Crystal structure of the clathrin adaptor protein 1 core

Abstract

The heterotetrameric adaptor proteins (AP complexes) link the outer lattice of clathrin-coated vesicles with membrane-anchored cargo molecules. We report the crystal structure of the core of the AP-1 complex, which functions in the trans-Golgi network (TGN). Packing of complexes in the crystal generates an exceptionally long (1,135-A) unit-cell axis, but the 6-fold noncrystallographic redundancy yields an excellent map at 4-A resolution. The AP-1 core comprises N-terminal fragments of the two large chains, beta1 and gamma, and the intact medium and small chains, micro1 and sigma1. Its molecular architecture closely resembles that of the core of AP-2, the plasma-membrane-specific adaptor, for which a structure has been determined. Both structures represent an "inactive" conformation with respect to binding of cargo with a tyrosine-based sorting signal. TGN localization of AP-1 depends on the small GTPase, Arf1, and the phosphoinositide, PI-4-P. We show that directed mutations of residues at a particular corner of the gamma chain prevent recruitment to the TGN in cells and diminish PI-4-P-dependent, but not Arf1-dependent, liposome binding in vitro.

Fig. 1.

Structure of the AP-1 core. (a) Ribbon representation. The γ chain is blue; β1, red; μ1N, green; μ1C, yellow–orange; and σ1, magenta. Each chain is color-ramped, from the N to the C terminus. The large chains go from a lighter to a darker shade: μ1 goes from green in μ1N to yellow in μ1C. Disordered segments are dotted lines of appropriate color. The disordered connecting link from μ1N to μ1C is a purple dotted line. Locations of the proposed PI-4-P-binding site on γ and of the YxxΦ site on μ1 are shown by black ellipses. A pseudo-2-fold axis, running diagonally from upper left to lower right, relates the two large-chain trunks to each other and μ1N to σ1. (b) Molecular surface representation. Colors as in a.(c) Diagram of AP-1. The drawing applies also to AP-2 (4) and presumably to AP-3 and AP-4 as well. The core comprises the large-chain trunks (γ, blue; β1, red), the medium chain (μ1N, yellow–green; μ1C, yellow–orange), and the small chain (σ1, magenta). The appendages of the large chains are linked to the trunks by extended flexible connectors. Yellow star, binding site for tyrosine-based sorting signal; white star, binding site for inositol-phosphate headgroup (proposed).

Fig. 2.

Comparison of the helix 2–helix 3 corner in the α chain (Left) of AP-2 and the γ chain (Right) of AP-1. (a) View as if looking from the left in Fig. 1a toward the corner enclosed in the oval; the top of the present figure corresponds to the bottom of Fig. 1. (b) The same as a, but rotated by 90° as shown. The α-adaptin of AP-2 is in yellow; the γ-adaptin of AP-1 is in cyan. The InsP₆ molecule and residues involved in binding phosphoinositides are in ball-and-stick representation (nitrogen, blue; oxygen, red; phosphorus, magenta; carbon, pale yellow in AP-2, pale cyan and gray in AP-1, and gray in InsP₆). (c) Amino acid sequences in this region and their secondary structures. The secondary-structure symbols are colored to match the ribbon diagram. Residues involved in phosphoinositide binding are in magenta (γ chain) and red (α chain); the residue R6 of γ chain is in green.

Fig. 3.

Effect of mutations in γ chain on the intracellular distribution of AP-1. HeLa cells transiently expressing wild-type or mutant (R6E, Y45A, R48A, and K52A) mouse γ-adaptin fused to enhanced GFP (green, Right) were fixed and fluorescently labeled with a sheep polyclonal antibody to TGN46 (yellow, Left) or a mouse monoclonal antibody 100/3 to human (but not mouse) γ-adaptin (endogenous γ; red, Center).

Fig. 4.

Effect of mutations in γ chain on recruitment of AP-1 cores to liposomes. AP-1 cores (0.2 μM) containing either wild-type or mutant (Y45A or R48A) mouse γ subunits were incubated with liposomes and the amount bound was determined as described in Materials and Methods. The composition of the liposomes (mol %) was 57% phosphatidylcholine (PC), 28% phosphatidylethanolamine (PE), and 15% PS (lanes 1, 3, 4, 6, 7, and 9) or 57% PC, 28% PE, 10% PS, and 5% PI-4-P (lanes 2, 5, and 8). Myristoylated Arf1, preloaded with GTPγS, was included in the liposomes analyzed in lanes 3, 6, and 9. Histograms show the amount (average ± standard deviation from three to four experiments) of AP-1 core cosedimented with liposomes (expressed as percent of total). In the absence of liposomes or the presence of liposomes containing PC and PE only, the amount of AP-1 in the pellet was <1–2%.