Formation of a major histocompatibility complex class I tapasin disulfide indicates a change in spatial organization of the peptide-loading complex during assembly.
The assembly and peptide loading of major histocompatibility complex Class I molecules within the endoplasmic reticulum are essential for antigen presentation at the cell surface and are facilitated by the peptide-loading complex. The formation of a mixed disulfide between the heavy chain of Class I and components of the loading complex (ERp57, protein disulfide isomerase, and tapasin) suggests that these molecules are involved in the redox regulation of components during assembly and peptide loading. We demonstrate here that a disulfide formed between heavy chain and tapasin can occur between cysteine residues located in the cytosolic regions of these proteins following translation of heavy chain in an in vitro translation system. The formation of this disulfide occurs after assembly into the loading complex and is coincident with the stabilization of the alpha2 disulfide bond within the peptide binding grove. A ternary complex between heavy chain, ERp57, and tapasin was observed and shown to be stabilized by a disulfide between both tapasinheavy chain and tapasin-ERp57. No disulfides were observed between ERp57 and heavy chain within the loading complex. The results provide a detailed evaluation of the various transient disulfides formed within the peptide-loading complex during biosynthesis. In addition, the absence of the disulfide between tapasin and heavy chain in TAP-deficient cells indicates that a change in the spatial organization of tapasin and heavy chain occurs following assembly into the loading complex.
Faculty of Life Sciences, Michael Smith Building, University of Manchester, Manchester M13 9PT, United Kingdom.
Joseph E
JE
Chambers
Catherine E
CE
Jessop
Neil J
NJ
Bulleid
D017951:Antigen Presentation / Q000276:immunology*; D002460:Cell Line; D002474:Cell-Free System; D004220:Disulfides / Q000276:immunology; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000276:immunology*; D015395:Histocompatibility Antigens Class I / Q000235:genetics / Q000276:immunology*; D006801:Humans; D026901:Membrane Transport Proteins / Q000235:genetics / Q000276:immunology*; D046912:Multiprotein Complexes / Q000235:genetics / Q000276:immunology*; D010084:Oxidation-Reduction; D014176:Protein Biosynthesis; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000276:immunology*
D004220:Disulfides; D015395:Histocompatibility Antigens Class I; D026901:Membrane Transport Proteins; D046912:Multiprotein Complexes; C107762:tapasin; D019704:Protein Disulfide-Isomerases; C085868:PDIA3 protein, human