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, 26 (2), 788-807

AUTOPHAGY-RELATED11 Plays a Critical Role in General Autophagy- And Senescence-Induced Mitophagy in Arabidopsis

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AUTOPHAGY-RELATED11 Plays a Critical Role in General Autophagy- And Senescence-Induced Mitophagy in Arabidopsis

Faqiang Li et al. Plant Cell.

Abstract

Autophagy-mediated turnover removes damaged organelles and unwanted cytoplasmic constituents and thus plays critical roles in cellular housekeeping and nutrient recycling. This "self eating" is tightly regulated by the AUTOPHAGY-RELATED1/13 (ATG1/13) kinase complex, which connects metabolic and environmental cues to the vacuolar delivery of autophagic vesicles. Here, we describe the Arabidopsis thaliana accessory proteins ATG11 and ATG101, which help link the ATG1/13 complex to autophagic membranes. ATG11 promotes vesicle delivery to the vacuole but is not essential for synthesizing the ATG12-ATG5 and ATG8-phosphatidylethanolamine adducts that are central to autophagic vesicle assembly. ATG11, ATG101, ATG1, and ATG13 colocalize with each other and with ATG8, with ATG1 tethered to ATG8 via a canonical ATG8-interacting motif. Also, the presence of ATG11 encourages starvation-induced phosphorylation of ATG1 and turnover of ATG1 and ATG13. Like other atg mutants, ATG11-deficient plants senesce prematurely and are hypersensitive to nitrogen and fixed-carbon limitations. Additionally, we discovered that the senescence-induced breakdown of mitochondria-resident proteins and mitochondrial vesicles occurs via an autophagic process requiring ATG11 and other ATG components. Together, our data indicate that ATG11 (and possibly ATG101) provides important scaffolds connecting the ATG1/13 complex to both general autophagy and selective mitophagy.

