title: In vitro unfolding of retinol-binding protein by dithiothreitol. Endoplasmic reticulum-associated factors.
abstract: HepG2 cells in the presence of DTT synthesize fully reduced serum retinol-binding protein (RBP) within the endoplasmic reticulum (ER). Upon removal of DTT, RBP forms disulfide bonds and a folding intermediate, compact II, accumulates within the ER. Compact II RBP co-migrates on nonreducing gel electrophoresis with the secreted form of RBP but is differentiated from secreted RBP by its sensitivity to DTT-induced unfolding (see accompanying article; Kaji, E. H., and Lodish, H. F. (1993) J. Biol. Chem. 268, 22188-22194). Here, we have reconstituted DTT-induced unfolding of compact II RBP in a broken cell system and demonstrate that ER-associated factors enhance the unfolding of RBP by DTT. Protein disulfide isomerase is likely to be one such factor since it enhances the rate of RBP unfolding by DTT in vitro; protein disulfide isomerase-induced unfolding requires the absence of retinoids, similar to the DTT-induced unfolding in vivo. ATP enhances the unfolding of RBP in the absence but not in the presence of retinol, both in intact and broken cells. Thus, protein disulfide isomerase and other ATP-dependent factors can unfold partly folded (or misfolded) RBP in the ER, suggesting how improperly folded proteins might be correctly refolded in vivo.
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142.
authors.forename: E H
authors.initials: EH
authors.lastname: Kaji
authors.forename: H F
authors.initials: HF
authors.lastname: Lodish
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D002384:Catalysis; D003600:Cytosol / Q000378:metabolism; D004229:Dithiothreitol / Q000737:chemistry*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006801:Humans; D007535:Isomerases / Q000378:metabolism; D007700:Kinetics; D010084:Oxidation-Reduction; D019704:Protein Disulfide-Isomerases; D017510:Protein Folding; D012177:Retinol-Binding Proteins / Q000737:chemistry*; D014407:Tumor Cells, Cultured
chemical_list: D012177:Retinol-Binding Proteins; D000255:Adenosine Triphosphate; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D004229:Dithiothreitol