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

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␾ and dileucine-based [DE]XXXL[LI]. Although it is well established that Yxx␾ motifs bind to the ␮ subunits of AP-1 or AP-2, dileucine motifs have been reported to bind to either the ␮ or ␤ subunits of these adaptors as well as the ␥/␴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 ␥/␴1 or ␣/␴2 hemicomplexes bound the dileucine-based motifs of several proteins quite strongly, whereas binding by the ␤1/␮1 and ␤2/␮2 hemicomplexes, and the individual ␤ or ␮ subunits, was extremely weak or undetectable. The ␥/␴1 and ␣/␴2 hemicomplexes displayed substantial differences in their preference for particular dileucine-based motifs. Most strikingly, an aspartate at position ؊4 compromised binding to the ␥/␴1 hemicomplex, whereas minimally affecting binding to ␣/␴2. There was an excellent correlation between binding to the ␣/␴2 hemicom-plex 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.