MHC class I molecules form ternary complexes with calnexin and TAP and undergo peptide-regulated interaction with TAP...

Newly assembled heavy chain-beta 2m heterodimers of class I histocompatibility molecules associate with the endoplasmic reticulum (ER) peptide transporter, TAP, and subsequently dissociate from TAP in parallel with their transport from the ER to the Golgi apparatus. It appears that TAP-associated class I molecules are waiting to bind appropriate peptides before they dissociate from TAP and leave the ER since binding of high affinity peptides to class I molecules in vitro leads to dissociation of TAP-class I complexes. In further support of this notion, we report that limiting peptide supply through inhibition of proteasome activities prolongs the association of mouse class I molecules with TAP and concomitantly slows their transport to the Golgi apparatus. By using a series of deletion mutants and hybrid class I molecules we demonstrate that the extracellular domains of class I molecules are sufficient for their peptide-regulated interaction with TAP. Furthermore, based on the inability of an alpha 3 domain-specific mAb to recognize TAP-class I complexes and the fact that a point mutant of the Dd molecule at residue 222 is unable to bind to TAP, it is likely that a major site of interaction with TAP resides in the membrane-proximal region of the heavy chain alpha 3 domain. Finally, we examined the relationship between the interaction of mouse heavy chain-beta 2m heterodimers with TAP and with the resident ER chaperone, calnexin. Most heterodimers that bound to TAP were found to associate simultaneously with calnexin. Upon delivery of peptide to class I molecules in permeabilized cells, dissociation from TAP was observed but the interaction with calnexin was largely maintained. Therefore, both TAP and calnexin may participate in the ER retention of peptide-deficient class I molecules. However, since release from calnexin occurs after dissociation from TAP, it appears that calnexin ultimately determines if a class I molecule is to be exported from the ER.