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, 114 (46), E10009-E10017

MPSR1 Is a Cytoplasmic PQC E3 Ligase for Eliminating Emergent Misfolded Proteins in Arabidopsis thaliana

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MPSR1 Is a Cytoplasmic PQC E3 Ligase for Eliminating Emergent Misfolded Proteins in Arabidopsis thaliana

Jong Hum Kim et al. Proc Natl Acad Sci U S A.

Abstract

Ubiquitin E3 ligases are crucial for eliminating misfolded proteins before they form cytotoxic aggregates that threaten cell fitness and survival. However, it remains unclear how emerging misfolded proteins in the cytoplasm can be selectively recognized and eliminated by E3 ligases in plants. We found that Misfolded Protein Sensing RING E3 ligase 1 (MPSR1) is an indispensable E3 ligase required for plant survival after protein-damaging stress. Under no stress, MPSR1 is prone to rapid degradation by the 26S proteasome, concealing its protein quality control (PQC) E3 ligase activity. Upon proteotoxic stress, MPSR1 directly senses incipient misfolded proteins and tethers ubiquitins for subsequent degradation. Furthermore, MPSR1 sustains the structural integrity of the proteasome complex at the initial stage of proteotoxic stress. Here, we suggest that the MPSR1 pathway is a constitutive mechanism for proteostasis under protein-damaging stress, as a front-line surveillance system in the cytoplasm.

