The γ/σ1 and α/σ2 hemicomplexes of clathrin adaptors AP-1 and AP-2 harbor the dileucine recognition site

The clathrin adaptors AP-1 and AP-2 bind cargo proteins via two types of motifs: tyrosine-based Yxx phi and dileucine-based [DE]XXXL[LI]. Although it is well established that Yxx phi motifs bind to the mu subunits of AP-1 or AP-2, dileucine motifs have been reported to bind to either the mu or beta subunits of these adaptors as well as the gamma/sigma 1 hemicomplex of AP-1. To clarify this controversy, the various subunits of AP-1 and AP-2 were expressed individually and in hemicomplex form in insect cells, and they were used in glutathione S-transferase pull-down assays to determine their binding properties. We report that the gamma/sigma 1 or alpha/sigma 2 hemicomplexes bound the dileucine-based motifs of several proteins quite strongly, whereas binding by the beta 1/mu 1 and beta 2/mu 2 hemicomplexes, and the individual beta or mu subunits, was extremely weak or undetectable. The gamma/sigma 1 and alpha/sigma 2 hemicomplexes displayed substantial differences in their preference for particular dileucine-based motifs. Most strikingly, an aspartate at position -4 compromised binding to the gamma/sigma 1 hemicomplex, whereas minimally affecting binding to alpha/sigma 2. There was an excellent correlation between binding to the alpha/sigma 2 hemicomplex and in vivo internalization mediated by the dileucine-based sorting signals. These findings provide new insights into the trafficking mechanisms of D/EXXXL[LI]-mediated sorting signals.