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, 23 (10), 3761-79

The ATG1/ATG13 Protein Kinase Complex Is Both a Regulator and a Target of Autophagic Recycling in Arabidopsis

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The ATG1/ATG13 Protein Kinase Complex Is Both a Regulator and a Target of Autophagic Recycling in Arabidopsis

Anongpat Suttangkakul et al. Plant Cell.

Abstract

Autophagy is an intracellular recycling route in eukaryotes whereby organelles and cytoplasm are sequestered in vesicles, which are subsequently delivered to the vacuole for breakdown. The process is induced by various nutrient-responsive signaling cascades converging on the Autophagy-Related1 (ATG1)/ATG13 kinase complex. Here, we describe the ATG1/13 complex in Arabidopsis thaliana and show that it is both a regulator and a target of autophagy. Plants missing ATG13 are hypersensitive to nutrient limitations and senesce prematurely similar to mutants lacking other components of the ATG system. Synthesis of the ATG12-ATG5 and ATG8-phosphatidylethanolamine adducts, which are essential for autophagy, still occurs in ATG13-deficient plants, but the biogenesis of ATG8-decorated autophagic bodies does not, indicating that the complex regulates downstream events required for autophagosome enclosure and/or vacuolar delivery. Surprisingly, levels of the ATG1a and ATG13a phosphoproteins drop dramatically during nutrient starvation and rise again upon nutrient addition. This turnover is abrogated by inhibition of the ATG system, indicating that the ATG1/13 complex becomes a target of autophagy. Consistent with this mechanism, ATG1a is delivered to the vacuole with ATG8-decorated autophagic bodies. Given its responsiveness to nutrient demands, the turnover of the ATG1/13 kinase likely provides a dynamic mechanism to tightly connect autophagy to a plant's nutritional status.

