The answer was aided by analysis of mutant cells that had a defect in antigen presentation by MHC class I molecules. These cells expressed far fewer MHC class I proteins than normal on their surface despite normal synthesis of these molecules in the cytoplasm. This defect could be corrected by adding synthetic peptides to the culture medium, suggesting that the supply of peptides to the MHC class I molecules in the endoplasmic reticulum might be the limiting factor. Analysis of the DNA of the mutant cells identified the problem responsible for this phenotype to be in genes for members of the ATP-binding cassette (ABC) family of proteins; the ABC proteins mediate the ATP-dependent transport of ions, sugars, amino acids, and peptides across membranes.
Missing from the mutant cells were two ABC proteins, called transporters associated with antigen processing-1 and -2 (TAP1 and TAP2), that are normally associated with the endoplasmic reticulum membrane. Transfection of the mutant cells with the missing genes restored the presentation of peptides by the cell’s MHC class I molecules. The two TAP proteins form a heterodimer in the membrane (Fig. 6.7), and mutations in either TAP gene can prevent antigen presentation by MHC class I molecules. The genes TAP1 and TAP2 are located in the MHC locus (see Section 6-10), near the PSMB9 and PSMB8 genes, and their basal level of expression is further enhanced by interferons produced in response to viral infection, similar to MHC class I and β1, β2, and β5 subunits of the proteasome. This induction results in increased delivery of cytosolic peptides into the endoplasmic reticulum.
Microsomal vesicles from non-mutant cells can mimic the endoplasmic reticulum in assays in vitro, by internalizing peptides that then bind to MHC class I molecules present in the microsome lumen. In contrast, vesicles from TAP1or TAP2-deficient cells do not take up peptides. Peptide transport into normal microsomes requires ATP hydrolysis, confirming that the TAP1:TAP2 complex is an ATP-dependent peptide transporter. The TAP complex has limited specificity for the peptides it will transport, transporting peptides of between 8 and 16 amino acids in length and preferring peptides that have hydrophobic or basic residues at the carboxy terminus—the precise features of peptides that bind MHC class I molecules (see Section 4-15). The TAP complex has a bias against proline in the first three amino-terminal residues, but lacks in any true peptide-sequence specificity. The discovery of TAP explained how viral peptides from proteins synthesized in the cytosol gain access to the lumen of the endoplasmic reticulum and are bound by MHC class I molecules.