Figure 3.
Atg4 activity is dependent on the redox potential. Atg4 activity was monitored as in Figure 2. (A) Atg4 activity is irreversibly inhibited by alkylating agents. Atg4 was pretreated with 1 mM DTT for 2 h (lane 1); then, incubated for 1 h with 10 mM iodoacetamide (IAM, lane 2) or 10 mM N-ethylmaleimide (NEM, lane 3); next, Atg4 was again incubated with 50 mM DTT for 30 min (lanes 4 and 5). (B) Atg4 activity is dependent on the DTTred/DTTox ratio. Atg4 activity was determined after incubation during 2 h in the presence of various DTTred/DTTox ratio (1/1, 1/10, 1/100, 10/1, 100/1 in mM). (C) Atg4 is not activated by GSH. Atg4 activity was analyzed after incubation for 2 h in the presence of 1 mM DTT (lane 2), 2 mM GSH (lane 3) or 5 mM GSH (lane 4), or in the absence of reducing agent (lane 1). (D) Redox titration of Atg4 activity. Atg4 activity was determined after incubation for 2 h at indicated Eh poised by 20 mM DTT in various dithiol/disulfide ratios. C: control untreated. The −∞ sample was used as reference for quantification. (E) Atg4 activities monitored as in (D) were interpolated by nonlinear regression of the data using a Nernst equation for 2 electrons exchanged (n = 2) and one redox component. The average midpoint redox potential (Em,7) of 4 independent experiments is reported in the figure as mean ± standard deviation.