Fig. 6: Vacuolar ionophore monensin triggers tonoplast ATG8ylation.

a, Confocal micrographs displaying the localization of all nine GFP-tagged ATG8 isoforms (ATG8A to ATG8I) of A. thaliana under monensin (0.5 h, 200 µM) treatment. Representative images from ten seedlings were analysed under each treatment. Scale bars, 10 µm. b, Confocal micrographs of the GFP–ATG8A-G117A mutant, highlighting its localization in response to monensin (0.5 h, 200 µM) treatment. The images include single optical slices and maximum intensity projections. Representative images from ten seedlings were analysed under each treatment. Scale bars, 10 µm. c, Confocal micrographs of mCh–ATG8A (magenta) colocalized with the tonoplast marker VAMP711–YFP illustrating the recruitment of ATG8 to the tonoplast upon monensin treatment. The panel includes a single optical slice alongside a merged image with VAMP711–YFP and a corresponding bright field image. The images follow a time course treatment (0, 10, 20, 30 and 60 min) with monensin (200 µM). Representative images from ten seedlings were analysed under each treatment. Scale bars, 10 µm. d, Venn diagram summarizing the results of a TurboID-based proximity-labelling proteomics experiment with TurboID–ATG8A under Torin (1.5 h, 9 µM), ES20-1 (8 h, 100 µM) and monensin (2 h, 200 µM) treatments, highlighting the overlap and unique proteins identified across conditions. TurboID alone is used as a negative control.