Functional domain mapping of the clathrin-associated adaptor medium chains μ1 and μ2

Abstract

The clathrin-associated adaptors AP-1 and AP-2 are heterotetrameric complexes involved in the recognition of sorting signals present within the cytosolic domain of integral membrane proteins. The medium chains of these complexes, μ1 and μ2, have been implicated in two types of interaction: assembly with the β1 and β2 chains of the corresponding complexes and recognition of tyrosine-based sorting signals. In this study, we report the results of a structure-function analysis of the μ1 and μ2 chains aimed at identifying regions of the molecules that are responsible for each of the two interactions. Analyses using the yeast two-hybrid system and proteolytic digestion experiments suggest that μ1 and μ2 have a bipartite structure, with the amino-terminal one-third (residues 1-145 of μ1 and μ2) being involved in assembly with the β chains and the carboxyl-terminal two-thirds (residues 147-423 of μ1 and 164-435 of μ2) binding tyrosine-based sorting signals. These observations support a model in which the amino-terminal one- third of μ2 is embedded within the core of the AP-2 complex, while the carboxyl-terminal two-thirds of the protein are exposed to the medium, placing this region in a position to interact with tyrosine-based sorting signals.