Serine 331 and tyrosine 333 are both involved in the interaction between the cytosolic domain of TGN38 and the mu2 subunit of the AP2 clathrin adaptor complex.
TGN38 is a type I integral membrane protein that cycles between the trans-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 mu2 subunit of the AP2 adaptor complex. We have studied the interaction between the entire cytosolic domain of TGN38 and mu2 using the yeast two hybrid system, in vitro binding of recombinant fusion proteins and IAsys optical biosensor technology. A specific interaction has been demonstrated in each of the systems we have employed. We have shown an absolute requirement for Tyr-333 of TGN38 in binding to mu2. In addition we found that mutation of Ser-331 to alanine reduces the affinity of the interaction. By measuring tryptophan fluorescence at equilibrium, we have also determined the dissociation constant for the interaction between the entire cytosolic tail of TGN38 and mu2 as 58 nM. In contrast to previously published work, our data suggest that not only Tyr-333 but also its context is important in determining the specificity of binding of TGN38 to mu2.
Stephens
D J
DJ
Department of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom.
Crump
C M
CM
Clarke
A R
AR
Banting
G
G
D033961:Adaptor Protein Complex 1; D033962:Adaptor Protein Complex 2; D033963:Adaptor Protein Complex 3; D033965:Adaptor Protein Complex alpha Subunits; D033983:Adaptor Protein Complex mu Subunits; D033942:Adaptor Proteins, Vesicular Transport; D001665:Binding Sites; D006023:Glycoproteins; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D012694:Serine / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
C510070:AP3M2 protein, human; D033961:Adaptor Protein Complex 1; D033962:Adaptor Protein Complex 2; D033963:Adaptor Protein Complex 3; D033965:Adaptor Protein Complex alpha Subunits; D033983:Adaptor Protein Complex mu Subunits; D033942:Adaptor Proteins, Vesicular Transport; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C499065:adaptor protein complex 1, mu 2 subunit; C438886:adaptor protein complex 2, mu 2 subunit; D014443:Tyrosine; D012694:Serine