EVIDENCE THAT TRANSPORTERS ASSOCIATED WITH ANTIGEN-PROCESSING TRANSLOCATE A MAJOR HISTOCOMPATIBILITY COMPLEX CLASS-I-BINDING PEPTIDE INTO THE ENDOPLASMIC-RETICULUM IN AN ATP-DEPENDENT MANNER

We have investigated the role of the putative peptide transporters ass ociated with antigen processing (TAP) by using a permeabilized-cell sy stem. The main objective was to determine whether these molecules, whi ch bear homology to the ATP-binding cassette family of transporters, t ranslocate antigenic peptides across the endoplasmic reticulum membran e for assembly with major histocompatibility complex (MHC) class I mol ecules and beta2-microglobulin light chain. The pore-forming toxin str eptolysin O was used to generate permeabilized cells, and peptide tran slocation was determined by measuring the amount of added radiolabeled peptide bound to endogenous class I molecules. No radiolabeled peptid e was associated with MHC class I glycoproteins from unpermeabilized c ells. We found that efficient peptide binding to MHC class I molecules in permeabilized cells is both transporter dependent and ATP dependen t. In antigen-processing mutant cells lacking a functional transporter , uptake occurs only through a less-efficient transporter and ATP-inde pendent pathway. In addition, short peptides (8-10 amino acids) known to bind MHC class I molecules compete efficiently with a radiolabeled peptide for TAP-dependent translocation, whereas longer peptides and a peptide derived from an endoplasmic reticulum signal sequence do not compete efficiently. This result indicates that the optimal substrates for TAP possess the characteristics of MHC-binding peptides.