Fig. 3
From: Atg4 proteolytic activity can be inhibited by Atg1 phosphorylation
The phosphorylation state of S307 modulates Atg4 interaction with Atg8. a Atg4 and Atg8 were fused to the activation domain (AD) and/or the DNA-binding domain (BD) of Gal4, respectively. Plasmids were transformed into the PJ69-4A strain and colonies were spotted on medium lacking leucine and uracil (control plate) or leucine, uracil, and adenine (test plate). Growth on the test plate indicates interaction. The control was cells expressing BD-Atg8 alone. b Phosphorylation of S307 blocks the interaction of Atg4 with conjugated Atg8. The atg4Δ (SAY084) mutant transformed with WT Atg4-13xmyc and the atg4Δ strain with stably integrated pCuGFPAtg8ΔR(305) (JSY151) carrying a plasmid expressing the 13xmyc-tagged WT or the indicated Atg4 variants, were exponentially grown and nitrogen starved in SD-N for 1 h. Cell lysates were subjected to pull-down experiments using GFP-trap sepharose beads. Isolated proteins, 4% of cell lysate (input) or 100% of the pull-down material (IP), were resolved by SDS-PAGE and analyzed by western blot using anti-myc and anti-Atg8 antibodies
