A Novel Subtype of AP-1-binding Motif within the Palmitoylated trans-Golgi Network/Endosomal Accessory Protein Gadkin/γ-BAR

Tanja Maritzen,Michael R Schmidt,Viktoria Kukhtina,Victoria A Higman,Holger Strauss,Rudolf Volkmer,Hartmut Oschkinat,Carlos G Dotti,Volker Haucke

doi:10.1074/jbc.m109.049197

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.

Highlights

Membrane traffic within eukaryotic cells is mediated in part by coated transport vesicles or tubules that deliver transmembrane cargo proteins and lipids to multiple intracellular destinations along the secretory and endocytic pathways (1, 2)
We identify a novel subtype of AP-1-binding motif within Gadkin that associates with the ␥1-adaptin ear domain
We show that Gadkin is a peripheral S-palmitoylated membrane protein and that this modification is absolutely required for its recruitment to trans-Golgi network (TGN)/endosomal membranes in living cells

Summary

EXPERIMENTAL PROCEDURES

Mutagenesis, and Antibodies—The full-length Gadkin coding sequence or truncated versions thereof were cloned into pEGFP-N (Clontech) for mammalian expression of C-terminally eGFP-tagged proteins. Protein extracts from cultured mammalian cells were prepared in lysis buffer containing 20 mM HEPES, pH 7.4, 2 mM MgCl2, 100 mM NaCl, and 1% Triton X-100 supplemented with 1 mM phenylmethylsulfonyl fluoride and mammalian protease inhibitor mixture (Sigma). Beads were extensively washed in 50 mM Tris buffer, pH 7.4, containing 150 mM NaCl, 2 mM MgCl2, and 0.1% Triton X-100 and treated with 25 mM N-ethylmaleimide (diluted from a freshly prepared 1 M stock solution in EtOH) in buffer for 30 min at room temperature to quench free sulfhydryl (SH)-groups. His6-tagged Gadkin in pET28a(ϩ), transformed in E. coli BL21(DE3) strain was expressed in 2ϫYT medium (16 g/liter tryptone, 10 g/liter yeast extract, 5 g/liter NaCl) and purified using the same procedures as for ␥-ear protein. B, 20 ␮g of GST (control), GST-tagged Gadkin WT, or its truncation mutants ⌬51 and 1–100 (bound to glutathione beads) were incubated with 1.5 mg of Triton X-100-solubilized rat brain extract.

RESULTS

Protein sAB

DISCUSSION