IDENTIFICATION OF A CONTACT REGION FOR PEPTIDE ON THE TAP1 CHAIN OF THE TRANSPORTER ASSOCIATED WITH ANTIGEN-PROCESSING

The transporter associated with Ag processing (TAP) translocates cytos olic peptides into the endoplasmic reticulum for presentation by MHC c lass I molecules, Recently, the actual peptide translocation step has been suggested to be preceded by binding of the peptide to TAP, In thi s study, we investigated the peptide binding site of TAP and its relev ance for peptide selection by cross-linking of translocatable peptides , Our data demonstrate, first, that for a TAP heterodimer containing t he rat TAP2(u) allelic product, which selects peptides on basis of the ir C terminus, the translocation efficiency correlates with the peptid e binding efficiency, Second, peptides having the cross-linker at diff erent positions all label both the TAP1 and the TAP2 subunit after bin ding to the heterodimer, indicating that both TAP subunits contribute directly to the peptide binding site and contact most or all amino aci ds of a bound peptide, Third, by enzymatic digestion and the use of sp ecific antisera, we identified a domain of human TAP1 that contributes to the peptide binding site. This domain contains the two hydrophobic and thus putative transmembrane regions closest to the ATP binding si te, We conclude that the peptide binding site controls the selectivity of TAP and is composed of domains of both TAP1 and TAP2, which each c ontact the bound peptide over all or most of its length, Moreover, the major contact site(s) for peptide on TAP1 are located within or close to the two putative transmembrane regions adjacent to the ATP binding site.