Fig. 4: The Mnl1–Pdi1 complex interacts with misfolded polypeptides.
a, Schematic of different versions of misfolded RB. The S-peptide is colored in green and disulfide bonds are shown as red lines. b, Mannosidase reactions were performed with the Mnl1–Pdi1 complex and the indicated substrates labeled with biotin. After the mannosidase reaction, the substrates were retrieved with streptavidin beads and the binding of DyLight 800-labled MRH–IgM was determined. The amount of substrate in the assays was monitored by Coomassie blue staining. c, Wild-type or mutant Mnl1–Pdi1 complex was incubated with DyLight 680-labeled RBΔS or RB. The complexes were retrieved with beads containing anti-FLAG antibodies and bound substrate analyzed by SDS–PAGE and fluorescence scanning. The amounts of Mnl1–FLAG in the assays were determined by immunoblotting for FLAG. A fraction of the input was analyzed directly. d, As in c, but with RBΔS and addition of a synthetic S-peptide at 10-fold or 20-fold molar excess. A control was performed with a mutant S-peptide carrying three substitutions (F8W, H12A and D14A) that prevent binding to RBΔS. e, As in c, but with folded and misfolded RA versions (which lack a glycan).