Figures

Figure 1.
Figure 1.
Phylogenetic Distribution of ATG11/FIP200 and ATG17 Proteins among Eukaryotes. The representatives from plants, metazoa, Ichthyosporea, amoebozoa, fungi, and stramenopiles were clustered based on their amino acid sequences and the presence/absence and arrangement of the signature ATG17, ATG17-like, coiled-coil, and ATG11 domains. See Supplemental Table 4 for the species names and UniProt accession numbers.
Figure 2.
Figure 2.
Interactions of Arabidopsis ATG11 with Itself, Subunits of the ATG1/13 Kinase Complex, and ATG8-Decorated Autophagic Structures. (A) Domain organization of ATG11. Lines represent introns, and colored and white boxes represent coding and untranslated regions, respectively. Positions of the signature ATG17-like, coiled-coil, and ATG11 domains are indicated. aa, amino acids. (B) and (C) Interaction of ATG11 with ATG1 and ATG13 in planta by BiFC. (B) Demonstration that each possible orientation of the NY and CY constructions for ATG1a, ATG11, and ATG13a could be expressed in N. benthamiana. Crude extracts prepared from leaves 36 h after infiltration were immunoblotted with anti-GFP antibodies. (C) BiFC using N. benthamiana leaf epidermal cells. Leaves were coinfiltrated with plasmids expressing the N- and C-terminal fragments of YFP fused to ATG1a, ATG11, and ATG13a. To detect indirect interactions between ATG1a and ATG11, the cells were also infiltrated with a plasmid expressing ATG13a tagged with an N-terminal Myc epitope. Shown are reconstituted BiFC signals as detected by confocal fluorescence microscopy of leaf epidermal cells 36 h after coinfiltration along with a bright-field (BF) image of the cells. Bar = 10 μm. (D) ATG11 and ATG13a colocalize with ATG8-decorated autophagic structures. The leaves of 4-d-old transgenic Arabidopsis seedlings stably expressing mCherry-ATG8a were coinfiltrated with plasmids expressing the NY-ATG13a and CY-ATG11 BiFC constructs and examined by confocal fluorescence microscopy as in (C). Arrowheads indicate colocalized structures. (E) and (F) Localization of the homodimerization domain within ATG11 by BiFC. (E) Diagram of the ATG11 truncations. (F) BiFC signals in N. benthamiana leaf epidermal cells from paired ATG11 truncations fused to the N- and C-terminal fragments of YFP along with a bright-field image of the cells. Bar = 10 μm.
Figure 3.
Figure 3.
Interaction of ATG101 with Other Subunits of the ATG1/13 Kinase Complex. (A) Structure of the ATG101 locus (At5g66930). Lines represent introns, and the colored and white boxes represent coding and untranslated regions, respectively. Amino acid (aa) sequence length is indicated on the right. (B) Y2H interactions of ATG101 with other subunits of the ATG1/13 complex. Full-length ATG101 designed as N-terminal fusions to either the GAL4 activating (AD) or binding (BD) domain was coexpressed with complementary AD or BD fusions of ATG1a, ATG11, and ATG13a. Shown are cells grown on selection medium lacking Trp and Leu (−L−W) or lacking Trp, Leu, and His and containing 3-amino-1,2,4-triazole (−L−W−H+3AT). (C) Interaction of ATG101 with ATG11 or ATG13a in planta by BiFC. N. benthamiana leaf epidermal cells were coinfiltrated with plasmids expressing the N- and C-terminal fragments of YFP fused to ATG101, ATG11, and ATG13a. Reconstituted BiFC signals, as detected by confocal fluorescence microscopy of leaf epidermal cells 36 h after infiltration, are shown along with a bright-field (BF) image of the cells. Bar = 10 μm.
Figure 4.
Figure 4.
Genotypic and Phenotypic Analyses of Arabidopsis atg11 Mutants. (A) Diagram of the ATG11 locus showing domain organization and the positions of relevant T-DNA insertion mutations. Lines represent introns, and the colored and white boxes represent coding and untranslated regions, respectively. Positions of the signature ATG17-like, coiled-coil, and ATG11 domains are indicated. Red triangles identify the T-DNA insertion sites for the atg11-1 and atg11-2 alleles. Half-arrows at bottom and top indicate positions of the primers used for RT-PCR in (B) and quantitative RT-PCR in (C), respectively. (B) RT-PCR analysis of the ATG11 transcript in the atg11-1 and atg11-2 mutants. Total RNA isolated from wild-type or homozygous mutant plants was subjected to RT-PCR using the primer pairs indicated in (A). RT-PCR with primers specific for UBC9 was included to confirm the analysis of equal amounts of RNA. (C) Quantitative real-time RT-PCR analysis of transcripts arising 5′ and 3′ to the T-DNA insertion site in atg11-1. Primer locations are indicated in (A). (D) Enhanced sensitivity to nitrogen starvation. Plants were germinated and grown for 1 week on nitrogen-containing liquid medium and then transferred to either nitrogen-containing (+N) or nitrogen-deficient (−N) medium for an additional 2 weeks. Lines tested include the wild type, the atg11-1 and atg11-2 alleles shown in (A), and previously described autophagy-defective mutants atg5-1 and atg7-2 (Thompson et al., 2005; Chung et al., 2010). (E) Premature senescence under an SD photoperiod. Plants were grown on soil at 21°C under SD conditions for 10 weeks. (F) Accelerated senescence of detached leaves. The first pair of true leaves was cut from 2-week-old seedlings grown with 1% Suc and incubated for 7 d in the dark at 24°C. (G) Relative chlorophyll content of detached leaves shown in (D). Each bar represents the mean ± sd from three independent experiments analyzing at least 30 leaves each. (H) Enhanced sensitivity to fixed-carbon starvation. Seedlings were grown under an LD photoperiod without added Suc for 2 weeks and then placed in darkness for 10 or 13 d before returning to LD conditions for a 12-d recovery. (I) Survival of fixed-carbon–deprived plants shown in (E). Each bar represents the mean percentage survival ± sd of three independent experiments examining at least 15 seedlings each.
