title: Interaction of amphiphysins with AP-1 clathrin adaptors at the membrane.
abstract: The assembly of clathrin/AP (adaptor protein)-1-coated vesicles on the trans-Golgi network and endosomes is much less studied than that of clathrin/AP-2 vesicles at the plasma membrane for endocytosis. In vitro, the association of AP-1 with protein-free liposomes had been shown to require phosphoinositides, Arf1 (ADP-ribosylation factor 1)-GTP and additional cytosolic factor(s). We have purified an active fraction from brain cytosol and found it to contain amphiphysin 1 and 2 and endophilin A1, three proteins known to be involved in the formation of AP-2/clathrin coats at the plasma membrane. Assays with bacterially expressed and purified proteins showed that AP-1 stabilization on liposomes depends on amphiphysin 2 or the amphiphysin 1/2 heterodimer. Activity is independent of the SH3 (Src homology 3) domain, but requires interaction of the WDLW motif with γ-adaptin. Endogenous amphiphysin in neurons and transfected protein in cell lines co-localize perinuclearly with AP-1 at the trans-Golgi network. This localization depends on interaction of clathrin and the adaptor sequence in the amphiphysins and is sensitive to brefeldin A, which inhibits Arf1-dependent AP-1 recruitment. Interaction between AP-1 and amphiphysin 1/2 in vivo was demonstrated by co-immunoprecipitation after cross-linking. These results suggest an involvement of amphiphysins not only with AP-2 at the plasma membrane, but also in AP-1/clathrin coat formation at the trans-Golgi network.
authors.affiliation: Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland.
authors.forename: Sonja
authors.initials: S
authors.lastname: Huser
authors.forename: Gregor
authors.initials: G
authors.lastname: Suri
authors.forename: Pascal
authors.initials: P
authors.lastname: Crottet
authors.forename: Martin
authors.initials: M
authors.lastname: Spiess
mesh_terms: D033961:Adaptor Protein Complex 1 / Q000737:chemistry / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism*; D002522:Chlorocebus aethiops; D002966:Clathrin / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D008081:Liposomes; D051379:Mice; D041681:NIH 3T3 Cells; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D021601:trans-Golgi Network / Q000378:metabolism
chemical_list: D033961:Adaptor Protein Complex 1; D048868:Adaptor Proteins, Signal Transducing; C101410:BIN1 protein, human; D002966:Clathrin; D008081:Liposomes; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; C107640:SH3GL2 protein, human; D025521:Tumor Suppressor Proteins; C075637:amphiphysin