Dileucine-based sorting signals bind to the β chain of AP-1 at a site distinct and regulated differently from the tyrosine-based motif-binding site

In previous work, we showed that peptides from endocytosed proteins containing the tyrosine YXX phi sorting motif are recognized by the mu 2 subunit of AP-2, the plasma membrane clathrin adaptor protein complex. This interaction is activated phosphoinositide lipids that are phosphorylated at the D-3 position of the inositol ring, and is also enhanced by the formation of clathrin-AP-2 coats, Here, we describe the detection of a specific interaction between peptides containing a second sorting motif, the dileucine motif, and AP-1, the clathrin adaptor complex responsible for sorting proteins at the trans-Golgi network (TGN). Surprisingly the site of dileucine binding is the beta 1 subunit, not mu 1. A YXX phi-containing peptide from a protein trafficked within the TGN does bind to mu 1, however. Phosphatidylinositol 3,4-diphosphate and 3,4,5-triphosphate did not activate the interaction between dileucine-containing peptides and AP-1 but instead inhibited it, and clathrin-AP-1 coat formation did not alter the interaction, Thus, there are at least two physically separate binding sites far sorting signals on APs, which are also regulated independently.