The appendage domain of the AP-2 subunit is not required for assembly or invagination of clathrin-coated pits.
Coated pits contain a resident membrane molecule(s) that binds clathrin AP-2 with high affinity. AP-2 binding to this site is likely to be the first step in coated pit assembly because this subunit functions as a template for the polymerization of clathrin into flat polygonal lattices. Integral membrane proteins involved in receptor mediated endocytosis cluster in the newly assembled pits as they invaginate and bud from the membrane. The AP-2 subunit is a multi-domain, molecular complex that can be separated by proteolysis into a brick-shaped core and ear-like appendage domains. We have used this property to identify the domain involved in the various stages of coated pit assembly and budding. We found that the core of AP-2 is the domain that binds both to membranes and to triskelions during assembly. Triskelions are perfectly capable of forming lattices on the membrane bound cores. Clathrin lattices bound only to core domains were also able to invaginate normally. Limited proteolysis was also useful for further characterizing the AP-2 binding site. Elastase treatment of the inside membrane surface released a peptide fraction that is able to bind AP-2 in solution and prevent it from interacting with membranes. Affinity purification of binding activity yielded a collection of peptides that was dominated by a 45-kD species. This is the candidate peptide for containing the AP-2-binding site. Therefore, the appendage domain does not directly participate in any of the assembly or invagination events required for coated pit function.
Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, Dallas 75235.
J S
JS
Peeler
W C
WC
Donzell
R G
RG
Anderson
D033942:Adaptor Proteins, Vesicular Transport; D002478:Cells, Cultured; D002966:Clathrin / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism / Q000648:ultrastructure*; D004705:Endocytosis; D005613:Freeze Etching; D006801:Humans; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000378:metabolism*; D008854:Microscopy, Electron; D010196:Pancreatic Elastase / Q000494:pharmacology; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013329:Structure-Activity Relationship
D033942:Adaptor Proteins, Vesicular Transport; D002966:Clathrin; D008565:Membrane Proteins; D010750:Phosphoproteins; D011956:Receptors, Cell Surface; D010196:Pancreatic Elastase