Characterization of the ERp57-Tapasin complex by rapid cellular acidification and thiol modification.
Major histocompatibility complex (MHC) class I molecules bind and present short peptides to cells of the immune system. The oxidoreductase ERp57 is involved in the assembly of MHC class I molecules and is a component of the peptide loading complex, where it is found disulfide-bonded to tapasin. We have studied ERp57 and the ERp57-tapasin conjugate by rapid acidification of the intracellular environment with trichloroacetic acid (TCA), followed by thiol modification with the alkylating agent 4'-maleimidylstilbene-2,2'-disulfonic acid (AMS). By using TCA/AMS treatment, non-tapasin-associated ERp57 is shown to exist almost exclusively in a reduced state, suggesting that both thioredoxin-like CXXC motifs are exposed and reduced. A 110-kDa product is readily detected with this TCA/AMS protocol and is confirmed as an ERp57-tapasin conjugate by its absence from the tapasin-deficient .220 cell line and by immunoblotting with both ERp57- and tapasin-specific antisera. The ERp57-tapasin conjugate can also be modified with the oxidizing agent diamide, indicating that within the pool of ERp57-tapasin complexes the free, non-tapasin-linked CXXC motif exists in both oxidized and reduced states, suggesting availability to undergo redox reactions.
D000143:Acids / Q000737:chemistry; D000478:Alkylation; D000818:Animals; D017920:Antiporters / Q000737:chemistry* / Q000378:metabolism*; D002460:Cell Line; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007136:Immunoglobulins / Q000737:chemistry* / Q000378:metabolism*; D007535:Isomerases / Q000737:chemistry* / Q000378:metabolism*; D026901:Membrane Transport Proteins; D010084:Oxidation-Reduction; D019704:Protein Disulfide-Isomerases; D051381:Rats; D013438:Sulfhydryl Compounds / Q000737:chemistry*
D000143:Acids; D017920:Antiporters; D006360:Heat-Shock Proteins; D007136:Immunoglobulins; D026901:Membrane Transport Proteins; D013438:Sulfhydryl Compounds; C107762:tapasin; D007535:Isomerases; C513196:PDIA3 protein, rat; D019704:Protein Disulfide-Isomerases; C085868:PDIA3 protein, human