Figure 6. Redox states of CPY and PDI.

(A) BY4742 wild-type (WT) cells were radiolabelled for 10 min in the absence (lanes 1 and 2) or presence of 10 mM DTT (lanes 3 and 4) to detect the fully oxidized and fully reduced p1CPY respectively. BY4742 (WT), ero1Δ P_GAL1-hQSOX1a (QSOX) and ero1Δ P_GAL1-ERV2 (Erv) were radiolabelled for 10 min in the presence of 5 mM DTT followed by a 30 min chase in the absence of DTT (lanes 5, 6 and 7). Cells were isolated at the end of the labelling and chase periods and proteins from cell pellets were TCA precipitated and solubilized in the presence (lanes 2 and 4–7) or absence (lanes 1 and 3) of 25 mM AMS prior to immunoisolation of CPY. Samples were resolved by non-reducing SDS/PAGE. The reduced (red) and oxidized (ox) forms of p1CPY are indicated. (B) TCA (20%) was added to logarithmically growing cultures to quench thiol/disulfide exchange and precipitate proteins. Free sulfhydryl groups were modified by resuspension in 5 mM Mal-PEG under denaturing conditions [3% SDS and 0.2 M Tris/HCl, (pH 8)]. Proteins were separated by SDS/PAGE and visualized by Western blotting using an anti-yeast PDI antibody. Apparent molecular mass shifts above the unmodified protein indicate the number of free sulfhydryl groups modified. The redox states of PDI in the parental BY4742 (WT), ero1Δ P_GAL1-hQSOX1a (QSOX) and ero1Δ P_GAL1-ERV2 (Erv) are compared with reduced (Red) and oxidized (Ox) controls that were treated with 10 mM DTT or 1 mM diamide before Mal-PEG modification.