MHC CLASS-I MOLECULES FORM TERNARY COMPLEXES WITH CALNEXIN AND TAP AND UNDERGO PEPTIDE-REGULATED INTERACTION WITH TAP VIA THEIR EXTRACELLULAR DOMAINS

Newly assembled heavy chain-beta(2)m heterodimers of class I histocomp atibility molecules associate with the endoplasmic reticulum (ER) pept ide transporter, TAP, and subsequently dissociate from TAP in parallel with their transport from the ER to the Golgi apparatus. It appears t hat TAP-associated class I molecules are waiting to bind appropriate p eptides before they dissociate from TAP and leave the ER since binding of high aiffinity peptides to class I molecules in vitro leads to dis sociation of TAP-class I complexes. In further support of this notion, we report that limiting peptide supply through inhibition of proteaso me 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 su fficient for their peptide-regulated interaction with TAP. Furthermore , based on the inability of all alpha(3) domain-specific mAb to recogn ize TAP-class I complexes and the fact that a point mutant of the D-d molecule at residue 222 is unable to bind to TAP, it is likely that a major site of interaction with TAP resides in tile membrane-proximal r egion of the heavy chain alpha(3) domain. Finally, we examined tile re lationship between the interaction of mouse heavy chain-beta(2)m heter odimers with TAP and with the resident ER chaperone, calnexin. Most he terodimers that bound to TAP were found to associate simultaneously wi th calnexin. Upon delivery of peptide to class I molecules in permeabi lized cells, dissociation from TAP was observed but the interaction wi th calnexin was largely maintained. Therefore, both TAP and calnexin m ay participate ill the: Ea retention of peptide-deficient class I mole cules. However, since release from calnexin occurs after dissociation from TAP, it appears that calnexin ultimately determines ifa class I m olecule is to be exported from the ER.