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 associated with antigen processing (TAP) by using a permeabilized-cell system. The main objective was to determine whether these molecules, which bear homology to the ATP-binding cassette family of transporters, translocate antigenic peptides across the endoplasmic reticulum membrane for assembly with major histocompatibility complex (MHC) class I molecules and beta 2-microglobulin light chain. The pore-forming toxin streptolysin O was used to generate permeabilized cells, and peptide translocation was determined by measuring the amount of added radiolabeled peptide bound to endogenous class I molecules. No radiolabeled peptide was associated with MHC class I glycoproteins from unpermeabilized cells. We found that efficient peptide binding to MHC class I molecules in permeabilized cells is both transporter dependent and ATP dependent. In antigen-processing mutant cells lacking a functional transporter, uptake occurs only through a less-efficient transporter and ATP-independent 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.
Androlewicz
M J
MJ
Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06510.
Anderson
K S
KS
Cresswell
P
P
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000187:drug effects; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D015395:Histocompatibility Antigens Class I / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism*; D013301:Streptolysins / Q000494:pharmacology; D014162:Transfection
D001426:Bacterial Proteins; D002352:Carrier Proteins; D015395:Histocompatibility Antigens Class I; D010455:Peptides; D013301:Streptolysins; C025964:streptolysin O; D000255:Adenosine Triphosphate