Competition for access to the rat major histocompatibility complex class I peptide-loading complex reveals optimization of peptide cargo in the absence of transporter associated with antigen processing (TAP) association

Major histocompatibility complex (MHC) class I molecules load peptides in the endoplasmic reticulum in a process during which the peptide cargo is normally optimized in favor of stable MHC-peptide interactions. A dynamic multimolecular assembly termed the peptide-loading complex ( PLC) participates in this process and is composed of MHC class I molecules, calreticulin, ERp57, and tapasin bound to the transporter associated with antigen processing ( TAP) peptide transporter. We have exploited the observation that the rat MHC class I allele RT1-A(a), when expressed in the rat C58 thymoma cell line, effectively competes and prevents the endogenous RT1-A(u) molecule from associating with TAP. However, stable RT1-A(u) molecules are assembled efficiently in competition with RT1-A(a), demonstrating that cargo optimization can occur in the absence of TAP association. Defined mutants of RT1-A(a), which do not allow formation of the PLC, fail to become thermostable in C58 cells. Wild-type RT1-A(a), which does allow PLC formation, also fails to become thermostable in this cell line, which carries the rat TAPB transporter that supplies peptides incompatible for RT1-A(a) binding. Full optimization of RT1-A(a) requires the presence of the TAP2A allele, which is capable of supplying suitable peptides. Thus, formation of the PLC alone is not sufficient for optimization of the MHC class I peptide cargo.