Figure 6.

HsLC3A directly binds to yeast, Arabidopsis, and human ATG4s. (A) C-HsLC3A-ShR and catalytic resistant C-HsLC3A^G120A-ShR were used for in vitro affinity isolation with various ATG4 homologs. The yeast and Arabidopsis ATG4s affinity isolated C-HsLC3A-ShR but HsATG4B failed to affinity isolate HsLC3A due to kinetic activity of HsATG4B (left, middle panel). All tested ATG4 homologs affinity isolated the catalytic resistant C-HsLC3A^G120A-ShR (right, middle panel). Anti-MBP and Coomassie staining were used for inputs of ATG4 and the ATG8 substrates, respectively. Asterisks indicate degraded ATG4s. (B) C-HsLC3A^G120A-ShR is not affinity isolated by the MBP-alone control (bottom panel). Same amount of C-HsLC3A^G120A-ShR was used for affinity isolation as shown in (A). Anti-MBP was used for inputs. Asterisks indicate nonspecific binding products with amylose resin.