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Abstract

Antigenic peptides are translocated into the lumen of the endoplasmic reticulum by the action of the transporter associated with antigen processing (TAP), where they are subsequently needed for the correct assembly of major histocompatability complex molecules. The transport function was reconstituted in insect cells by expression of both TAP genes. On the basis of this overexpression system, substrate selection was analyzed in detail by a direct bimolecular peptide binding assay. Competition assays with peptide variants, including substitutions of residues with alanine or structurally related amino acids, underline the broad peptide specificity of the human TAP complex. Steric requirements of the substrate-binding pocket were mapped using elongated peptides and scans with bulky, hydrophobic amino acids. Complex nonapeptide libraries were used to determine the contribution of each residue to stabilize peptide-TAP complexes. For the first time, this ap proach lets us directly evaluate the importance of peptide selection for the overall process of antigen presentation on the level of the peptide transporter. [References: 25]