The role of ERp57 in disulfide bond formation during the assembly of major histocompatibility complex class I in a synchronized semipermeabilized cell translation system.
We have established a semipermeabilized cell system that reproduces the folding and assembly of a major histocompatibility complex (MHC) class I complex as it would occur in the intact cell. The translation of the MHC class I heavy chain (HLA-B27) in this system was synchronized allowing the folding and assembly of polypeptide chains synthesized within a short time frame to be analyzed. This has enabled us to dissect the time course of interaction of both disulfide and nondisulfide-bonded heavy chain with various molecular chaperones during its assembly in a functionally intact endoplasmic reticulum. The results demonstrate that unassembled, nondisulfide-bonded forms of heavy chain interact initially with calnexin. A later and more prolonged interaction of calreticulin, specifically with assembled, disulfide-bonded heavy chain, highlights distinct differences in the roles of these two proteins in the assembly of MHC class I molecules. We also demonstrate that the thiol-dependent reductase ERp57 initially interacts with nondisulfide-bonded heavy chain, but this rapidly becomes disulfide-bonded and indicates that heavy chain folding occurs during its interaction with ERp57. In addition, we also confirm a direct interaction between MHC class I heavy chain and tapasin, emphasizing the role that this protein plays in the later stages of MHC class I assembly.
Farmery
M R
MR
School of Biological Sciences, University of Manchester, Manchester M13 9PT, United Kingdom.
Allen
S
S
Allen
A J
AJ
Bulleid
N J
NJ
D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism; D037281:Calnexin; D037282:Calreticulin; D002460:Cell Line; D002463:Cell Membrane Permeability; D002474:Cell-Free System; D003001:Cloning, Molecular; D004220:Disulfides / Q000378:metabolism; D005805:Genes, MHC Class I; D015796:HLA-B27 Antigen / Q000235:genetics* / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D007535:Isomerases / Q000378:metabolism*; D007700:Kinetics; D018832:Molecular Chaperones / Q000378:metabolism; D014176:Protein Biosynthesis; D019704:Protein Disulfide-Isomerases; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis; D012156:Reticulocytes / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism; D013601:T-Lymphocytes; D001613:beta 2-Microglobulin / Q000378:metabolism
D002135:Calcium-Binding Proteins; D037282:Calreticulin; D004220:Disulfides; D015796:HLA-B27 Antigen; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D001613:beta 2-Microglobulin; D037281:Calnexin; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; C085868:PDIA3 protein, human