Fig. 2: Interactions between Mnl1 and Pdi1.
a, C579 in the Mnl1 loop forms a disulfide bond with the first cysteine (C406) of the CGHC motif of the Trx a′ domain of Pdi1 (encircled with a dashed line). b, As in a, but for cysteine C644 of the Mnl1 loop and the first cysteine (C61) of the CGHC motif of the Trx a domain. c, Purified complexes of Pdi1 with wild-type Mnl1 or the indicated cysteine mutants were incubated with different concentrations of DPS to induce disulfide bridge formation between Pdi1 and Mnl1. The samples were subjected to nonreducing SDS–PAGE and staining with Coomassie blue. Mw, molecular weight. d, Residues of the Mnl1 loop inserted into the hydrophobic pocket of the Trx b′ domain. e, ERAD of CPY*–HA was determined by CHX chase experiments in cells lacking Mnl1 (mnl1Δ). The cells were transformed with an empty vector, expressed wild-type Mnl1 or the indicated cysteine mutants. The samples were analyzed by SDS–PAGE and immunoblotting for HA. The intensities of the CPY*–HA bands were quantified. The fractions of CPY*–HA remaining at different time points are shown (means and s.d. of three experiments). f, As in e, but with other residues substituted at the interface between Mnl1 and Pdi1. g, As in f, but with additional interface mutants. h, The indicated FLAG-tagged Mnl1 mutants were expressed from the endogenous locus. A membrane fraction was solubilized in Nonidet P-40 and the extract was subjected to IP with anti-FLAG antibodies. The samples were analyzed by SDS–PAGE and immunoblotting for FLAG and Pdi1. A sample of the cell lysate was analyzed directly for Pdi1. i, As in h, but with overexpressed Mnl1–FLAG constructs. A sample of the cell lysate was analyzed by immunoblotting for FLAG and Pdi1. j, As in i, but cell lysates were separated into supernatant and membrane fractions. Membrane fractions were incubated with Nonidet P-40 and centrifuged again. The supernatants and detergent extracts were subjected to IP with anti-FLAG antibodies. All samples were analyzed by SDS–PAGE and immunoblotting for FLAG and Pdi1.
