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.
Highlights
HepG2 cells in the presence of DTT synthesize fully nism for this quality control function of the ER is unclear
Compact I, 11, and secreted retinol-binding protein (RBP) co-migrate on nonreducing gels, but they are differentiated by their sensitivity to unfolding by DTT, compact I is more sensitive toDTTinduced unfolding than compact 11.Both compact I andI1 are more resistant to DTT-induced unfolding in the presence of retinol thain its absence
We study the unfolding of compact I1 RBP
Summary
In the accompanying article [40],we reported that RBP forms disulfide bonds rapidly, yielding the folding intermediate compact I; this matures over the 15 min to compact 11. When cytosolic extracts were prepared from metabolically labeled cells and digested by proteinase K, two forms of RBP were observed compact and clipped (Fig. 2, panel A, lane 2 ). When the cytosolic extracts were treated with DTT, we observed the compact RBP unfolding to an unfolding intermediate (UFI) form similar in mobility to the UFI from RBP in intact cells (Fig. 2, panel A, lanes 3 and I ). Detergent solubilized, immunoprecipitated with anti-RBP antibody, and analyzed by nonreducing SDS-PAGE (13%).For samples in lanes 2-8 (panel A and C) and lanes 10-16 (panelA),cytosolic extracts were made from HepG2 cells pulse-labeled for 1 h and chased for 15 min, as detailed under“Experimental Procedures.”.
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