SPECIFICITY OF INTERACTION BETWEEN ADAPTER-COMPLEX MEDIUM CHAINS AND THE TYROSINE-BASED SORTING MOTIFS OF TGN38 AND LGP120

Multiple sorting steps within eukaryotic cells are mediated by tyrosin e-based sorting motifs. Motifs conforming to the consensus -YXX empty set - (where empty set indicates a bulky hydrophobic residue) have bee n shown to specify high-efficiency internalization from the plasma mem brane, targeting from the plasma membrane to the trans-Golgi network a nd targeting to lysosomal compartments as well as being involved in ba solateral sorting in polarized cells. These motifs are recognized by t he medium-chain subunits of heterotetrameric adaptor complexes. Wherea s these motifs have been shown to be sufficient to mediate interaction with the mu-chains, we and others have shown that their context is im portant in determining the affinity of interaction. In this study we h ave investigated the interaction between the tyrosine motifs of the ty pe-1 integral membrane proteins TGN38 and lgp120 with medium-chain sub units using the yeast two-hybrid system. Whereas the wild-type version of the cytosolic domain of TGN38 interacts with highest affinity with mu 2, we show that the cytosolic domain of lgp120 interacts almost ex clusively with mu 3A. The specificity of binding of tyrosine-based sor ting motifs to mu-chains is shown to be highly sensitive to the contex t in which the motif lies. For example, the -YQTI- motif of lgp120 is effectively nonfunctional with regard to mu-chain binding when placed in the context of the TGN38 cytosolic domain. Deletion of four amino a cids (NLKL) at the extreme C-terminus of TGN38, leaving the YXX empty set motif as the C-terminus, greatly enhances the affinity of interact ion with mu 2. Furthermore, addition of these same residues to the ext reme C-terminus of lgp120 effectively abolishes the interaction of the cytosolic domain of lgp120 with mu-chains. We also show that the newl y identified mu-adaptin-related protein 2 (mu 4) only interacts weakly with tyrosine-based sorting motifs.