Fig. 1.

Schematics of BRET-based AtAtg8 synthetic substrate and NASCA assay. (A) The conserved glycine (G) residue at the C terminus of AtAtg8s is shown. Arrow indicates the site of cleavage by AtAtg4 cysteine proteases. (B) In the BRET-based AtAtg8 substrates, Citrine and modified Renilla luciferase, SuperhRLUC (ShR) were fused to the amino and the carboxyl terminus of AtAtg8, respectively. Cleavage of this substrate by AtAtg4 will generate Citrine-AtAtg8 and ShR byproducts. In addition, some of the substrate may remain uncleaved. The amount of cleaved ShR byproduct can be separated from the uncleaved AtAtg8 substrate on a native polyacrylamide gel electrophoresis followed by direct detection in the presence of a luciferase substrate, coelenterazine (CLZ). XXX indicates additional amino acids after the conserved G of AtAtg8s except AtAtg8h and AtAtg8i.