An unstable β2-microglobulin: Major histocompatibility complex class I heavy chain intermediate dissociates from calnexin and then is stabilized by binding peptide

Abstract

Proper assembly of the class I heavy chain (HC), β2-microglobulin (β2m), and peptide must occur in the endoplasmic reticulum (ER) in order for MHC class I molecules to be expressed on the cell surface. Newly synthesized class I HC bind calnexin, an ER resident chaperone. These calnexin-associated class I HC appeared to lack the stable association with β2m in peptide transporter-deficient T2 cells since β2m-unassociated class I HC-specific HC10 antibody, but not β2m-associated class I HC- specific W6/32 antibody, coimmunoprecipitated calnexin. To determine the precursor-product relationship of the pool of HC that bind peptide, class I- restricted peptides were added to lysates of T2 cells in vitro. These peptides stabilized preexisting β2m-associated HC complexes (β2m- :HC:pep-), but had no significant effect on the preexisting pool of calnexin-associated HC that lack β2m. Release of HC from calnexin appeared to be controlled by the binding of β2m, since β2m-deficient FO-1 cells showed a prolonged association of class I HC with calnexin, while β2m- transfected FO-1 cells displayed a more rapid dissociation of class I HC from calnexin. Consistent with this result, the dissociation of class I HC from calnexin did not appear to be dependent on peptide binding since the dissociation rates were similar in peptide transporter-deficient T2 cells and in wild-type T1 cells. From these observations, we speculate that in the stepwise assembly of class I molecules, calnexin may mediate dimerization of class I HC with β2m, and that the unstable β2m+:HC:pep- complexes, after dissociation from calnexin, subsequently bind peptide to complete the assembly.