Anti-ATG8 | Autophagy-related protein

FREE1 interacts with ATG conjugation system and ESCRT-III under starvation conditions.a Transgenic Arabidopsis seedlings expressing YFP-FREE1 upon autophagy induction by carbon starvation for at least 18 h were subjected to GFP-Trap assay for IP-MS analysis. Identified FREE1-interacting partners were analyzed using STRING. b Immunofluorescence labeling of the endogenous ATG conjugation system proteins ATG5, ATG12b or ATG16 in Arabidopsis protoplasts transfected with GFP-FREE1 for 24 h before confocal laser scanning microscopy (CLSM) observation. White dash boxes indicate the 5 X enlarged areas. Scale bars, 10 μm. c GFP-Trap and co-IP analysis of GFP-FREE1 with endogenous ATG5, ATG12b, or ATG16 using native promoter driven GFP-FREE1 transgenic plants. Arabidopsis transgenic plants expressing GFP or GFP-FREE1 driven by the native promoter were subjected to protein extraction and IP with GFP-Trap, followed by western blot analysis with indicated antibodies. d Structural TEM analysis of high-pressure freezing/frozen substituted atg5-1 mutant plant root tips upon BTH treatment for 8 h. Black dash boxes indicated the 5 X zoom-in areas. Arrowheads indicated examples of the unsealed autophagosomal structures. Scale bars, 500 nm. e 5 X zoom-in areas from d. Arrowheads indicated examples of the unsealed autophagosomal structures. Scale bars, 500 nm. f Quantification analysis of the ratio of the unclosed autophagosomal structures (APs) in Col-0 and atg5-1 mutants per cross-cell section. Means±SD; n = 10 (Col-0) and n = 17 (atg5-1) cells, two-tailed unpaired t test; ***p < 0.001. All the imaging analysis and immunoblots were repeated at least for three times with similar results.

KIN10 phosphorylates FREE1 to orchestrate ESCRT machinery function in autophagosome closure.a Transient expression and confocal analysis of the spatial relationship between FREE1 and KIN10 in Arabidopsis leaf protoplasts. The transfected leaf protoplasts were cultured for 24 h before CLSM observation. Scale bars, 10 μm. b FRET analysis of CFP-FREE1 with KIN10-YFP. Arabidopsis protoplasts co-expressing CFP and YFP (negative control), CFP-YFP (positive control), or CFP-FREE1 and KIN10-YFP were cultured for 24hr before FRET analysis. Means±SD; n = 16 (CFP/YFP), n = 11 (CFP-YFP), and n = 16 (CFP-FREE1/KIN10-YFP) individual puncta per experimental group, one-way analysis of variance (ANOVA), followed by Tukey’s multiple test; ***p < 0.001. The middle lines of the boxes represent the medians of datasets. The upper and bottom lines of the boxes are respectively the upper quantile and the lower quantile of the data. The whiskers mark the upper and lower limits of these datasets, respectively. c Y2H analysis between AD-KIN10 and BD-FREE1231–601 (the truncated FREE1 without self-activation on BD vector). d GFP-Trap and co-immunoprecipitation (co-IP) analysis of KIN10-6HA and YFP-FREE1 in Arabidopsis protoplasts. Arabidopsis protoplasts co-expressing KIN10-6HA with YFP-FREE1, or YFP only were subjected to protein extraction and IP with GFP-Trap, followed by immunoblotting with indicated antibodies. e GFP-Trap and co-IP of KIN10 and FREE1 using transgenic plants expressing KIN10-GFP under nitrogen starvation (-N) for at least 18hrs, followed by immunoblotting using GFP and FREE1 antibodies. f Quantification analysis of the ratio of co-immunoprecipitated FREE1 by KIN10 shown in e. Means±SD; n = 4 individual experiments, two-tailed unpaired t test, ***p < 0.001. g In vivo phosphorylation of FREE1 upon nitrogen starvation. GFP-FREE1/free1 transgenic plants were subjected to mock or nitrogen starvation for at least 18 h, followed by protein extraction and IP with GFP-Trap, and subsequent immunoblotting detection by Phos-tag biotin and the indicated antibodies. h Semi-in vitro phosphorylation assay of FREE1, FREE1S530A and FREE1S530AS533A proteins by KIN10-GFP purified from the transgenic plants expressing KIN10-GFP. KIN10 was enriched from transgenic seedlings expressing KIN10-GFP by immunoprecipitation with anti-GFP and then used to phosphorylate FREE1, FREE1S530A or FREE1S530AS533A tagged with His-sumo, which was further detected by Phos-tag biotin. i Semi-in vitro phosphorylation assay of FREE1, FREE1S530A and FREE1S530AS533A proteins by KIN10-GFP purified from the transgenic plants expressing KIN10-GFP in snrk2.2/2.3 mutants. KIN10 was enriched from transgenic seedlings expressing KIN10-GFP in snrk2.2/2.3 mutants background by immunoprecipitation with anti-GFP and then used to phosphorylate FREE1 or FREE1S530A or FREE1S530AS533A tagged with His-sumo, which was further detected by Phos-tag biotin. j In vivo interaction between ATG8 and FREE1, FREE1S530A, or FREE1S530AS533A mutant variants. GFP-FREE1, GFP-FREE1S530A, and GFP-FREE1S530AS533A transgenic plants were subjected to nitrogen starvation for at least 18 h, followed by protein extraction and IP with GFP-Trap, and subsequent immunoblotting with indicated antibodies. k Transient co-expression of GFP-FREE1 or GFP-FREE1S530A with mCherry-ATG8i in Arabidopsis protoplasts. The transfected protoplasts were cultured for 24 h before CLSM observation. Scale bars, 10 μm. All the imaging analysis and immunoblots were repeated at least for three times with similar results.

