Extended Data Fig. 6: Cell fractionation and cycloheximide-chase experiments.
(a) The indicated FLAG-tagged Mnl1 mutants were expressed in S. cerevisiae cells from the endogenous locus. A membrane fraction was collected in the presence of DTT to reduce all disulfides. The membranes were then solubilized in Nonidet P-40 and the extract subjected to immunoprecipitation (IP) with 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 FLAG and Pdi1. (b) ERAD of CPY*-HA was determined by cycloheximide (CHX) chase experiments in cells lacking Mnl1 (mnl1Δ). The cells were transformed with either an empty vector or expressed wild-type Mnl1 or the indicated cysteine mutants. The samples were analyzed by SDS-PAGE and immunoblotting for HA. Blotting for Pgk1 served as a loading control. Three similar experiments were used for the quantification shown in Fig. 2e. (c) As in (b), but with other residues mutated at the interface between Mnl1 and Pdi1. Quantification of three similar experiments is shown in Fig. 2f. (d) As in (b), but with additional interface mutants. Quantification of three similar experiments is shown in Fig. 2g. (e) As in (b), but with Mnl1 mutants lacking the CTD or carrying mutations in the hydrophobic groove. Quantification of three similar experiments is shown in Fig. 3c.
