The assembly of H2-Kb class I molecules translated in vitro requires oxidized glutathione and peptide

Abstract

Association of the mouse major histocompatibility complex (MHC) class I heavy chain H2-Kb with mouse beta 2-microglobulin (beta 2m) was studied in an in vitro translation system. Formation of stable class I complexes was found to be dependent on the presence of presentable peptides and oxidized glutathione, which promotes the formation of disulfide bridges. Translocation of peptides into microsomes was demonstrated by showing that a radioiodinated peptide containing an N-glycosylation acceptor site became glycosylated. Class I complex formation was observed only when heavy chains and beta 2m were translated simultaneously, and thus occurs in the microsomes and not after their solubilization. However, peptide binding takes place only after solubilization of the microsomes. The class I complexes translated in vitro show the same specificity and length preference for peptides as their counterparts in RMA-S cells. Assembly of in vitro translated class I complexes was found to occur also in the absence of peptides, resulting in the formation of unstable molecules that are stabilized by incubation with peptides.