Figures

Figure 1.
Figure 1.
Structure and Genetic Analysis of Arabidopsis ATG1 and ATG13 Gene Families. (A) Gene diagrams of the ATG1 gene family. White and shaded boxes indicate UTRs and coding regions, respectively. Black boxes identify the N-terminal kinase and C-terminal regulatory domains. The catalytic site Lys (K) and ATP binding site in the kinase domain are indicated by the arrowheads. Lines denote introns. Triangles mark T-DNA insertion sites and are labeled with allele names. The positions of primers used for RT-PCR are located by the arrows. aa, amino acids. (B) Comparison of the ATG1a and ATG1t transcript structures. The coding region for ATG1t terminates immediately distal to the kinase domain (black box) and is followed out of frame by a portion of the transcript encoding the DNA polymerase B δ subunit (cross-hatched box). Y locates the catalytic Tyr required for δ subunit activity. aa, amino acids. (C) Nucleotide and derived amino acid sequence of the ATG1t gene surrounding the junction of the ATG1t transcript with a portion of DNA polymerase B δ coding sequence. The asterisks denote stop codons. (D) Gene diagrams of the ATG13 gene family. White and shaded boxes indicate UTRs and coding regions, respectively. Lines denote introns. Triangles mark T-DNA insertion sites and are labeled with allele names. The positions of primers used for RT-PCR are located by the arrows. aa, amino acids. (E) RT-PCR analysis of the atg1a, atg13a, and atg13b T-DNA insertion mutants. Total RNA from wild-type or homozygous mutant plants was subjected to RT-PCR using the indicated primer pairs in (A) and (D). RT-PCR with PAE2-specific primers was used to confirm the analysis of equal amounts of RNA.
Figure 2.
Figure 2.
ATG1a and ATG13a Interact and Associate with Autophagic Compartments. (A) Y2H analysis of ATG1-ATG13 interactions. Full-length ATG1a and ATG13a expressed as N-terminal fusions to either the GAL4 DNA activator (AD) or binding domains (BD) were coexpressed in yeast on selection medium containing 3-amino-1,2,4-triazole and lacking Trp, Leu, and His. The known interaction between ATG7 and ATG8a was included as a positive control. AD and BD indicated empty AD and BD plasmids. (B) Localization of ATG1a and ATG13a to autophagy-type compartments in Arabidopsis protoplasts. Free GFP and GFP fused to ATG8a, ATG1a, or ATG13a were transiently expressed in leaf protoplasts and their localization observed 18 h after transformation by fluorescence confocal microscopy. Green, GFP fluorescence; red, chlorophyll fluorescence. Arrowheads locate possible autophagosomes in the cytoplasm. Asterisks mark potential autophagic bodies inside the vacuole. (C) BiFC analysis of ATG1a and ATG13a in Arabidopsis. The split nYFP and cYFP reporters simultaneously expressed alone or fused to the N terminus of ATG1a and ATG13a were detected by fluorescence confocal microscopy in leaf protoplasts 18 h after transformation. Images obtained from YFP and chlorophyll (Chloro) fluorescence and bright-field (BF) microscopy are shown. Bars = 5 μm.
Figure 3.
Figure 3.
Expression Patterns of ATG1 and ATG13. (A) Transcript abundance patterns for the ATG1 and ATG13 families obtained from the Genevestigator database (https://www.genevestigator.ethz.ch/). The EST number for each locus is listed at the bottom. (B) Immunoblot detection of the ATG1a and ATG13a proteins in crude extracts from various Arabidopsis tissues dissected from soil-grown plants. Immunoblot analysis with anti-PBA1 antibodies was performed to assess protein loading. The migration positions of ATG1a and the various ATG13a species, as confirmed by their absence in extracts from atg1a-1 and atg13a-1 atg13b-2 plants, are indicated. The asterisk identifies a protein nonspecifically cross-reacting with the anti-ATG1a antibodies. Ros, rosette; Sen, senescing; Yg, young.
Figure 4.
Figure 4.
atg13a atg13b Plants Display Phenotypes Characteristic of Autophagy Mutants. Lines tested include wild-type (WT) Col-0, homozygous atg5-1, atg7-2, atg13a-1, atg13a-2, atg13b-1, atg13b-2 single mutants, and atg13a-1 atg13b-2 and atg13a-2 atg13b-2 double mutants (A) Sensitivity to N starvation. Seedlings were grown for 1 week on MS+N liquid medium and then transferred to N-rich (+N) or N-deficient (–N) media for an additional 2 weeks. (B) Accelerated senescence. Plants were soil grown at 22°C in an SD photoperiod for 10 weeks. (C) Sensitivity to fixed-C starvation. Plants were grown under LD on MS-containing agar without added Suc for 2 weeks, transferred to darkness for 10 or 13 d, and then allowed to recover for 12 d in LD. (D) Percentage of plant survival after the fixed-C starvation shown in (C). Each bar represents the average survival (±sd) of three replicates examining at least 15 seedlings each.
Figure 5.
Figure 5.
atg13a atg13b Mutants can Generate ATG12-ATG5 and ATG8-PE Adducts but not Autophagic Bodies (A) Immunoblot detection of ATG1a, ATG13a, ATG8, ATG5, and the ATG12-ATG5 conjugate in single and double homozygous seedlings bearing mutations in ATG1a, ATG5, ATG7, ATG13a, and ATG13b. Arrowheads locate ATG1a, ATG5, and the ATG12-ATG5 conjugate. The apparent molecular masses of the various forms of ATG13a are indicated. Asterisks identify proteins nonspecifically cross-reacting with the anti-ATG1a or ATG13a antibodies. Immunoblot analysis with anti-PBA1 antibodies was used to confirm equal protein loading. WT, wild type. (B) Immunoblot detection of the ATG8-PE adducts in membrane fractions. CE, crude seedling extract prior to fractionation; S, soluble fraction obtained after centrifugation; Mem, membrane fraction obtained after centrifugation and solubilized in Triton X-100. Fractions were treated for 1 h at 37°C with PLD prior to SDS-PAGE in 6 M urea. Dashed lines, free ATG8; solid lines, ATG8-PE adducts. (C) Detection of free GFP generated by vacuolar breakdown of GFP-ATG8a. Seven-day-old mutant and wild-type seedlings expressing GFP-ATG8a were exposed to N-deficient medium for the indicated time before extraction. Crude extracts were subjected to SDS-PAGE and immunoblot analysis with anti-GFP antibodies. GFP-ATG8a and free GFP are located by the closed and open arrowheads, respectively. Anti-PBA1 antibodies were used to confirm equal protein loading. (D) atg13a atg13b mutant plants cannot generate autophagic bodies. Seven-day-old mutant and wild-type seedlings expressing GFP-ATG8a were exposed to N-deficient medium with or without CA for 16 h and then visualized by fluorescence confocal microscopy. Bar = 10 μm [See online article for color version of this figure.]
