Abstract
Adaptor protein (AP) complexes bind to transmembrane proteins destined for internalization and to membrane lipids, so linking cargo to the accessory internalization machinery. This machinery interacts with the appendage domains of APs, which have platform and beta-sandwich subdomains, forming the binding surfaces for interacting proteins. Proteins that interact with the subdomains do so via short motifs, usually found in regions of low structural complexity of the interacting proteins. So far, up to four motifs have been identified that bind to and partially compete for at least two sites on each of the appendage domains of the AP2 complex. Motifs in individual accessory proteins, their sequential arrangement into motif domains, and partial competition for binding sites on the appendage domains coordinate the formation of endocytic complexes in a temporal and spatial manner. In this work, we examine the dominant interaction sequence in amphiphysin, a synapse-enriched accessory protein, which generates membrane curvature and recruits the scission protein dynamin to the necks of coated pits, for the platform subdomain of the alpha-appendage. The motif domain of amphiphysin1 contains one copy of each of a DX(F/W) and FXDXF motif. We find that the FXDXF motif is the main determinant for the high affinity interaction with the alpha-adaptin appendage. We describe the optimal sequence of the FXDXF motif using thermodynamic and structural data and show how sequence variation controls the affinities of these motifs for the alpha-appendage.
Highlights
Clathrin-mediated endocytosis is the process whereby proteins and lipids destined for internalization from the plasma membrane are packaged into vesicles with the aid of a clathrin coat
Using electron microscopy it was shown that the AP2 complex can be subdivided into the following: (i) a trunk domain, which interacts with cargo proteins and PtdIns[4,5]P2, and (ii) two appendage domains made from the C termini of the ␣- and -subunits, which interact with a large number of accessory proteins by binding to short motifs in these proteins
When AP2 complexes are bound to polymerized clathrin, the 2-adaptin appendage domain is predicted to be largely occupied by clathrin, and steric hindrance is thought to exclude most accessory proteins
Summary
Constructs and Protein Expression—The ␣-adaptin appendage domain (residues 701–938) and the appendage-plus hinge domain (residues 653–938), the human 2-adaptin appendage domain (residues 701–937), rat amphiphysin1AB (Amph1AB; residues 1–378), and rat amphiphysin2AB (Amph2AB; residues 1– 422) were expressed as N-terminal glutathione S-transferase (GST) fusion proteins (in pGex4T2) in BL21 cells following overnight isopropyl 1-thio--D-galactopyranoside induction at 22 °C. The appendage domain of human 2-adaptin (residues 701–937) was expressed in BL21 cells as an N-terminal His fusion protein (in pET-15b) and purified by passage over nickel-nitrilotriacetic acid, followed by Q-Sepharose and gel filtration chromatography. Pull Downs from COS-7 Cell or Rat Brain Extracts with GST Appendages—For interaction experiments, the extracts described above were incubated with 30 –50 g of GST fusion protein on glutathione-Sepharose beads for 1 h at 4 °C, and the bead-bound proteins were washed four times with 150 mM NaCl, 20 mM HEPES, pH 7.4, 2 mM DTT, protease inhibitors, and 0.1% Triton X-100. The validated coordinates and structure factors for the crystal structure containing the synaptojanin WVXF and the amphyphysin FEDNFVP have been deposited in the Protein Data Bank [36] (PDB code 2vj0). Figures were generated using Aesop, and the peptide interaction map was generated using the output from LIGPLOT [37] as a starting point
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