Extended Data Fig. 7: Purification of mutant Hmt1-Pdi1 complexes and disulfide bond formation between the components.
(a) The indicated mutant Mnl1-Pdi1 complexes were purified and analyzed by SDS-PAGE in the presence or absence of DTT, followed by staining with Coomassie blue. (b) As in (a), but with a complex lacking the CTD of Mnl1 (Mnl1ΔC-Pdi1). (c) Mnl1ΔC-Pdi1 or Mnl1-Pdi1 were incubated with different concentrations of DPS to induce disulfide bridge formation between Mnl1 and Pdi1. The samples were analyzed by non-reducing SDS-PAGE. (d) As in (a), but for the purified complex of Pdi1 and Mnl1 carrying mutations in the hydrophobic pocket of the CTD (mCTD). (e) A fusion of maltose binding protein (MBP) and CTD or mCTD (MBP-CTD or MBP-mCTD) was purified from mammalian tissue culture cells and analyzed as in (a). (f) Citrate synthase (CiS) was incubated with different proteins at a 1:3 molar ratio at different temperatures for 20 min. The samples were analyzed by dynamic light scattering and the percentage of CiS in particles larger than 200 nm (aggregates) was determined. Shown are means and standard deviations of three experiments. Note that MBP-CTD, but not the other proteins, prevent the aggregation of CiS and that MBP-CTD does not form aggregates. (g) As in (f), but for luciferase (Luc). (h) Increasing concentrations of unfolded RNase B (RBun) were incubated with wild-type or mutant Mnl1-Pdi1 complex. Disulfide bond formation was induced by addition of DPS and the samples were analyzed by non-reducing SDS-PAGE and Coomassie blue staining. (i) As in (h), but with Mnl1-Pdi1 complexes in which Mnl1 loop cysteines were mutated.
