DILEUCINE-BASED SORTING SIGNALS BIND TO THE BETA-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 co ntaining 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 phosp horylated at the D-3 position of the inositol ring, and is also enhanc ed by the formation of clathrin-AP-2 coats, Here, we describe the dete ction of a specific interaction between peptides containing a second s orting motif, the dileucine motif, and AP-1, the clathrin adaptor comp lex 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 t he TGN does bind to mu 1, however. Phosphatidylinositol 3,4-diphosphat e and 3,4,5-triphosphate did not activate the interaction between dile ucine-containing peptides and AP-1 but instead inhibited it, and clath rin-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.