INTERMEDIATES IN THE ASSEMBLY AND DEGRADATION OF CLASS-I MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) MOLECULES PROBED WITH FREE HEAVY CHAIN-SPECIFIC MONOCLONAL-ANTIBODIES

Unassembled (free) heavy chains appear during two stages of the class I MHC molecule's existence: immediately after translation but before a ssembly with peptide and beta(2)-microglobulin, and later, upon disint egration of the heterotrimeric complex. To characterize the structures of folding and degradation intermediates of the class I heavy chain, three monoclonal antibodies have been produced that recognize epitopes along the H-2K(b) heavy chain which are obscured upon proper folding and subsequent assembly with beta(2)-microglobulin (KU1: residues 49-5 4; KU2: residues 23-30; KU4: residues 193-198). The K-b heavy chain is inserted into the lumen of the endoplasmic reticulum in an unfolded s tate reactive with KU1, KU2, and KU4. Shortly after completion of the polypeptide chain, reactivity with KU1, KU2 and KU4 is lost synchronou sly, suggesting that folding of the class I heavy chain is a rapid, co operative process. Perturbation of the folding environment in intact c ells with the reducing agent dithiothreitol or the trimming glucosidas e inhibitor N-7-oxadecyl-deoxynojirimycin prolongs the presence of mAb -reactive K-b heavy chains. At the cell surface, a pool of free K-b he avy chains appears after 60-120 min of chase, whose subsequent degrada tion but not their initial appearance, is impaired in the presence of concanamycin B, an inhibitor of vacuolar acidification. Thus, free hea vy chains that arise at the cell surface are destroyed after internali zation.