Figure 5. Formation of Rad6-Uba1 disulfide depends on free ubiquitin and controls K63 ubiquitination.

(A) Representative immunoblot anti-Rad6 from in vitro oxidation reactions of recombinant Rad6 (100 nM) and UBA1 (100 nM) in the presence of 2 mM H₂O₂. Lane labels describe the sequential order of events and which protein was oxidized prior to mixing. Quantification was performed using iBright Analysis Software, and graph shows mean ± standard deviation for three biological replicates. Quantification of Rad6-E1 band was normalized by the amount present in lane 2 compared with the levels of DTT-reduced Rad6 for each sample. *p ≤ 0.005, **p ≤ 0.025 determined by Student’s t test.

(B) Spectroscopic analysis of 75 μg of purified Rad6 treated in the presence or absence of 2 mM H₂O₂ for 30 min at RT. Samples were then incubated with 100 μM 4-chloro-7-nitrobenzofurazan (NBD) for an additional 30 min, followed by removal of excess reagent by successive cycles of washes and filtration. Rad6 sulfenic acid peak is exhibited at 420 nm.

(C) Close-up view of Rad6 active site (PDB: 1AYZ) highlighting its catalytic cysteine (pink) and vicinal residues (blue) as sticks.

(D) Immunoblots of Rad6 mutants for amino-acid residues interacting with catalytic Cys88 show differential ubiquitination and disulfide formation. Anti-actin was used as loading control. *Rad6 charged with ubiquitin through thioester bond.

(E) Immunoblots anti-Rad6, anti-K63 ubiquitin chains, and anti-ubiquitin show dynamics of Rad6-Uba1 complex and polyubiquitin chain formation as well as depletion of the pool of monomeric ubiquitin over time after cellular exposure to 0.6 mM H₂O₂. Anti-GAPDH was used as loading control.

(F) Accumulation of Rad6-Uba1 complex and depletion of the ubiquitin pool is dose dependent. Immunoblot from Rad6-HA-expressing cells subjected to increased H₂O₂ concentrations for 30 min. Anti-GAPDH was used as loading control.

(G) Immunoblot from in vitro incubation of yeast recombinant Rad6 (100 nM)and UBA1 (100 μM) in the presence of increased ubiquitin concentration for 30 min followed by 2 mM H₂O₂ treatment for 30 min. Proteins were also pre-incubated with the E1 inhibitor PYR-41 (75 μM) or alkylated with iodoacetamide (20 mM) for 30 min prior to exposure to H₂O₂.

(H) Yeast cells at stationary phase do not show Rad6-Uba1 disulfide formation and have high levels of K63 ubiquitin chains. Immunoblot of cells treated with indicated H₂O₂ concentrations for 30 min in mid-log (optical density [OD]₆₀₀ = 0.5) or late (OD₆₀₀ = 3.2) stationary phase. Anti-GAPDH was used as loading control.

(I) Schematic of regulation of Rad6 activity in response to stress. Oxidation of Rad6 catalytic cysteine leads to the formation of a Rad6-Uba1 disulfide complex, which inhibits Rad6 activity and controls the levels of K63 ubiquitination in a negative feedback mechanism. Created with BioRender.com.

(J) Immunoblot shows higher levels of K63-linked ubiquitin chain accumulation in Rad6^R7A/R11A cells over time. Yeast cells expressing WT Rad6 and a Rad6^R7A/R11A mutant generated through CRISPR-Cas9 method were incubated with 0.6 mM H₂O₂ for the respective times.

(K) Immunoblot from cells expressing WT Rad6 and a Rad6^R7A/R11A mutant subjected to increasing H₂O₂ concentrations. Rad6^R7A/R11A cells show reduced formation of Rad6-Uba1 complex and higher levels of K63 ubiquitin chains. Anti-GAPDH was used as loading control.