Gamma subunit of the AP-1 adaptor complex binds clathrin: implications for cooperative binding in coated vesicle assembly.
The heterotetrameric AP-1 adaptor complex is involved in the assembly of clathrin-coated vesicles originating from the trans-Golgi network (TGN). The beta 1 subunit of AP-1 is known to contain a consensus clathrin binding sequence, LLNLD (the so-called clathrin box motif), in its hinge segment through which the beta chain interacts with the N-terminal domains of clathrin trimers. Here, we report that the hinge region of the gamma subunit of human and mouse AP-1 contains two copies of a new variant, LLDLL, of the clathrin box motif that also bind to the terminal domain of the clathrin heavy chain. High-affinity binding of the gamma hinge to clathrin trimers requires both LLDLL sequences to be present and the spacing between them to be maintained. We also identify an independent clathrin-binding site within the appendage domain of the gamma subunit that interacts with a region of clathrin other than the N-terminal domain. Clathrin polymerization is promoted by glutathione S-transferase (GST)-gamma hinge, but not by GST-gamma appendage. However, the hinge and appendage domains of gamma function in a cooperative manner to recruit and polymerize clathrin, suggesting that clathrin lattice assembly at the TGN involves multivalent binding of clathrin by the gamma and beta1 subunits of AP-1.
Doray
B
B
Washington University School of Medicine, Department of Internal Medicine, St. Louis, Missouri 63110, USA.
Kornfeld
S
S
D033942:Adaptor Proteins, Vesicular Transport; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002966:Clathrin / Q000378:metabolism*; D022163:Clathrin-Coated Vesicles / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012995:Solubility
D033942:Adaptor Proteins, Vesicular Transport; D002352:Carrier Proteins; D002966:Clathrin; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase