SUBSTRATE SELECTION BY TRANSPORTERS ASSOCIATED WITH ANTIGEN-PROCESSING OCCURS DURING PEPTIDE BINDING TO TAP

Presentation of antigenic peptides by major histocompatibility complex (MHC) class I molecules depends on translocation of cytosolic peptide s into the endoplasmic reticulum (ER) by transporters associated with antigen processing (TAP). Peptide transport by TAP is thought to inclu de at least two steps: initial binding of peptide to TAP, and its subs equent translocation requiring ATP hydrolysis. These events can be mon itored in peptide binding and transport assays. Previous studies have shown that the efficiency of peptide transport by human, mouse and rat transporters varies according to the C-terminals of peptide substrate s in an allele and species-specific manner. However, it has not been c lear during which step of peptide interaction with TAP selection occur s. We used an assay monitoring the peptide binding step to study the b inding affinity of a library of 199 peptides for human TAP and the two major allelic rat TAP complexes. We observed a dominant influence of the C-terminus on peptide binding affinity for all transporters, and h ighly restrictive selection of peptides with aliphatic and aromatic C- terminals by rat TAP1/TAP2(u) complexes. The selectivity of peptide bi nding to rat TAP complexes is in full accordance with published data o n selective peptide transport and on control of antigen presentation b y rat TAP. These results strongly suggest that (i) peptide selection b y TAP occurs exclusively in the initial binding step; (ii) all factors involved in peptide selection by TAP are present in insect cells. (C) 1998 Elsevier Science Ltd. All rights reserved.