Figure 4.

Arabidopsis ATG4a Cleaves Chlamydomonas ATG8.

ATG4 activity of the recombinant AtATG4a was assayed by monitoring the cleavage of CrATG8 from the unprocessed (CrATG8) to processed (pCrATG8) forms (indicated by arrowheads) using SDS-PAGE followed by Coomassie Blue staining and quantification of protein band intensities. The ATG4 activity (relative units) was determined as the ratio of the band intensity of the processed CrATG8 to the sum of the intensities of the unprocessed and processed CrATG8. Activity value of 1 corresponds to the maximum. Processed pCrATG8 (lane 1) and unprocessed CrATG8 (lane 2) were loaded as controls. Representative images are shown. Data are from three independent experiments and evaluated by two-factor ANOVA. Same letters indicate no significant differences. P < 0.05.

(A) Effect of incubation time. AtATG4a was incubated with CrATG8 in the absence or in the presence of 10 mM DTT for the indicated times.

(B) Effect of DTT concentration. AtATG4a was incubated with CrATG8 in the absence or in the presence of increasing concentrations of DTT for 4 h.

(C) Effect of TCEP concentration. AtATG4a was incubated with CrATG8 in the absence or in the presence of increasing concentrations of TCEP for 4 h.