Figure 1. Sil1 regulates BiP oxidation state in cells.

(A) Yeast strains (CSY289, 290, 612, 689) were spotted onto SMM plates containing 0–1.5 mM diamide and incubated for 2 d at 30°C. (B) Schematic for the biotin-switch procedure. (C) Yeast strains deleted for endogenous BiP (kar2∆) containing plasmids encoding FLAG-tagged BiP were assayed for oxidized BiP levels using the biotin-switch protocol. Oxidative stress was generated by overexpression of Ero1*. BiP was immunoprecipitated, and total and oxidized BiP were detected by Western blotting. The relative levels of oxidized BiP are expressed as the ratio of the intensity of the avidin and anti-BiP signals. The signal ratio was set to 1.0 for wild-type cells grown in the absence of Ero1*. (D) Lysates were prepared from the indicated yeast after Ero1* induction. Oxidized BiP levels were detected and quantified as in C. (E) Yeast strains (CSY5, 275, 448, 449) were spotted onto YPD plates containing 0–2.0 mM diamide and were incubated for 2 d at 30°C. (F) Cells were treated with 5 mM diamide for 15 min, diamide was removed, and cells were returned to 30°C until harvest. Oxidized BiP levels were determined as in C. (G) Plot of the averaged quantified data ± SEM from F and a second independent experiment using the same protocol. For each strain, the signal ratio was set to 1.0 for cells grown without diamide.

DOI: http://dx.doi.org/10.7554/eLife.24141.002

Figure 1—figure supplement 1. BiP-Sil1 structure.

Sil1-BiP complex structure (PBD ID: 3QML) encompassing the armadillo repeats from yeast Sil1 (residues 113–421) and the yeast BiP ATPase domain (residues 43–426) (Yan et al., 2011). BiP is shown in wheat; Sil1 is colored green. The redox-active BiP cysteine is shown as a red sphere. BiP lysine-314 is shown as a cyan sphere.

DOI: http://dx.doi.org/10.7554/eLife.24141.003

Figure 1—figure supplement 2. A BiP K314E mutation disrupts Sil1 binding.

(A) GST-Sil1, bound to glutathione-agarose beads, was incubated with recombinant wild-type BiP or mutant BiP-K314E proteins for 1 hr. Bound proteins were eluted from the agarose beads with SDS, and proteins were resolved by SDS-PAGE and visualized using a Coomassie blue stain. (B) CSY214 (kar2∆ [pCS623]), CSY594 (kar2∆ sil1∆ [pCS623]), and CSY595 (kar2∆ lhs1∆ [pCS623]) strains were transformed with LEU2-marked plasmids coding for the indicated BiP alleles (pCS681, pKP37, pCS685, pKP97) or empty vector. Transformants were spotted onto SMM plates containing 5-FOA (to select against wild-type BiP plasmid pCS623) or SMM minus leucine plates (to confirm successful transformation with the LEU2-marked plasmid). A sil1∆ lhs1∆ yeast strain is inviable; the observed inviability of the BiP-K314E mutant in the lhs1∆ strain background is consistent with an inability of the BiP-K314E mutant to associate with Sil1, which phenocopies a sil1 null strain.

DOI: http://dx.doi.org/10.7554/eLife.24141.004