Figure 2.

Atg4 is activated by reduction and reversibly inhibited by oxidation. Atg4 (4.5 μM) activity was monitored by following the cleavage of Atg8 (5 μM) from the unprocessed (Atg8) to the processed (pAtg8) form (indicated by arrowheads) by SDS-PAGE and Coomassie brilliant blue staining. (A) Effect of DTT on Atg4 activity. Atg4 was incubated for 2 h in the presence of DTT concentrations ranging from 10 μM to 5 mM. (B) Effect of H₂O₂ on the activity of prereduced Atg4. After pretreatment with 0.1 mM DTT for 1 h, Atg4 was incubated in the presence of increasing H₂O₂ concentrations for 2 h. Quantification of Atg4 activity from (A and B) is shown in (C and D), respectively. The reference sample for quantification (Atg4 activity = 1 in arbitrary units) was 5 mM DTT and 0 mM H₂O₂ (prereduced with DTT) in (A and B), respectively. (E) Inhibition of Atg4 activity by H₂O₂ is reversed by DTT. Atg4 protein was pretreated with 1 mM DTT for 2 h (lane 2); then, Atg4 was incubated with 2 mM H₂O₂ for 30 min (lane 3); finally, Atg4 was newly treated with 10 mM DTT for 30 min (lane 4). Control (lane 1): incubation with no addition. For all lanes, Atg8 was added to the reaction mixture for 1 h after each specific treatment to assess Atg4 activity.