2015 Jun 18
Autophagic Degradation of the 26S Proteasome Is Mediated by the Dual ATG8/Ubiquitin Receptor RPN10 in Arabidopsis
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Autophagic Degradation of the 26S Proteasome Is Mediated by the Dual ATG8/Ubiquitin Receptor RPN10 in Arabidopsis
Autophagic turnover of intracellular constituents is critical for cellular housekeeping, nutrient recycling, and various aspects of growth and development in eukaryotes. Here we show that autophagy impacts the other major degradative route involving the ubiquitin-proteasome system by eliminating 26S proteasomes, a process we termed proteaphagy. Using Arabidopsis proteasomes tagged with GFP, we observed their deposition into vacuoles via a route requiring components of the autophagy machinery. This transport can be initiated separately by nitrogen starvation and chemical or genetic inhibition of the proteasome, implying distinct induction mechanisms. Proteasome inhibition stimulates comprehensive ubiquitylation of the complex, with the ensuing proteaphagy requiring the proteasome subunit RPN10, which can simultaneously bind both ATG8 and ubiquitin. Collectively, we propose that Arabidopsis RPN10 acts as a selective autophagy receptor that targets inactive 26S proteasomes by concurrent interactions with ubiquitylated proteasome subunits/targets and lipidated ATG8 lining the enveloping autophagic membranes.
Copyright © 2015 Elsevier Inc. All rights reserved.
Arabidopsis 26S Proteasome Abundance Is Controlled by Autophagy
(A) Proteasome subunit levels rise in autophagy mutants. Shown is immunoblot detection of various proteasome subunits and accessory factors in total protein extracts from the wild-type (WT) or a collection of autophagy mutants. (B) 26S proteasome activity is unchanged in autophagy mutants. Total protein extracts were assayed for CP peptidase activity using succinyl-leucyl-leucyl-valyl-tyroyl-7-amido-4-methylcoumarin (succinyl-LLVY-AMC). Bars represent the mean (± SD) of three biological replicates, each generated with three technical replicates. (C) Proteasome subunits are delivered to the vacuole upon nitrogen starvation. Seedlings expressing PAG1-GFP or RPN5a-GFP were either kept on nitrogen (N)-rich medium or switched to N-free medium plus 1 μM ConA for 16 hr. Root cells were imaged by confocal microscopy. Scale bar, 10 μm. Nuc, nucleus; V, vacuole. (D) PAG1-GFP co-localizes with mCherry-ATG8a in autophagic bodies. Seedlings were exposed to N starvation and 1 μM ConA for either 16 or 4 hr (left and right, respectively). Scale bars, 5 μm (left) and 10 μm (right). (E) Assembled proteasomes accumulate in autophagic vesicles. WT seedlings were incubated for 16 hr with 20 μM of the fluorescent epoxomycin (Epo) probes MVB003 or MVB072, which label active proteasomes. Where indicated, seedlings were treated simultaneously with 1 μM ConA or pre-treated with 20 μM Epo for 4 hr. Scale bar, 5 μm. See also Figures S1 and S2.
Figure 2. 26S Proteasomes Are Delivered to the Vacuole upon Nitrogen Starvation
(A) Delivery of vesicles containing PAG1-GFP and RPN5a-GFP to the vacuole is blocked in strong autophagy mutants. WT,
atg7-2, atg10-1, and nbr1-2 seedlings were switched to N-free medium containing 1 μM ConA for 16 hr. Root cells were examined for autophagic bodies by confocal microscopy. Scale bars, 10 μm. (B) Time course of free GFP release from PAG1-GFP and RPN5a-GFP during N starvation. WT and atg7-2 seedlings expressing each reporter were starved for the indicated times, and total protein extracts were immunoblotted with anti-GFP antibodies. The GFP fusions and free GFP are indicated by closed and open arrowheads, respectively. (C) Effect of autophagy mutants on the cleavage of PAG1-GFP and RPN5a-GFP during N starvation. WT and mutant seedlings were either kept on N-rich medium or switched to N-free medium for 16 hr. Immunoblotting was performed as in (B). (D) Quantification of the free GFP/PAG1-GFP ratio during N starvation by densitometric scans of the immunoblots shown in (B). Bars represent the mean (± SD) of three biological replicates. (E) N starvation induces the loss of many 26S proteasome components. WT and atg7-2 seedlings were N-starved for the indicated times. Total protein extracts were probed with antibodies against various proteasome subunits.
