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Abstract
By resolving immunoprecipitates on nonreducing sodium dodecyl sulfate gels, we have detected several disulfide-bonded intermediates in folding within the endoplasmic reticulum of newly made H1 subunits of the asialoglycoprotein receptor. H1 in the endoplasmic reticulum (ER) can be partially unfolded by treatment of cells with dithiothreitol, but H1 in Golgi or post-Golgi organelles is resistant to such unfolding. This defines a late step in H1 folding that occurs just prior to exit from the ER. Depletion of calcium from the endoplasmic reticulum, either by treatment with A23187 or thapsigargin, has no effect on folding or secretion of newly made albumin, but totally blocks H1 maturation from the ER. No ER intermediates in H1 folding are formed in cells treated with A23187 or thapsigargin, indicating that at least an early step in H1 folding requires a high Ca2+ concentration in the ER lumen. As judged by cross-linking experiments, formation of H1 dimers and trimers occurs immediately after biosynthesis of the peptide chain, before monomer folding, and occurs normally in cells in which ER Ca2+ is reduced and where the monomer never folds properly. Calcium is essential for the asialoglycoprotein receptor to bind galactose, and our results suggest that Ca2+ is also essential for the receptor polypeptides to fold in the ER.
Highlights
Calcium Is Required for Folding ofNewly Made Subunits of the Asialoglycoprotein Receptor within the Endoplasmic Reticulum*
N o endoplasmic reticulum (ER) intermediates in H1 folding are formed in cells treated with A23187 or thapsigargin, indicating that at least an early step in receptor, the influenza hemagglutinin, and the vesicular stomatitis virus G glycoprotein, occurs within the ER
Different proteins exit at different times after their synthesis, most likely because different periods of time are required to fold different proteins made in the same cell [15,16,17,18,19] and the same protein made in different cells [20]
Summary
ER of newly made n,-antitrypsin in HepG2 cells was com- lycosidases H or D,asdetailedin Ref. 28, before analysis bygel pletely blocked by treatment of cells with theCa'+ ionophores electrophoresis.Immunoprecipitation of HepG2 extracts with an. After a further 5 min of incubation, the cell monolayers were again washed three times in phosphate-buffered saline asnodlubilized ates in the folding within the ERof the exoplasmic, disulfide- in detergent in preparation for immunoprecipitation. Of the -230 amino acids in the exoplasmic domain of H1, the carboxyl-termina1l 50 constitute theCa"-depende n t galactose-binding domain (reviewed in Ref. 44).This domaincontains -30 conserved aminoacids,including 4 conserved cysteine residues, which are characteristic of the C-class of animal lectins. This class, which containsboth secreted andcell- surface proteins, areall thought tobe Ca'+dependent oligosaccharide-binding proteins. Our results suggest that these calcium ions are essential for folding of the polypeptide within the ER, althoughwe cannot eliminate othereffects of calcium on protein folding, perhaps mediated through one or more of the Ca"-dependent ER chaperonins
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