Assembly of HLA DR1 molecules translated in vitro: binding of peptide in the endoplasmic reticulum precludes association with invariant chain.
MHC class II molecules usually bind peptides in the endocytic pathway, but can also present endogenous peptides from newly synthesized proteins in a chloroquine-insensitive manner, suggesting that peptide binding might occur in the endoplasmic reticulum (ER). We used in vitro translation of HLA-DR1 class II molecules in the presence of microsomes to study peptide binding in the ER. Formation of functional class II molecules in vitro depends on formation of disulfide bridges in alpha and beta chains. The class II alpha beta heterodimers made by in vitro translation resemble class II molecules synthesized in cells in (i) their reactivity with conformation-specific antibodies, (ii) their assembly with Ii chain homotrimers, (iii) the generation of SDS-stable dimers upon peptide binding and (iv) their specificity of peptide binding. The assembly of class II molecules occurs via an alpha beta intermediate and can occur post-translationally, but only in intact microsomes. Class II alpha beta heterodimers are able to bind peptides in ER-derived microsomes, a process that precludes subsequent association of class II molecules with Ii chain. This mechanism might explain presentation of endogenous peptides by class II molecules.
Center for Cancer Research, Massachusetts Institute of Technology, Cambridge 02139.
M J
MJ
Bijlmakers
P
P
Benaroch
H L
HL
Ploegh
D000911:Antibodies, Monoclonal; D000944:Antigens, Differentiation, B-Lymphocyte; D000951:Antigens, Neoplasm / Q000235:genetics / Q000378:metabolism*; D003432:Cross-Linking Reagents; D004229:Dithiothreitol; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005978:Glutathione / Q000031:analogs & derivatives; D019803:Glutathione Disulfide; D015798:HLA-DR1 Antigen / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral / Q000378:metabolism; D000949:Histocompatibility Antigens Class II / Q000235:genetics / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012333:RNA, Messenger / Q000096:biosynthesis; D013388:Succinimides; D014759:Viral Envelope Proteins / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000944:Antigens, Differentiation, B-Lymphocyte; D000951:Antigens, Neoplasm; D003432:Cross-Linking Reagents; D015798:HLA-DR1 Antigen; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral; D000949:Histocompatibility Antigens Class II; D010446:Peptide Fragments; D012333:RNA, Messenger; D013388:Succinimides; D014759:Viral Envelope Proteins; C045084:invariant chain; C011240:dithiobis(succinimidylpropionate); D005978:Glutathione; D004229:Dithiothreitol; D019803:Glutathione Disulfide