Interaction of Class I major histocompatibility molecules with the transporter associated with antigen processing (TAP) and the molecular chaperone calnexin

grantor: University of TorontoClass I major histocompatibility molecules consist of a ~45 kDa transmembrane glycoprotein termed the heavy chain, a ~12 kDa soluble protein termed ß 2-microglobulin, and a peptide ligand of 8 to 10 residues. These three subunits assemble in the endoplasmic reticulum (ER) and then are transported to the cell surface. Cytotoxic T lymphocytes of the immune system specifically recognize and eliminate cells, bearing class I molecules containing peptides derived from foreign (e.g., viral) proteins. The bulk of class I-binding peptides are generated in the cytosol by the proteasome and then are transported across the ER membrane via a peptide transporter termed TAP. Within the ER, the molecular chaperone calnexin facilitates folding of class I heavy chains and their assembly with ß2m and retains class I assembly intermediates until assembly of the ternary complex with peptide is complete. This thesis documents the discovery, characterization, and functional consequences of an interaction between the TAP and class I molecules in mouse cells. Newly assembled class I H chain-ß2m heterodimers but not free H chains associate with TAP in a ternary complex that also contains calnexin. Dissociation of both TAP and calnexin from class I molecules occurs in parallel with the movement of class I molecules out of the ER. In permeabilized cells, binding of peptide to class I molecules leads to disruption of the TAP-class I association but not the calnexin-class I association. When peptide supply is limited through inhibition of proteasome activities, the TAP-class I association is prolonged and class I movement out of the ER is slowed. Furthermore, mutant class I molecules assembling without interacting with TAP appear to obtain peptides that dissociate rapidly at the cell surface causing lower cell surface class I levels and less efficient antigen presentation to T cells relative to their wild type counterparts. These findings suggest a model whereby the TAP-class I interaction facilitates loading of stably-bound peptides onto class I molecules by providing a high local concentration of peptides and, also, together with calnexin, by retaining class I molecules until they obtain optimal, stably-bound peptides.Ph.D