FREE1 specifically interacts with the distinct paralogs of ATG8.a Y2H analysis of BD-FREE1231–601 (the truncated FREE1 without self-activation on BD vector) with AD-ATG8 isoforms. b Evolutionary relationships of ATG8 isoforms. The optimal tree with the sum of branch length=4.32642842 is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. c Transient expression of CFP-FREE1 with specific ATG8 isoforms YFP-ATG8a and YFP-ATG8i in Arabidopsis protoplasts, the transfected protoplasts were cultured for 24 h before CLSM observation. White dash boxes indicate the 2.5 X enlarged insets. Scale bars, 10 μm. dArabidopsis protoplasts transfected with FREE1, ATG5 and ATG8a or ATG8i were subjected to structure illuminated microscopy (SIM) analysis. Scale bars, 1 μm. e GFP-Trap and co-immunoprecipitation (co-IP) analysis of 3Myc-FREE1 with YFP-ATG8a or YFP-ATG8i using Arabidopsis protoplasts. Arabidopsis protoplasts co-expressing 3Myc-FREE1 with YFP-ATG8a, YFP-ATG8i, or YFP only were subjected to protein extraction and co-IP with GFP-Trap, followed by immunoblotting with indicated antibodies. f FRET analysis between CFP-FREE1 and YFP-ATG8a or YFP-ATG8i in Arabidopsis protoplasts. Arabidopsis protoplasts were transfected with CFP and YFP (negative control), CFP-YFP (positive control), CFP-FREE1 and YFP-ATG8a, or CFP-FREE1 and YFP-ATG8i, followed by cultured for 24 h before FRET analysis. Means±SD; n = 14 (CFP/YFP), n = 14 (CFP-YFP), n = 28 (CFP-FREE1/YFP-ATG8a), and n = 33 (CFP-FREE1/YFP-ATG8i) individual puncta per experimental group, one-way analysis of variance (ANOVA), followed by Dunnett’s multiple comparisons test; *p < 0.05; **p < 0.01. The middle lines of the boxes represent the medians of datasets. The upper and bottom lines of the boxes are the upper quantile and the lower quantile of the data, respectively. The whiskers mark the upper and lower limits of these datasets, respectively. All the imaging analysis and immunoblots were repeated at least for three times with similar results.

Effects of different H2S donors or H2S biosynthesis inhibitors on cell autophagy capability of C. reinhardtii under MC-LR treatment. (A) Effects of H2S biosynthesis scavenger or inhibitor on MC-LR-induced expression of ATG1, AGT7, AGT8, and ATG101. Here, 3-d-old C. reinhardtii cells were treated with 100 nM MC-LR with or without H2S artificial donor NaHS (100??M), GYY4137 (GYY, 10??M), H2S scavenger hypotaurine (HY, 10??M), or H2S biosynthesis enzyme inhibitor aminooxyacetic acid (AOA, 1?mM) for 12 h, with transcriptional levels of ATG1, AGT7, AGT8, and ATG101 then measured by qRT-PCR analysis. Values are means ± SD of three biological replicates. (B) Effects of H2S biosynthesis scavenger or inhibitor on MC-LR-induced protein accumulation of ATG8. Here, 3-d-old C. reinhardtii cells were treated with 100 nM MC-LR with or without H2S artificial donor for 3 d, with protein accumulation of ATG8 measured by Western blotting using anti-ATG8 antibody. Anti-Tubulin was used as the loading control.