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, 10 (1), e1004116
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E3 Ubiquitin Ligase CHIP and NBR1-mediated Selective Autophagy Protect Additively Against Proteotoxicity in Plant Stress Responses

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E3 Ubiquitin Ligase CHIP and NBR1-mediated Selective Autophagy Protect Additively Against Proteotoxicity in Plant Stress Responses

Jie Zhou et al. PLoS Genet.

Erratum in

  • PLoS Genet. 2014 Jun;10(6):e1004478

Abstract

Plant stress responses require both protective measures that reduce or restore stress-inflicted damage to cellular structures and mechanisms that efficiently remove damaged and toxic macromolecules, such as misfolded and damaged proteins. We have recently reported that NBR1, the first identified plant autophagy adaptor with a ubiquitin-association domain, plays a critical role in plant stress tolerance by targeting stress-induced, ubiquitinated protein aggregates for degradation by autophagy. Here we report a comprehensive genetic analysis of CHIP, a chaperone-associated E3 ubiquitin ligase from Arabidopsis thaliana implicated in mediating degradation of nonnative proteins by 26S proteasomes. We isolated two chip knockout mutants and discovered that they had the same phenotypes as the nbr1 mutants with compromised tolerance to heat, oxidative and salt stresses and increased accumulation of insoluble proteins under heat stress. To determine their functional interactions, we generated chip nbr1 double mutants and found them to be further compromised in stress tolerance and in clearance of stress-induced protein aggregates, indicating additive roles of CHIP and NBR1. Furthermore, stress-induced protein aggregates were still ubiquitinated in the chip mutants. Through proteomic profiling, we systemically identified heat-induced protein aggregates in the chip and nbr1 single and double mutants. These experiments revealed that highly aggregate-prone proteins such as Rubisco activase and catalases preferentially accumulated in the nbr1 mutant while a number of light-harvesting complex proteins accumulated at high levels in the chip mutant after a relatively short period of heat stress. With extended heat stress, aggregates for a large number of intracellular proteins accumulated in both chip and nbr1 mutants and, to a greater extent, in the chip nbr1 double mutant. Based on these results, we propose that CHIP and NBR1 mediate two distinct but complementary anti-proteotoxic pathways and protein's propensity to aggregate under stress conditions is one of the critical factors for pathway selection of protein degradation.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Identification and analysis of chip mutants for heat and oxidative stress tolerance.
(A) Diagram of the CHIP gene and insertion sites. (B) Transcript levels of CHIP in Col-0 wild type (WT) and chip mutants as determined using qRT-PCR. Error bars indicate SE (n = 3). (C) Assays of heat tolerance of young seedlings. Approximately 70 two-weeks old seedlings were placed in a 45°C growth chamber for 9 hours. The heat-treated plants were then moved to a 22°C growth chamber for recovery. The picture was taken at three days after the heat treatment. (D) Survival rates of heat-stressed young seedlings. Approximately 70 two-weeks seedlings were placed in a 45°C growth chamber for 9 hours. The heat-treated plants were then moved to a 22°C growth chamber for recovery. The survival rates were determined at three days after the heat treatment. Error bars indicate SE (n = 3). (E) Assays of heat tolerance of mature plants. Six-weeks old mature plants were placed in a 45°C growth chamber for 9 hours. The heat-treated plants were then moved to a 22°C growth chamber for recovery. The picture was taken at three days after the heat treatment. (F) Assays of tolerance to oxidative stress. Six-weeks old mature plants were sprayed with 20 µM methyl viologen (MV) and kept under light for two days before the picture of representative plants was taken. The experiments were repeated three times with similar results.
Figure 2
Figure 2. Response of the chip mutants to ABA (germination) and salt.
Approximately 100 seeds each for Col-0 wild type (WT) and chip mutants were sterilized and sown on 1/2× MS medium (A) or on 1/2× MS medium containing 0.5 µM ABA (B) or 150 mM NaCl (C). Greening cotyledons were scored at the indicated days after sowing. Error bars indicate SE (n = 3).
Figure 3
Figure 3. Increased accumulation of insoluble ubiquitinated proteins in the chip mutants under heat stress.
(A) Accumulation of insoluble proteins. Leaf tissues from wild-type (WT) and chip mutants collected at indicated hours (h) at 45°C for preparation of total, soluble and insoluble proteins as described in Materials and Methods. Total proteins in the starting homogenates and insoluble proteins in the last pellets were determined for the percentages of insoluble proteins to total proteins were calculated. (B) Ubiquitination of insoluble protein aggregates in the chip mutants under heat stress. Proteins from the first supernatants (S) and last pellets (P) were subjected to SDS-PAGES and probed with anti-ubiquitin monoclonal antibody. The experiment was repeated three times with similar results.
Figure 4
Figure 4. Phenotypic analysis of the chip nbr1 double mutants in heat tolerance.
