SELECTION AND BINDING OF PEPTIDES TO HUMAN TRANSPORTERS ASSOCIATED WITH ANTIGEN-PROCESSING AND RAT CIM-A AND CIM-B

Cytotoxic T lymphocytes recognize antigenic peptides presented by MHC class I molecules. The peptides are generated in the cytosol by protea somes, and probably also other proteases, and are then translocated in to the endoplasmic reticulum (ER) lumen. The transporters associated w ith Ag processing (TAP) are key molecules for transporting peptides fr om the cytosol to the lumen of the ER. Using semipermeabilized cells, TAP-dependent peptide translocation was demonstrated, and the selectiv ity of peptide translocation was based on the carboxyl-terminal amino acid of peptides. We have examined peptide binding proteins in the ER membrane and the selection of peptides for binding to TAP by using a p anel of peptides of different sequences and carboxyl-termini as well a s peptides containing D amino acids. Peptides bound to TAP molecules i n the absence of ATP. The presence of ATP induced binding of peptides to two additional membrane proteins (58 and 43 kDa). The selection of peptides by TAP molecules was based on peptide sequence and the carbox yl- terminal amino acid. Peptides containing D amino acid did not bind to TAP molecules. Rat cim-a and -b selected peptides differently, and selection was not only dependent on the carboxyl-terminal residue of the peptide, but included an influence of the peptide sequence. The di fferent off-rates after peptide binding to TAP, indicated a dual bindi ng step of peptide to TAP. ATP regulated the off-rate of peptides at a high affinity binding step. Our results demonstrate that the binding of peptides to TAP molecules is specific and most likely involves a mu ltiple step pathway.