protein, human; D008565:Membrane Proteins; D011994:Recombinant Proteins; D006639:Histidine; D005982:Glutathione Transferase; D014357:Trypsin