Fig. 2: Translocation of ATG8 to tonoplast requires the ATG conjugation system.
From: ATG8ylation-mediated tonoplast invagination mitigates vacuole damage
a Schematic depiction of the core protein complexes that regulate the formation of autophagosomes, encompassing the ATG1 complex, ATG9 vesicles, PI3K complex, ATG2-ATG18 complex, and ATG conjugation systems. b The impact of monensin on the subcellular localization of GFP-ATG8a in atg1abct, atg11-1, atg9-4 and atg2-1 mutants, as well as inhibition of PI3K activity with wortmannin. c Analysis of the impact of ATG4 on the subcellular localization of YFP-ATG8a. d Effect of mutation in ATG5, ATG7, and ATG16 genes on the subcellular localization of ATG8 under monensin treatment. Five-day-old GFP-ATG8a/atg1abct, GFP-ATG8a/atg11-1, GFP-ATG8a/atg9-4, GFP-ATG8a/atg5-1, YFP-ATG8e/atg7-2 and YFP-ATG8a(G132A) x mCherry-ATG8f double transgenic seedlings were treated with 20 μM monensin for 1 h, followed by confocal microscopy observation. Wortmannin (16.5 μM) was pre-incubated for 10 min and then added with 20 μM monensin for 1 h, followed by confocal imaging. Scale bars in (b–d), 20 μm. Similar confocal imaging results were obtained in at least six individual roots with three replicates. e–h The impact of monensin on ATG8 lipidation in wild-type Col-0 and autophagy mutants including atg11-1, atg7-2, atg5-1 and atg16-c1. The ratios (n = 3 biological replicates) between the lipidation form of ATG8 and actin was quantified with ImageJ (f and h). An asterisk indicates an unknown band. Mon, Monensin. Data are mean ± SD. Significance analysis using an unpaired two-sided Student’s ttest. The immunoblotting assays were independently replicated three times with consistent results. Source data are provided as a Source Data file.
