SERINE-331 AND TYROSINE-333 ARE BOTH INVOLVED IN THE INTERACTION BETWEEN THE CYTOSOLIC DOMAIN OF TGN38 AND THE MU-2 SUBUNIT OF THE AP2 CLATHRIN ADAPTER COMPLEX

TGN38 is a type I integral membrane protein that cycles between the tr ans-Golgi network and the plasma membrane, Internalization at the cell surface and targeting back to the trans-Golgi network is dependent on a hexapeptide motif, SDYQRL, in the cytosolic tail of the protein, It was recently demonstrated that this motif specifically interacts with the mu 2 subunit of the AP2 adaptor complex, We have studied the inte raction between the entire cytosolic domain of TGN38 and mu 2 using th e yeast two hybrid system, in vitro binding of recombinant fusion prot eins and IAsys optical biosensor technology, A specific interaction ha s been demonstrated in each of the systems we have employed, We have s hown an absolute requirement for Tyr-333 of TGN38 in binding to mu 2. In addition we found that mutation of Ser-331 to alanine reduces the a ffinity of the interaction, By measuring tryptophan fluorescence at eq uilibrium, we have also determined the dissociation constant for the i nteraction between the entire cytosolic tail of TGN38 and mu 2 as 58 n M. In contrast to previously published work, our data suggest that not only Tyr-333 but also its context is important in determining the spe cificity of binding of TGN38 to mu 2.