Fig. 2
From: Atg4 proteolytic activity can be inhibited by Atg1 phosphorylation
Atg4 phosphorylation on serine 307 blocks Atg4 function. a The atg4Δ cells carrying the integration plasmid pCuATG8GFP(403) (SAY114) and an empty vector (atg4Δ) or plasmids expressing the indicated 13xmyc-tagged Atg4 variants were grown in SMD and nitrogen starved in SD-N medium for 3 h. Proteins were precipitated with TCA and analyzed by western blot. b The atg4Δ strain carrying the integration plasmid pCuGFPAtg8ΔR(305) (JSY151) or plasmids expressing the indicated 13xmyc-tagged Atg4 variants were grown in SMD, nitrogen starved in SD-N for 3 h, labeled with the vacuole-specific dye FM 4-64 and imaged. Differential interference contrast (DIC). Scale bar, 5 µm. Fluorescence intensity plots of representative vacuoles are shown (indicated by white lines). c Quantification of GFP-Atg8∆R distribution on vacuole, i.e., rim vs. lumen, in cells imaged in a was performed as described in Methods. Results are mean ± SD (n = 50). d Atg8ΔR was conjugated to SUVs via the addition of Atg7, Atg3, Atg12–Atg5, MgCl2, and ATP. Deconjugation was initiated by the addition of WT Atg4 or the indicated mutants. Samples were collected after 5, 10, 30, and 60 min, and separated on a 11% SDS gel supplemented with 4.5 M urea. The graph on the right shows the quantification of three independent experiments
