Association of ERp57 with mouse MHC class I molecules is tapasin dependentand mimics that of calreticulin and not calnexin

Before peptide binding in the endoplasmic reticulum, the class I heavy (H) chain-beta (2)-microglobulin complexes are detected in association with TAP and two chaperones, TPN and CRT. Recent studies have shown that the thiol- dependent reductase, ERp57, is also present in this peptide-loading complex . However, it remains controversial whether the association of ERp57 with M HC class I molecules precedes their combined association with the peptide-l oading complex or whether ERp57 only associates with class I molecules in t he presence of TPN. Resolution of this controversy could help determine the role of ERp57 in class I folding and/or assembly. To define the mouse clas s I H chain structures involved in interaction with ERp57, we tested chaper one association of L-d mutations at residues 134 and 227/229 (previously im plicated in TAP association), residues 86/88 (which ablate an N-linked glyc an), and residue 101 (which disrupts a disulfide bond). The association of ERp57 with each of these mutant If chains showed a complete concordance wit h CRT, TAP, and TPN but not with calnexin. Furthermore, ERp57 failed to ass ociate with H chain in TPN-deficient .220 cells. These combined data demons trate that, during the assembly of the peptide-loading complex, the associa tion of ERp57 with mouse class I is TPN dependent and parallels that of CRT and not calnexin.