The medium subunits of adaptor complexes recognize distinct but overlapping sets of tyrosine-based sorting signals

Abstract

Tyrosine-based sorting signals conforming to the motif YXXφ (Y is tyrosine, X is any amino acid, and φ is an amino acid with a bulky hydrophobic side chain (leucine, isoleucine, phenylalanine, methionine, valine)) interact with the medium (μ) subunits of clathrin adaptor (AP) complexes. We have analyzed the selectivity of interaction between YXXφ signals and the μ1, μ2, and μ3 (A or B) subunits of the AP-1, AP-2, and AP-3 complexes, respectively, by screening a combinatorial XXXYXXφ library using the yeast two-hybrid system. All the medium subunits were found to prefer proline at position Y+2, suggesting that YXXφ signals are stabilized by a bend in the polypeptide backbone. Other than for this common preference, each medium subunit favored specific sets of residues at the X and φ positions; these preferences were consistent with the proposed roles of the different adaptor complexes in rapid endocytosis and lysosomal targeting. A considerable specificity overlap was also revealed by these analyses, suggesting that additional factors, such as the context of the signals, must be important determinants of recognition.