(A) Assays of heat tolerance of mature plants. Six-weeks old Col-0 wild type (WT), chip-1, and nbr1-1 single mutants and chip-1 nbr1-1 double mutant plants were placed in a 45°C growth chamber for 9 hours. The heat-treated plants were then moved to a 22°C growth chamber for recovery. The picture was taken at three days after the heat treatment. (B) Assays of heat tolerance of young seedlings. Approximately 70 two-weeks old seedlings were placed in a 45°C growth chamber for 9 hours. The heat-treated plants were then moved to a 22°C growth chamber for recovery. The picture was taken at three days after the heat treatment. (C) Survival rates of heat-stressed young seedlings. Approximately 70 two-weeks seedlings were placed in a 45°C growth chamber for 9 hours. The heat-treated plants were then moved to a 22°C growth chamber for recovery. The survival rates were determined at three days after the heat treatment. Error bars indicate SE (n = 3).
Figure 5
Figure 5. Accumulation and ubiquitination of insoluble proteins in the chip, nbr1 single and double mutants under heat stress.
(A) Accumulation of insoluble proteins. Leaf tissues from wild-type (WT), chip, nbr1 single and double mutants collected at indicated hours (h) under 45°C for preparation of total, soluble and insoluble proteins as described in Materials and Methods. Total proteins in the starting homogenates and insoluble proteins in the last pellets were determined for the percentages of insoluble proteins to total proteins were calculated. (B) Ubiquitination of insoluble protein aggregates in wild type (WT), chip, nbr1 single and double mutants collected at indicated hours (h) under heat stress. Proteins from the last pellets were subjected to SDS-PAGES and probed with anti-ubiquitin monoclonal antibody. The experiment was repeated three times with similar results.
Figure 6
Figure 6. Functional categorization of heat-induced insoluble proteins.
Proteins identified from proteomic profiling of protein aggregates accumulated in heat-stressed chip and nbr1 mutants were grouped using the GO program based two gene product properties: Cellular component (A) and biological process (B).
Figure 7
Figure 7. Differential accumulation of heat-induced insoluble proteins in the chip and nbr1 mutants.
The relative abundances of the 10 proteins in the insoluble fraction of heat-stressed wild-type and mutant plants were estimated from their detected peptide numbers after normalizing them to the same amount for total protein (0.1 mg) used for isolation of insoluble proteins. Increased accumulation of an aggregated protein in the mutants after 6 (A) and 9 (B) hours of heat stress was determined by calculating the ratio of the normalized peptide number in the mutants to that in wild type. At1g29910, LHCB1; At2g34420, LHCB1B2; At2g05070, LHCB2; At2g39730, RCA; At5g01530, LHCB4; At1g20620, CAT3; At4g35090, CAT2; At3g62030, ROC4; At4g10340, LHCB5; At4g20360, RABE1B.
Figure 8
Figure 8. Changes in RCA and catalase proteins in response to heat stress.
Proteins from the first supernatants (S) and last pellets (P) isolated from wild type (WT) and chip, nbr1 and rpn1a mutants collected at indicated hours (h) under heat stress were subjected to SDS-PAGES and probed with anti-RCA (A) or anti-catalase (B) monoclonal antibody.
Figure 9
Figure 9. Changes in catalase proteins and activity in response to heat stress.
(A) Catalase proteins in wild type (WT) and chip, nbr1 double mutant collected at indicated hours (h) under heat stress. Total proteins (T), proteins from the first supernatants (S) and last pellets (P) were subjected to SDS-PAGES and probed with anti-catalase monoclonal antibody 3B6. Both a shorter (upper panel) and a longer (lower panel) exposure of the blot were shown so that the relative levels of both low and high molecular weight catalase proteins could be better visualized. (B) Catalase activity in wild type (WT) and chip, nbr1 double mutant collected at indicated hours (h) under heat stress. Total proteins (T) and proteins from the first supernatants (S) were assayed for catalase activity using a spectrophotometric procedure. Error bars indicate SE (n = 3).
Figure 10
Figure 10. Determination of accumulation of autophagosomes using GFP-ATG8a.
(A) Five-weeks old transgenic wild-type Col-0 (WT), chip-1 and atg7-2 mutant plants expressing GFP-ATG8a were treated with (45°C) or without (22°C) heat shock for indicated hours (h) and then placed at room temperature for 0.5 h. The leaves were visualized by fluorescence confocal microscopy of GFP signal. (B) Numbers of punctate GFP-ATG8a spots representing autophagosomes per 10,000 µm2 section. Means and SE were calculated from three experiments. According to Duncan's multiple range test (P = 0.05), means do not differ significantly if they are indicated with the same letter. Bar = 20 µm.
Figure 11
Figure 11. A proposed model for CHIP- and NBR1-mediated antiproteotoxic pathways.
Stress conditions such as high temperature cause generation of misfolded and damaged proteins. Chaperone-associated E3 ubiquitin ligase CHIP ubiquitinates soluble misfolded proteins that are associated with chaperone molecules and targets their degradation by the 26S proteasomes. Misfolded proteins or protein aggregates are recognized by an unknown E3 ubiquitin ligase and targeted for degradation by NBR1-mediated selective autophagy. Those soluble misfolded proteins that CHIP-mediated 26S proteasomes fail to degrade due to limited capacity will also aggregate and become targets of NBR1-mediated selective autophagy.

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This work was supported by the Natural Science Foundation of China (grant 2013C150203) and the U.S. National Science Foundation (grant IOS–0958066). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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