Fig. 4.

Decreased yCul1 rubylation linked to intra-cellular ROS. (A) WT, rpn11-m1 and rpn11^D122/A mutants in logarithmic phase growth were treated with 4.4 mM of H₂O₂ for 2 h and yCul1 rubylation status was determined by immunoblotting. (B) Mutants of ROS scavengers, Δtrx2, Δgsh1 and Δglr1 were grown in YPD at 28 °C, and yCul1 rubylation status evaluated in whole cell extract by immunoblotting (see also Fig. S3) (C) Wild type cells growth for 16 h were diluted to 0.5OD₆₀₀ in YPD and incubated for 6 h at 28 °C before addition of 2 mM DTT to growth media. Rubylation status of yCul1 was examined by immunoblotting at 6 h, and at 8, 10 h. yCul1 modification ratio was quantified (A, B, C bottom) (D) Cells were sorted by FACS according to their oxidation status into oxidized, reduced, or “mixed” (i.e., unsorted) populations (as assessed by the Grx1-roGFP2 sensor). (E) 10⁶ WT cells were collected during sorting for each repeat of the different subpopulations (n = 3 repeats), followed by examination of the yCul1 Rubylation status by immunoblotting and quantification (see also Fig. S4). (F) schematic representation of the correlation between the oxidation state and yCul1 rubylation status in individual cells.