Figure 3. Proteaphagy Is Stimulated by Chemical or Genetic Inhibition of the Proteasome
(A) Time course of free GFP release from PAG1-GFP and RPN5a-GFP following MG132 exposure. WT and
atg7-2 seedlings were exposed to 50 μM MG132 (MG) for the indicated times, and total protein extracts were immunoblotted with anti-GFP antibodies. The GFP fusions and free GFP are indicated by closed and open arrowheads, respectively. (B) Effects of various autophagy mutants on the cleavage of PAG1-GFP and RPN5a-GFP during MG132 exposure. WT, atg7-2, atg10-1, atg13a-2 atg13b-2, and nbr1-2 seedlings were incubated with or without 50 μM MG132 for 16 hr. Immunoblotting was performed as in (A). (C) MG132 induces the delivery of autophagic vesicles to the vacuole. WT seedlings were either N-fed or starved and/or exposed to 50 μM MG132 and/or 1 μM ConA for 16 hr. Root cells were examined for autophagic bodies by confocal microscopy. Scale bar, 10 μm. (D) Quantification of the free GFP/tagged-GFP ratio during N starvation or MG132 treatment of the seedlings analyzed in (C). Corresponding immunoblots are shown in Figure S6C. Bars represent the mean (± SD) of three biological replicates. Letters indicate values that are statistically different from one another (p < 0.05). (E) The proteasome mutants rpt2a-2 and rpt4b-2, but not rpn10-1, stimulate proteaphagy. Well fed seedlings were assayed for the release of free GFP by immunoblotting as in (A). (F) Quantification of the free GFP/PAG1-GFP ratio in the proteasome mutants by densitometric scans of the immunoblot shown in (E). Bars represent the mean (± SD) of three biological replicates. Letters indicate values that are statistically different from one another (p < 0.05). See also Figures S3–S6.
Figure 4. Proteasome Inhibition Stimulates Ubiquitylation of the Particle and Increased Association of RPN10
(A) Affinity purification of 26S proteasomes from
Arabidopsis seedlings expressing PAG1-FLAG. Total protein extracts from WT and pag1-1 PAG1-FLAG seedlings were incubated with anti-FLAG beads, washed, and eluted with FLAG peptide. Fractions were subjected to SDS-PAGE and stained for protein with silver. (B) Composition of 26S proteasomes upon inhibition. Proteasomes were affinity-purified from seedlings treated with 50 μM MG132 (MG) or clasto-lactacystin β-lactone (Lac). Preparations were separated by SDS-PAGE and stained for protein with silver (left) or immunoblotted with anti-ubiquitin (Ub) antibodies (center) or antibodies against proteasome subunits and the FLAG epitope (right). The arrowhead identifies PA200. (C) Analysis of proteasome preparations by native-PAGE. Proteasomes were affinity-purified from seedlings treated as in (B) and separated by native PAGE. Gels were either stained for total protein with silver (left) or immunoblotted with anti-ubiquitin antibodies (right). Migration positions of the CP, the CP-PA200 complex, the RP, singly and doubly capped 26S proteasomes (26S-RP(1) and 26S-RP(2), respectively), and dissociated Ub conjugates are indicated by the arrowheads and bracket. (D) Treatment with the deubiquitylating enzyme USP2 releases RPN10. Proteasomes were affinity-purified from control and MG132-treated seedlings as in (A), but, prior to elution, samples were incubated in the presence or absence of 10 nM USP2. Following elution, samples were separated by SDS-PAGE and analyzed as in (B). The amounts of associated ubiquitin and RPN10 were quantified and expressed as a percentage of the MG132-treated samples before digestion. (E) Treatment with MG132, but not N starvation, induces proteasome ubiquitylation and increased RPN10 association. Proteasomes were affinity-purified from pag1-1 PAG1-FLAG seedlings with or without the atg7-2 mutation after treatment with or without N starvation or 50 μM MG132. Samples were analyzed as in (B). Ubiquitylation levels were quantified and expressed as a percentage of the MG132-treated WT sample.
Figure 5. The 26S Proteasome Ubiquitin Receptor RPN10 Interacts with ATG8
(A) Y2H interactions between
Arabidopsis RPN10 and ATG8a. The indicated full-length proteins fused to either the GAL4 activating (AD) or binding (BD) domains were co-expressed and selected for growth on medium lacking tryptophan, leucine, and histidine and containing 3-AT. The known interactions between RPN10 and DSK2b, and ATG8a and ATG7 or NBR1, were included as positive controls, whereas combinations involving the GAL4 AD and BD domains alone were included as negative controls. (B and C) Arabidopsis RPN10 and ATG8a interactions detected in planta by BiFC. Tobacco leaf epidermal cells were co-infiltrated with the indicated plasmid combinations, and the fluorescent signals were detected by confocal microscopy 36 hr after infiltration. The known interaction between RPN10 and DSK2b was included as a control. For B, the infiltrated leaf sections were excised, vacuum-infiltrated with 1 μM ConA, and incubated in the dark for 16 hr prior to imaging. Scale bars, 10 μm (B) and 5 μm (C). (D) Phylogenetic analysis of the nine Arabidopsis ATG8 isoforms and their ortholog from Saccharomyces cerevisiae. The sequences were aligned in Clustal Omega and subjected to tree analysis using MrBayes. The expressed sequence tag (EST) value for each Arabidopsis isoform is indicated. (E) Y2H interactions between RPN10 and members from each ATG8 subclade. The assays were performed as in (A). (F) RPN10 interactions with members of the ATG8 family detected in planta by BiFC. Tobacco leaf epidermal cells were co-infiltrated with the indicated plasmid combinations and imaged as in (C). Scale bar, 10 μm.
