Enhanced folding and processing of a disulfide mutant of the human asialoglycoprotein receptor H2b subunit.
Unfolded forms of the H2b subunit of the human asialoglycoprotein receptor, a galactose-specific C-type lectin, are degraded in the endoplasmic reticulum (ER), whereas folded forms of the protein can mature to the cell surface (Wikström, L., and Lodish, H. F. (1993) J. Biol. Chem. 268, 14412-14416). There are eight cysteines in the exoplasmic domain of the protein, forming four disulfide bonds in the folded protein. We have constructed double cysteine to alanine mutants for each of the four disulfide bonds and examined the folding and metabolic fate of each of the mutants in transfected 3T3 fibroblasts. We find that mutation of the two cysteines nearest to the transmembrane region (C1) does not prevent proper folding of the protein, whereas mutations of the other three disulfides prevent proper folding of the protein and all of the mutant proteins are degraded in the ER. A normal (approximately 20%) fraction of the C1 mutant protein exists the endoplasmic reticulum and is processed in the Golgi complex, and it does so at a faster rate compared to the wild-type. Furthermore, the folded form of this mutant protein is more resistant to unfolding by dithiothreitol than the wild-type. The C1 mutant protein is expressed on the cell surface and can form a functional receptor with the H1 subunit with similar binding affinities for natural ligands as that of the wild-type receptor. The same fraction of newly made mutant and wild-type proteins (approximately 80%) remain in the ER, but the mutant protein is degraded more quickly. Thus, the presence of the C1 disulfide bond in the wild-type receptor both reduces the rate of protein folding and exit to the Golgi and slows the rate of ER degradation of the portion (approximately 80%) of the receptor that never folds properly.
Yuk
M H
MH
Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, USA.
Lodish
H F
HF
D016475:3T3 Cells; D000818:Animals; D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009961:Orosomucoid / Q000031:analogs & derivatives / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins; D004220:Disulfides; D009961:Orosomucoid; D011956:Receptors, Cell Surface; C017030:asialoorosomucoid; D003545:Cysteine