Abstract
Before peptide binding in the endoplasmic reticulum, the class I heavy (H) chain-beta(2)-microglobulin complexes are detected in association with TAP and two chaperones, TPN and CRT. Recent studies have shown that the thiol-dependent reductase, ERp57, is also present in this peptide-loading complex. However, it remains controversial whether the association of ERp57 with MHC class I molecules precedes their combined association with the peptide-loading complex or whether ERp57 only associates with class I molecules in the presence of TPN. Resolution of this controversy could help determine the role of ERp57 in class I folding and/or assembly. To define the mouse class I H chain structures involved in interaction with ERp57, we tested chaperone association of L(d) mutations at residues 134 and 227/229 (previously implicated in TAP association), residues 86/88 (which ablate an N-linked glycan), and residue 101 (which disrupts a disulfide bond). The association of ERp57 with each of these mutant H chains showed a complete concordance with CRT, TAP, and TPN but not with calnexin. Furthermore, ERp57 failed to associate with H chain in TPN-deficient.220 cells. These combined data demonstrate that, during the assembly of the peptide-loading complex, the association of ERp57 with mouse class I is TPN dependent and parallels that of CRT and not calnexin.
Publication types
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Comparative Study
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Amino Acid Substitution / genetics
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Animals
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Antiporters / antagonists & inhibitors
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Antiporters / genetics
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Antiporters / physiology*
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Calcium-Binding Proteins / antagonists & inhibitors
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Calcium-Binding Proteins / metabolism*
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Calnexin
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Calreticulin
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Carbohydrate Conformation
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Cell Line, Transformed
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Cysteine / genetics
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Disulfides / antagonists & inhibitors
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Disulfides / metabolism
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Endoplasmic Reticulum / genetics
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Endoplasmic Reticulum / metabolism*
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H-2 Antigens / genetics
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H-2 Antigens / metabolism*
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Heat-Shock Proteins / antagonists & inhibitors
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Heat-Shock Proteins / metabolism*
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Histocompatibility Antigen H-2D
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Humans
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Immunoglobulins / deficiency
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Immunoglobulins / genetics
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Immunoglobulins / physiology*
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Isomerases / antagonists & inhibitors
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Isomerases / metabolism*
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L Cells
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Membrane Transport Proteins
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Mice
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Mutagenesis, Site-Directed
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Polysaccharides / metabolism
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Protein Binding / genetics
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Protein Binding / immunology
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Protein Disulfide-Isomerases
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Ribonucleoproteins / antagonists & inhibitors
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Ribonucleoproteins / metabolism*
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Transfection
Substances
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Antiporters
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Calcium-Binding Proteins
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Calreticulin
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Disulfides
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H-2 Antigens
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Heat-Shock Proteins
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Histocompatibility Antigen H-2D
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Immunoglobulins
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Membrane Transport Proteins
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Polysaccharides
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Ribonucleoproteins
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tapasin
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Calnexin
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Isomerases
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Pdia3 protein, mouse
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Protein Disulfide-Isomerases
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PDIA3 protein, human
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Cysteine