Figure 6. ATG8 Interacts with the UIM2 Region of RPN10
(A) Diagram of the
Arabidopsis RPN10 protein. The positions of the vWA domain and the three UIMs are indicated. The residue numbers define the various C-terminal truncations used in (B). (B and C) Y2H interactions of Arabidopsis ATG8a with various truncations and site-directed substitutions of RPN10. The known interactions between the vWA domain and RPN9, UIM1 and DSK2b, UIM3 and RAD23c, and ATG8a and ATG7 were included as positive controls. (D) In vitro binding assays demonstrate an interaction between UIM2 of RPN10 and ATG8. Equal amounts (5 μg) of purified GST or GST-RPN10 variants (residues 201–386 bearing single and combination mutants of UIM1-3) were incubated together with His 6-ATG8e or His 6-ATG8f and pulled down with GST-Bind resin. Bound proteins were visualized by immunoblotting with anti-ATG8 or anti-GST antibodies. (E) Quantification of the binding affinity between RPN10 and ATG8. Varying concentrations of His 6-ATG8e were incubated with 1 μM RPN10(201–386) and pulled down with Ni-NTA beads. RPN10 remaining in the supernatant was quantified by SDS-PAGE and immunoblotting with anti-GST antibodies and expressed as a percentage of the RPN10 input. (F) ATG8a interacts with UIM2 of RPN10 in planta by BiFC. Tobacco leaf epidermal cells were co-infiltrated with the indicated plasmid combinations, and fluorescent signals were detected by confocal microscopy 36 hr after infiltration. Scale bar, 10 μm. See also Figure S7.
Figure 7. RPN10 Is Essential for Inhibitor-Induced, but Not Nitrogen Starvation-Induced, Proteaphagy
rpn10-1 mutation blocks autophagic transport of PAG1-GFP upon proteasome inhibition but not N starvation. WT and rpn10-1 seedlings expressing PAG1-GFP were treated as in Figure 3C, and root cells were examined for autophagic bodies by confocal fluorescence microscopy. Scale bar, 10 μm. (B and C) Release of free GFP from PAG1-GFP in N-starved or MG132-treated pag1-1 PAG1-GFP plants with or without the rpn10-1 mutation. Seedlings were treated as in Figures 2C or 3B. (B) Immunoblot detection of PAG1-GFP and free GFP in total protein extracts with anti-GFP antibodies. (C) Quantification of the free GFP/PAG1-GFP ratio following N starvation or MG132 or Lac treatment. Bars represent the mean (± SD) of three biological replicates. Letters indicate values that are statistically different from one another (p < 0.05). (D) A model for proteaphagy in Arabidopsis. The MG132-induced selective route involves initial ubiquitylation of inhibited proteasomes by one or more E3s, association of RPN10 with the ubiquitin moieties, and binding of RPN10 to ATG8-PE lining the enveloping autophagic vesicles. The N starvation-induced bulk route involves non-selective transport into the vacuole with or without the help of a hypothetical proteaphagy receptor responsive to N stress. (E) ATG8, RPN10, and ubiquitin form a tripartite complex. Equal amounts (1 μg) of purified His 6-ATG8e, either GST or GST-RPN10 harboring UIM1-3 mutations, and poly-ubiquitin chains were incubated together and pulled down with Ni-NTA beads. Input and bound proteins were visualized by immunoblotting with anti-ATG8, anti-GST, or anti-ubiquitin antibodies.
All figures (7)
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Research Support, U.S. Gov't, Non-P.H.S.
Arabidopsis / metabolism
Arabidopsis Proteins / genetics
Arabidopsis Proteins / metabolism
Autophagy-Related Protein 8 Family
Cysteine Proteinase Inhibitors / pharmacology
Green Fluorescent Proteins / genetics
Green Fluorescent Proteins / metabolism
Leupeptins / pharmacology
Microtubule-Associated Proteins / genetics
Microtubule-Associated Proteins / metabolism
Plants, Genetically Modified
Proteasome Endopeptidase Complex / genetics
Proteasome Endopeptidase Complex / metabolism
Protein Binding / drug effects
Sequence Homology, Amino Acid
Two-Hybrid System Techniques
Ubiquitination / drug effects
ATG8 protein, Arabidopsis
Autophagy-Related Protein 8 Family
Cysteine Proteinase Inhibitors
RPN10 protein, Arabidopsis
Green Fluorescent Proteins
Proteasome Endopeptidase Complex
ATP dependent 26S protease
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