Intracellular transport blockade caused by disruption of the disulfide bridge in the third external domain of major histocompatibility complex class I antigen.
The third external domain of major histocompatibility class I antigens has a highly conserved disulfide bridge between cysteine-203 and cysteine-259. To elucidate the functional significance of this disulfide bridge, we have produced a mutant H-2Ld gene by site-directed mutagenesis in which the codon for cysteine-203 is changed to a codon for serine, which is unable to form a disulfide bridge. The mutant H-2Ld gene was introduced into mouse L cells and its expression has been studied. No measurable expression of the H-2Ld antigen was detected on the cell surface of the transformants by antibody-binding assays. However, a large quantity of the mutant H-2Ld antigen was found in the cytoplasm of the transformants as observed by immunoprecipitation of metabolically labeled lysate and by immunocytochemistry of membrane-permeabilized cells, using an antibody specific for the first external domain of the H-2Ld antigen. The mutant antigen was glycosylated and associated, at least in part, with beta 2-microglobulin. Subcellular fractionation experiments indicated that the transport of the antigen was blocked between the endoplasmic reticulum and the plasma membrane. It is concluded that structural integrity of the third external domain is a prerequisite for intracellular transport of class I antigens. On the basis of these findings we suggest that the domain structure containing the disulfide bridge serves as a signal structure necessary for receptor-mediated intracellular transport and that this requirement is the evolutionary basis for high conservation of similar structures present throughout the immunoglobulin supergene family.
Miyazaki
J
J
Appella
E
E
Ozato
K
K
D000818:Animals; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005802:Genes, MHC Class II; D006183:H-2 Antigens / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008285:Major Histocompatibility Complex; D051379:Mice; D009154:Mutation
D004220:Disulfides; D006183:H-2 Antigens