We recently determined that fusion proteins containing tyrosine‐based endocytic signals bind to the mu 2 subunit of AP‐2, the complex that drives clathrin coat formation and mediates endocytosis from the plasma membrane. Here we analyze the selectivity of peptide recognition by mu 2 and by AP‐2 using combinatorial selection methods and surface plasmon resonance. Both mu 2 and AP‐2 are shown to interact with various sequences of the form tyrosine‐polar‐polar‐hydrophobic (Yppø) found on receptors that follow the clathrin pathway. The optimal sequence for interaction with mu 2 and with AP‐2 has tyrosine as an anchor and prefers arginine at position Y + 2 and leucine at position Y + 3. In contrast, no preferred sequence is detected surrounding the Yppø signal, indicating that recognition of the Yppø endocytic signal does not require a prefolded structure. We conclude that sorting into the endocytic pathway is governed by a surprisingly simple interaction between the mu 2 chain and a tyrosine‐containing tetrapeptide sequence.

Nef, a approximately 200 residue multifunctional regulatory protein of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), interacts with components of host cell signal transduction and clathrin-dependent protein sorting pathways. The downregulation of surface CD4 molecules and major histocompatibility complex (MHC) class I antigens by Nef is believed to be important in AIDS pathogenesis [1-7]. Nef contains a globular core domain and two disordered segments--a myristylated arm at the amino terminus and a carboxy-terminal loop projecting from the globular core [8,9]. Here, we aimed to determine the sorting signals in HIV-1 Nef that were responsible for its involvement in the clathrin-mediated pathway. We found that a sequence in the carboxy-terminal disordered loop of Nef is essential for downregulation of CD4. This sequence resembles the dileucine motif, one of two well-characterized sorting signals that target membrane proteins to clathrin-coated vesicles. The dileucine-motif-containing segment of Nef bound directly and specifically to the beta-adaptin subunit of the clathrin adaptor complexes AP-1 and AP-2, which are responsible for recruiting sorted proteins into coated pits. Unlike wild-type Nef, a mutant form of Nef that lacked the dileucine motif did not localize to clathrin-coated pits and did not downregulate CD4 expression, although it could downregulate MHC class I surface expression. Thus, the dileucine motif in HIV-1 is required for CD4 downregulation and for interaction with clathrin adaptor complexes.

In previous work, we showed that peptides from endocytosed proteins containing the tyrosine YXXphi sorting motif are recognized by the mu 2 subunit of AP-2, the plasma membrane clathrin adaptor protein complex. This interaction is activated by 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 beta1 subunit, not mu 1. A YXXphi-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 for sorting signals on APs, which are also regulated independently.