Figure 6.
Figure 6.
ATG1a and ATG13a Protein but Not Transcript Levels Disappear during Fixed-C/N Limitation. Five-day-old wild-type seedlings were either kept in fixed-C/N-rich (+Suc+N) medium or switched to fixed-C/N-limiting medium (−Suc−N) under continuous light and then incubated for 1 d before extraction of total protein and RNA. (A) ATG1a and ATG13a protein levels detected by immunoblot analysis. Anti-PBA1 antibodies were used to confirm equal protein loading. The apparent molecular masses of ATG1a and the three ATG13a isoforms are indicated. (B) ATG1a and ATG13a mRNA levels as detected by RT-PCR. RT-PCR with UBC21-specific primers was used to confirm the analysis of equal amounts of total RNA.
Figure 7.
Figure 7.
ATG1a and ATG13a Proteins Rapidly Disappear during Fixed-C/N Limitation. Five-day-old wild-type seedlings grown in LD were either kept in +Suc+N medium or switched to +Suc−N, −Suc+N, or −Suc−N media for the indicated times before extraction of total protein. (A) and (C) Immunoblot analysis of the starvation time course with anti-ATG1a (A) and ATG13a antibodies (C). Each lane contains an equivalent amount of tissue fresh weight. Immunoblot analyses with anti-PBA1 antibodies were used to confirm near equal protein loading. (B) and (D) Quantitation of ATG1a (B) and ATG13a (D) disappearance normalized using the signals from PBA1.
Figure 8.
Figure 8.
ATG1a and ATG13a Proteins Rapidly Reappear after Suc/N-Limited Plants Are Refed. Five-day-old wild-type seedlings were either kept in +Suc+N medium or switched to −Suc−N for 36 h and then switched back to +Suc+N medium for the indicated times before extraction of total protein. (A) Immunoblot analysis of the refeeding time course with anti-ATG1a or ATG13a antibodies. Each lane contains an equivalent amount of tissue fresh weight. Immunoblot analyses with anti-PBA1 antibodies were used to confirm near equal protein loading. (B) and (C) Quantitation of ATG1a (B) and ATG13a (C) reappearance normalized using the signals from PBA1.
Figure 9.
Figure 9.
Turnover and Phosphorylation States of ATG1a and ATG13a Proteins Are Regulated by Autophagy. (A) to (C) Effect of inhibitors and mutants on the turnover of ATG1a and ATG13a during starvation. Five-day-old seedlings of the various genetic backgrounds were either kept in +Suc+N medium (+) or switched to –Suc−N medium (−) at t = 0 and then incubated for the indicated times before extraction of total protein. ATG1a and ATG13a proteins levels were detected by immunoblot analysis with anti-ATG1a and ATG13a antibodies. Immunoblot analyses with anti-PBA1 antibodies were used to confirm near equal protein loading. Each lane contains an equivalent amount of tissue fresh weight. (A) Effect of various inhibitors. CA (0.5 μM), MG132 (50 μM), or an equivalent volume of DMSO were added at t = 0. (B) Effect of various mutations. WT, wild type. (C) Fixed-C/N limitation alters the SDS-PAGE mobility of ATG1a. Extracts from plants before and after 72 h of −Suc−N limitation were electrophoresed in adjacent lanes or mixed (Mix). The apparent molecular masses of the two ATG1a species are indicated. (D) Effect of λ protein phosphatase on the SDS-PAGE mobilities of ATG1a and ATG13a. For analysis of ATG1a, atg5-1 plants were incubated for 72 h in –Suc−N medium. For analysis of ATG13a, wild-type plants were grown in +Suc+N medium. Extracts were treated for 1 h with λ phosphatase (Ppase) with or without the phosphatase inhibitor PhosSTOP and then subjected to immunoblot analysis with anti-ATG1a and anti-ATG13a antibodies. UN, untreated extracts.
Figure 10.
Figure 10.
ATG1a Associates in the Vacuole with ATG8a-Decorated Autophagic Bodies. (A) Levels of YFP-ATG1a protein are reduced by N starvation–induced autophagy. Wild-type (WT) and atg7-2 seedlings were grown for 6 d on N-rich agar medium and then transferred to N-rich (+N) or N-deficient (–N) liquid media without or with 0.5 μM CA (+CA) for 16 h. YFP-ATG1a protein levels were measured by immunoblots with anti-ATG1a and anti-YFP antibodies. Immunoblot analysis with anti-PBA1 antibodies was used to confirm equal protein loading. (B) YFP-ATG1a associates with autophagic bodies in a process that requires ATG7. Seven-day-old wild-type and atg7-2 seedlings were exposed to N-containing or N-deficient liquid media with or without 0.5 μM CA. After 16 h, the root tip cells were imaged by fluorescence confocal microscopy. Bar = 10 μm. (C) YFP-ATG1a and mCherry-ATG8a colocalize in autophagic bodies. Seven-day-old wild-type plants expressing both reporters were grown on +Suc−N medium and then treated for 8 h with 0.5 μM CA. Root tip cells were imaged by fluorescence confocal microscopy for YFP and mCherry. Merged represents the superimposition of the two images. Bar = 10 μm. Higher magnifications of the vacuolar lumen (outline by dashed lines) demonstrating colocalization of the two proteins on autophagic bodies are shown below. Bar = 5 μm.

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