A novel subtype of AP-1-binding motif within the palmitoylated trans-Golgi network/endosomal accessory protein Gadkin/gamma-BAR

J Biol Chem. 2010 Feb 5;285(6):4074-4086. doi: 10.1074/jbc.M109.049197. Epub 2009 Dec 3.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites / genetics
  • COS Cells
  • Cell Line
  • Cell Membrane / metabolism
  • Chlorocebus aethiops
  • Circular Dichroism
  • Endosomes / metabolism*
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • HeLa Cells
  • Humans
  • Immunoblotting
  • Lipoylation
  • Magnetic Resonance Spectroscopy
  • Membrane Proteins / chemistry
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Microscopy, Fluorescence
  • Molecular Sequence Data
  • Mutation
  • Protein Binding
  • Protein Interaction Domains and Motifs
  • Protein Structure, Tertiary
  • Transcription Factor AP-1 / genetics
  • Transcription Factor AP-1 / metabolism*
  • Transfection
  • trans-Golgi Network / metabolism*

Substances

  • AP1AR protein, human
  • Membrane Proteins
  • Transcription Factor AP-1
  • Green Fluorescent Proteins