MODEL FOR THE IN-VIVO ASSEMBLY OF NASCENT L(D) CLASS-I MOLECULES AND FOR THE EXPRESSION OF UNFOLDED L(D) MOLECULES AT THE CELL-SURFACE

To characterize the process of class I assembly and maturation, we have studied the L(d) molecule of the mouse. Previous studies have shown that a significant proportion of intracellular and surface L(d) molecules can be detected in an alternative conformation designated L(d)alt1. Nascent L(d)alt molecules are non-peptide ligand associated and are weakly associated with beta2-microglobulin (beta2m). Unexpectedly, when monoclonal antibodies were added directly to the lysis buffer, significant amounts of L(d)alt/beta2m heterodimer were detected, suggesting that beta2m association is not necessarily sufficient to induce L(d) conformation. By contrast, addition of peptide to cell lysates rapidly induced the folding of beta2m-associated L(d)alt to conformed L(d). Furthermore, the time course and dynamics of this conversion correlated precisely with peptide binding to L(d). The precursor-product relationship of L(d)alt and conformed L(d) was also visualized in vivo by pulse-chase analysis of BALB/c splenocytes. To investigate the factors that regulate intracellular transport of class I molecules, expression of L(d) was studied in the peptide transport-deficient cell line, RMA.S-L(d), and in beta2m-/- splenocytes. In contrast to wild-type cell lines both L(d)alt and conformed L(d) are poorly expressed at the cell surface of RMA.S-L(d) and beta2m-/- splenocytes. Therefore, surface expression of L(d)alt is dependent upon the concomitant expression of conformed L(d) molecules. To determine whether surface L(d)alt molecules can result from melting of conformed L(d) molecules, surface L(d) molecules were loaded with several different known L(d) peptide ligands. Complexes of L(d) with different ligands were found to have dramatically disparate surface half-lives. Importantly, the L(d) peptide complexes that turned over the most rapidly resulted in the most gain in surface L(d)alt, implying that peptide dissociation can induce the accumulation of nonconformed L(d) heavy chains at the cell surface.