A novel subtype of AP-1-binding motif within the palmitoylated trans-Golgi network/endosomal accessory protein Gadkin/gamma-BAR.
Membrane traffic between the trans-Golgi network (TGN) and endosomes is mediated in part by the assembly of clathrin-AP-1 adaptor complex-coated vesicles. This process involves multiple accessory proteins that directly bind to the ear domain of AP-1gamma via degenerate peptide motifs that conform to the consensus sequence diameterG(P/D/E)(diameter/L/M) (with diameter being a large hydrophobic amino acid). Recently, gamma-BAR (hereafter referred to as Gadkin for reasons explained below) has been identified as a novel AP-1 recruitment factor involved in AP-1-dependent endosomal trafficking of lysosomal enzymes. How precisely Gadkin interacts with membranes and with AP-1gamma has remained unclear. Here we show that Gadkin is an S-palmitoylated peripheral membrane protein that lacks stable tertiary structure. S-Palmitoylation is required for the recruitment of Gadkin to TGN/endosomal membranes but not for binding to AP-1. Furthermore, we identify a novel subtype of AP-1-binding motif within Gadkin that specifically associates with the gamma1-adaptin ear domain. Mutational inactivation of this novel type of motif, either alone or in combination with three more conventional AP-1gamma binding peptides, causes Gadkin to mislocalize to the plasma membrane and interferes with its ability to render AP-1 brefeldin A-resistant, indicating its physiological importance. Our studies thus unravel the molecular basis for Gadkin-mediated AP-1 recruitment to TGN/endosomal membranes and identify a novel subtype of the AP-1-binding motif.
Maritzen
Tanja
T
From the Institute of Chemistry and Biochemistry, Department of Membrane Biochemistry, Freie Universität and Charité-Universitätsmedizin Berlin, Takustrasse 6, 14195 Berlin, Germany.
Schmidt
Michael R
MR
From the Institute of Chemistry and Biochemistry, Department of Membrane Biochemistry, Freie Universität and Charité-Universitätsmedizin Berlin, Takustrasse 6, 14195 Berlin, Germany.
Kukhtina
Viktoria
V
From the Institute of Chemistry and Biochemistry, Department of Membrane Biochemistry, Freie Universität and Charité-Universitätsmedizin Berlin, Takustrasse 6, 14195 Berlin, Germany.
Higman
Victoria A
VA
the Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Strasse 10, 13125 Berlin, Germany.
Strauss
Holger
H
the Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Strasse 10, 13125 Berlin, Germany.
Volkmer
Rudolf
R
the Institut für Medizinische Immunologie, Charité-Universitätsmedizin Berlin, Hessische Strasse 3-4, 10115 Berlin, Germany.
Oschkinat
Hartmut
H
the Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Strasse 10, 13125 Berlin, Germany.
Dotti
Carlos G
CG
the Vlanders Institute for Biotechnology, Molecular & Developmental Genetics Program and Department of Human Genetics, University of Leuven Medical School, Herestraat 49, 3000 Leuven, Belgium, and.
Haucke
Volker
V
From the Institute of Chemistry and Biochemistry, Department of Membrane Biochemistry, Freie Universität and Charité-Universitätsmedizin Berlin, Takustrasse 6, 14195 Berlin, Germany; the Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Strasse 10, 13125 Berlin, Germany. Electronic address: volker.haucke@fu-berlin.de.
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D002942:Circular Dichroism; D011992:Endosomes / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D054878:Lipoylation; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D017434:Protein Structure, Tertiary; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D021601:trans-Golgi Network / Q000378:metabolism*
C500885:AP1AR protein, human; D008565:Membrane Proteins; D018808:Transcription Factor AP-1; D049452:Green Fluorescent Proteins