Figure 5.
Figure 5.
ATG11 Associates with Autophagic Vesicles. (A) to (C) Rescue of the atg11-1 phenotype with the UBQ10:GFP-ATG11 transgene. (A) Growth of wild-type, atg11-1, and UBQ10:GFP-ATG11 atg11-1 plants under nitrogen starvation. Plants were germinated and grown for 1 week on nitrogen-containing (+N) liquid medium and then transferred to either nitrogen-containing or nitrogen-deficient (−N) liquid medium for an additional 2 weeks. (B) Senescence under an SD photoperiod. Plants were grown on soil at 21°C under an 8-h-light/16-h-dark cycle for 10 weeks. (C) Sensitivity to fixed-carbon starvation. Seedlings were grown under an LD photoperiod (16 h of light/8 h of dark) without added Suc for 2 weeks and then placed in darkness for 13 d before returning to LD conditions for a 12-d recovery. (D) Deposition of GFP-ATG11–containing vesicles in the vacuole by a process that requires ATG7. Wild-type and atg7-2 plants expressing UBQ10:GFP-ATG11 were grown for 6 d on nitrogen-containing medium and then transferred to nitrogen-deficient medium without or with 1 μM ConA for an additional 24 h. Root cells were imaged by confocal fluorescence microscopy. Bar = 10 μm. (E) GFP-ATG11 colocalizes with mCherry-ATG8a in autophagic bodies. Plants stably expressing both reporters were grown for 6 d on nitrogen-containing medium and then exposed for 8 h to nitrogen-deficient medium supplemented with 1 μM ConA before confocal microscopy. Boxes outlined in the left panels were magnified three times in the right panels. Bars = 10 and 5 μm for the left and right panels, respectively.
Figure 6.
Figure 6.
Lack of ATG11 Blocks Autophagic Body Deposition in the Vacuole but Not Modification of ATG8 or ATG12. (A) Deposition of autophagic bodies inside the vacuole. Transgenic seedlings expressing GFP-ATG8a were grown for 6 d on nitrogen-containing solid medium with 1% Suc and then exposed for 24 h to nitrogen-deficient liquid medium without or with the addition of 1 μM ConA before confocal fluorescence microscopic analysis of root cells. Lines tested include the wild type and atg7-2, atg11-1, and atg11-2 mutants each expressing GFP-ATG8a. Bar = 10 μm. (B) Detection of the free GFP released during the vacuolar degradation of GFP-ATG8a. Seven-day-old seedlings described in (A) were grown on nitrogen-containing liquid medium and then exposed to nitrogen-deficient medium for 16 h. Total protein was subjected to immunoblot analysis with anti-GFP antibodies. Closed and open arrowheads indicate GFP-ATG8a and free GFP, respectively. Immunoblotting with anti-PBA1 antibodies was used to confirm nearly equal loading in (B) and (C). (C) Immunoblot detection of the ATG12-ATG5 conjugate. Total protein from 7-d-old seedlings grown on MS solid medium with 1% Suc was subjected to immunoblot analysis with anti-ATG5 antibodies. Closed and open arrowheads indicate the ATG12-ATG5 conjugate and free ATG5, respectively. (D) Immunoblot detection of ATG8-PE adducts. Seedlings were grown on nitrogen-containing liquid medium for 7 d and then exposed to nitrogen-deficient medium for 2 d before extraction. Crude extracts (CE) were separated into the soluble (S) and membrane (Memb) fractions by centrifugation. The membrane fraction was solubilized in Triton X-100 and incubated with or without phospholipase D (PLD) for 1 h. Samples were then subjected to SDS-PAGE in the presence of 6 M urea and immunoblotted with antibodies against ATG8a. Dashed lines indicate free ATG8; solid lines indicate ATG8-PE adducts.
Figure 7.
Figure 7.
ATG11 Is Required for Proper Phosphorylation and Autophagic Turnover of ATG1a. (A) and (B) ATG11 regulates the phosphorylation of ATG1a and ATG13a/b. (A) Loss of ATG11 affects the phosphorylation status of ATG1a and ATG13a/b, as observed by changes in SDS-PAGE migration pattern and abundance of isoforms. Wild-type, atg5-1, and atg11-1 seedlings were grown for 7 d under continuous light on nitrogen-containing liquid medium and then transferred for 1 d to nitrogen-containing or nitrogen-deficient (+/−) medium. Crude extracts were subjected to immunoblot analysis with anti-ATG1a or anti-ATG13a antibodies. Immunoblot analysis with anti-PBA1 antibodies was used to confirm equal protein loading. (B) ATG1a is dephosphorylated in the atg11-1 mutant. Seven-day-old atg7-2 and atg11-1 plants were incubated for 3 d in MS liquid medium without nitrogen and Suc. Crude extracts (CE) were treated with λ-phosphatase (Ppase) in the presence or absence of phosphatase inhibitor for 1 h and then subjected to immunoblot analysis with anti-ATG1a antibodies. Positions of the 72-kD phosphorylated and 70-kD unphosphorylated forms of ATG1a are indicated. (C) Loss of ATG11 reduces the accumulation of ATG1a in autophagic bodies during nitrogen starvation. Wild-type, atg7-2, and atg11-1 seedlings expressing YFP-ATG1a were grown for 6 d on nitrogen-containing solid medium containing 1% Suc and then exposed to nitrogen-deficient liquid medium without or with 1 μM ConA for 1 d. Root cells were imaged by confocal fluorescence microscopy of root cells. Bar = 10 μm. (D) Loss of ATG11 reduces the nutrient stress–induced turnover of ATG1a. Wild-type, atg7-2, and atg11-1 plants expressing YFP-ATG1a were grown for 6 d on nitrogen-deficient liquid medium and then incubated in nitrogen-containing or nitrogen-deficient medium without or with 1 μM ConA for 16 h. YFP-ATG1a protein levels were revealed by immunoblots with anti-ATG1a and anti-GFP antibodies. Immunoblot analysis with anti-PBA1 antibodies was used to confirm equal protein loading.
