Class I major histocompatibility complex molecules present antigenic peptides to cytotoxic T lymphocytes, which leads to the elimination of virus infected cells. Class I molecules are heterotrimers consisting of a heavy chain, a light chain termed beta2-microglobulin, and a peptide ligand. Assembly of class I molecules begins in the endoplasmic reticulum where the heavy chain associates with beta2-microglobulin, and the heavy chain-beta2-microglobulin heterodimers enter a peptide loading complex where class I molecules acquire peptides. During the biogenesis of class I molecules, ERp57, a thiol oxidoreductase, associates with free class I heavy chains and, at a later stage, with the peptide loading complex. In this thesis, I show for the first time that ERp57 participates in oxidative folding of the heavy chain. Depletion of ERp57 by RNAi delayed heavy chain disulfide bond formation and slowed folding of the heavy chain alpha3 domain. Interestingly, depletion of another thiol oxidoreductase, ERp72, had no such effect. Since ERp57 associates with the lectin-chaperones calnexin and calreticulin, it is thought that ERp57 requires these chaperones to gain access to its substrates. To test this idea, I examined class I biogenesis in cells lacking calnexin or calreticulin or that express an ERp57 mutant that fails to bind to these chaperones. Remarkably, heavy chain disulfides formed at the same rate in these cells as in wild type cells, suggesting that ERp57 has the capacity to recognize its substrates directly in addition to being recruited through lectin-chaperones. ERp57 also forms a mixed disulfide with tapasin within the peptide loading complex and I found that the formation of this mixed disulfide is independent of its interaction with calnexin and calreticulin. I also found that calreticulin could be recruited into the peptide loading complex in the absence of interactions with both ERp57 and substrate oligosaccharides, demonstrating the importance of its polypeptide-binding site in substrate recognition. Finally, by inactivating the redox active sites of ERp57, I demonstrate that its enzymatic activity is dispensable in stabilizing the loading complex and in supporting efficient peptide loading. Thus, ERp57 plays a structural rather than catalytic role within the peptide loading complex.

Class I major histocompatibility complex molecules present antigenic peptides to cytotoxic T lymphocytes, which leads to the elimination of virus infected cells. Class I molecules are heterotrimers consisting of a heavy chain, a light chain termed beta2-microglobulin, and a peptide ligand. Assembly of class I molecules begins in the endoplasmic reticulum where the heavy chain associates with beta2-microglobulin, and the heavy chain-beta2-microglobulin heterodimers enter a peptide loading complex where class I molecules acquire peptides. During the biogenesis of class I molecules, ERp57, a thiol oxidoreductase, associates with free class I heavy chains and, at a later stage, with the peptide loading complex. In this thesis, I show for the first time that ERp57 participates in oxidative folding of the heavy chain. Depletion of ERp57 by RNAi delayed heavy chain disulfide bond formation and slowed folding of the heavy chain alpha3 domain. Interestingly, depletion of another thiol oxidoreductase, ERp72, had no such effect. Since ERp57 associates with the lectin-chaperones calnexin and calreticulin, it is thought that ERp57 requires these chaperones to gain access to its substrates. To test this idea, I examined class I biogenesis in cells lacking calnexin or calreticulin or that express an ERp57 mutant that fails to bind to these chaperones. Remarkably, heavy chain disulfides formed at the same rate in these cells as in wild type cells, suggesting that ERp57 has the capacity to recognize its substrates directly in addition to being recruited through lectin-chaperones. ERp57 also forms a mixed disulfide with tapasin within the peptide loading complex and I found that the formation of this mixed disulfide is independent of its interaction with calnexin and calreticulin. I also found that calreticulin could be recruited into the peptide loading complex in the absence of interactions with both ERp57 and substrate oligosaccharides, demonstrating the importance of its polypeptide-binding site in substrate recognition. Finally, by inactivating the redox active sites of ERp57, I demonstrate that its enzymatic activity is dispensable in stabilizing the loading complex and in supporting efficient peptide loading. Thus, ERp57 plays a structural rather than catalytic role within the peptide loading complex.