Figure 8.
Figure 8.
ATG1a and ATG11 Both Interact with ATG8e. (A) Schematic representation of Arabidopsis ATG1a and the sequence alignment of its putative AIM with those in other ATG1 family members from Arabidopsis and with orthologs from other eukaryotes. K and ATP indicate the catalytic site Lys and the ATP binding site in the kinase domain, respectively. Reg indicates the C-terminal early autophagy targeting/tethering regulatory domain. Residues targeted by mutagenesis are marked with arrows. The conserved aromatic/hydrophobic residues and the upstream and downstream acidic residues in the AIM are highlighted in green and red, respectively. The numbers on the right indicate the position of the first residue within the context of each full-length protein. aa, amino acids. (B) Interaction of Arabidopsis ATG8e with ATG1a and ATG11 in planta by BiFC. N. benthamiana leaf epidermal cells were coinfiltrated with plasmids expressing the N-terminal fragment of YFP (NY) fused to ATG8e and the C-terminal YFP (CY) fragment fused to ATG11, ATG1a, or an ATG1a mutant affecting the AIM motif (Y360-A/L363-A). Shown are confocal fluorescence microscopic images of coinfiltrated cells along with a bright-field (BF) image of the cells. Bar = 5 μm (C) Time-lapse images showing that ATG11 interacts with ATG8e in fast-moving particles. The NY-ATG8e and CY-ATG11 transgenes were coexpressed as in (B) and visualized over time by confocal fluorescence microscopy. The time duration in seconds is shown for each panel. Bar = 5 μm.
Figure 9.
Figure 9.
Dark-Induced Senescence Induces Mitochondrion Turnover via an Autophagic Process. The third and fourth rosette leaves of 4-week-old plants were individually darkened for 3 to 5 d prior to examination. (A) Representative IDLs from wild-type, atg7-2, and atg11-1 plants showing accelerated chlorosis. (B) Chlorophyll contents of IDLs shown in (A). Each bar represents the mean ± sd from three independent experiments measuring 12 leaves each. fw, fresh weight. (C) Loss of mitochondrial markers during IDL senescence. IDLs were harvested at the indicated times of dark exposure from wild-type, atg7-2, and atg11-1 plants expressing Mito-CFP, homogenized, and the crude extracts were subjected to immunoblot analyses with antibodies against ATG1a, VDAC, COX II, and GFP. Immunoblot analysis with anti-histone H3 antibodies was used to confirm nearly equal protein loading. (D) Quantification of mitochondrion numbers during IDL senescence. Mitochondria within representative 2500-μm2 regions of leaf cells from (C) were counted using the Particle Analysis function of ImageJ. Each bar represents the mean ± sd from two independent experiments counting at least eight regions each. Asterisks indicate statistically significant differences as determined by Student’s t test (P < 0.05).
Figure 10.
Figure 10.
Association of Mitochondria with Autophagic Bodies during Leaf Senescence. (A) Accumulation of Mito-CFP in vacuolar puncta during dark-induced leaf senescence (IDL) by a process that requires the ATG system. Four-week-old wild-type, atg7-2, and atg11-1 plants expressing Mito-CFP were subjected to IDL for 3 d followed by a 20-h incubation with 1 μM ConA or DMSO before confocal fluorescence microscopy of epidermal cells from the third and fourth rosette leaves. Bars = 10 μm and 3.4 μm in the inset. Insets show 3× magnifications of the vacuole. (B) Colocalization of Mito-YFP with the autophagic membrane marker mCherry-ATG8a in autophagic bodies. Wild-type plants expressing both reporters were subjected to IDL senescence as in (A). The vacuolar region of leaf epidermal cells was imaged by confocal fluorescence microscopy. Bar = 10 μm. A 3× magnification of the merged signals (outlined by the white box) is included to confirm colocalization of the two proteins in autophagic bodies (arrowheads). A free Mito-YFP–labeled mitochondrion and an autophagic body not containing mitochondria are indicated with the diamond and the star, respectively. (C) Colocalization of the mitochondrial stain MitoTracker Green FM with mCherry-ATG11. Arabidopsis leaf protoplasts stably expressing mCherry-ATG11 were treated for 30 min with MitoTracker Green FM, washed twice, and then incubated for 24 h with ConA in the absence of Suc before confocal fluorescence microscopy. Closed arrowheads indicate vacuolar puncta containing both fluorescent markers; open arrowheads are puncta preferentially containing mCherry-ATG11. BF, bright field; Vac, vacuole. Bar = 5 μm.
Figure 11.
Figure 11.
Schematic of the Interactions between ATG1, ATG8, ATG13, ATG11, and ATG101 as Determined by Y2H and/or BiFC Assays. The AIM sequences potentially involved in ATG8 binding are shown in red.

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