Keywords: E3 ligase; PQC; proteostasis.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
MPSR1 is essential for plant survival under proteotoxic stress. (A) Comparative transcriptome analysis in response to proteotoxic stress. (Upper) The heat map illustrates the up-and-down expression profile of genes. (Lower) The induced expression levels of three selected RING E3 ligases (mock vs. AZC). (B) Phenotypic screening of transgenic plants, overexpressing seven different RING E3 ligases, under proteotoxic stress. The numbers stand for: 1, Col-0; 2, 2xflag-At1g26800 (MPSR1); 3, At1g14200-sGFP; 4, At5g55970-sGFP; 5, At5g47610-3xmyc; 6, At1g55530-3xmyc; 7, At4g23450-sGFP (AtAIRP1); 8, At5g01520-sGFP (AtAIRP2). (C) Accumulation of insoluble pUb proteins in seven transgenic plants and wild-type seedlings upon AZC treatment (100 µM). The level of actin was used as a loading control. At Right, the predicted domains of the tested RING E3 ligases are shown. (D) Relative expression of MPSR1 transcript under AZC treatment (5 mM). AtHSP17.4, a small heat-shock protein gene, and AtBip3, an ER stress-responsive gene, were used as controls. The data are the average values of three biological samples ± SD (n = 3). (E) Cartoon showing schematically the structures of transgenic genes and the position of CRISPR-mediated mutation. (F) Accumulation of insoluble pUb proteins in 35S:MPSR1-OE, 35S:MPSR1-KD, 35S:MPSR1-DN, and mpsr1-cas lines under stress or nonstress condition. (G) Levels of ubiquitinated proteins in the soluble fraction, insoluble fraction, and whole samples. Protein samples were resolved by 8% SDS/PAGE. (H) Levels of free ubiquitins in WT, 35S:MPSR1-KD, and 35S:MPSR1-OE lines under proteotoxic stress condition or nonstress condition. Protein samples were resolved by 15% SDS/PAGE. Blue arrowheads indicate free ubiquitins. Red brackets indicate ubiquitinated proteins. (I) Phenotype analysis of 35S:MPSR1-OE, 35S:MPSR1-KD, 35S:MPSR1-DN, and mpsr1-cas lines upon AZC treatment (50, 100 µM).
Fig. 2.
Fig. 2.
MPSR1 is a PQC E3 ligase that targets misfolded proteins independently of chaperones. (A) In vitro self-ubiquitination assay of MPSR1. The GST-MPSR1 protein was incubated with ubiquitination reaction buffer in the presence or absence of ATP, ubiquitin, UBA1, and UBC8 for 1 h, resolved by SDS/PAGE on an 8% gel, and detected using α-GST or α-Ub antibodies. The cartoon shows schematically the structure of wild-type and dominant negative (DN) mutant MPSR1. The inverted red arrowhead indicates the substituted base in the MPSR1 RING domain mutant. (B) Substitution of a conserved cysteine to serine at the RING domain of MPSR1 (MPSR1-DN-GFP) abolished the self-ubiquitination activity of MPSR1-GFP in vitro. (C) Yeast two-hybrid assay of MPSR1 and cytoplasmic chaperones. HSP101, HSP90.1, HSP70.1, HSP40, and HSP17.4 were not associated with MPSR1. (D) In vitro pulldown assay of Δ2GFP (artificial misfolded protein) with GST-MPSR1. Δ2GFP or GFP was incubated with GST-MPSR1 and pulled down with GST-resin. Retrieved proteins were determined with an α-GFP antibody. GST was used as a negative control. (E) Yeast two-hybrid assay using full-length and deletion constructs of MPSR1. C-terminal disordered segment of MPSR1 interacted with Δ2GFP (Left). MPSR1 did not interact with GFP (Right). (F) In vitro ubiquitination of Δ2GFP by GST-MPSR1. Ubiquitinated Δ2GFP was determined with α-GFP an antibody (Left and Middle). GFP used as a negative control (Right). Red arrowhead indicates the nonspecific aggregation of misfolded GFP. (G) In vivo coimmunoprecipitation analysis of flag-tagged MPSR1 (flag-MPSR1) and catalase using 35S:MPSR1-OE seedlings, treated with the indicated chemicals. flag-MPSR1 and associated proteins were precipitated with α-flag resin. Coprecipitated catalase and flag-MPSR1 were determined with an α-catalase antibody and an α-flag antibody, respectively (Left and Middle). Ubiquitinated catalases were immunoprecipitated with an α-catalase antibody and determined with an α-Ub antibody (Right). The blue arrowheads indicate nonubiquitinated flag-MPSR1. The red arrowheads indicate nonubiquitinated catalase. The green arrowheads indicate IgG band. The red square brackets indicate ubiquitinated proteins. (H) Subcellular colocalization of MPSR1 and CAT. 35S:MPSR1-OE seedlings were used for an immunohistochemistry assay using α-flag for MPSR1 and α-CAT for CAT. After the AZC treatment, green fluorescence from MPSR1 and red fluorescence from CAT were colocalized as speckles near the nucleus. White dotted squares show the zoom-in area. White dotted circles indicate the nucleus. The nucleus was detected with DAPI staining.
Fig. 3.
Fig. 3.
Client-induced stabilization of MPSR1 under proteotoxic stress. (A) MPSR1 self-ubiquitination was blocked by AZC treatment. In vivo coimmunoprecipitation assay using 35S:MPSR1-OE plantlets treated with AZC (5 mM), MG132 (20 µM), or AZC (5 mM)/MG132 (20 µM). Ubiquitinated and nonubiquitinated flag-MPSR1 were precipitated with α-flag resin and detected with an α-flag antibody or with an α-Ub antibody. The red square brackets indicate ubiquitinated MPSR1. Blue arrowhead indicates nonubiquitinated MPSR1. Green arrowhead indicates IgG band, which used for immunoprecipitation. (B) flag-MPSR1 and flag-MPSR1-DN expression levels in 35S:MPSR1-OE and 35S:MPSR1-DN seedlings, respectively, treated with or without AZC (5 mM) for the indicated times were determined with an α-flag antibody. RT-PCR analysis showing flag-MPSR1 and flag-MPSR1-DN transcript levels. (C) Effect of proteolysis inhibitor MG132 (10, 20 µM) or protease inhibitor mixture (PI) (0.1, 0.2×) on flag-MPSR1 degradation. RT-PCR analysis showing flag-MPSR1 transcript levels. (D) In vitro cell-free GST-MPSR1 degradation assay using Col-0 cytosolic extracts. Time-dependent GST-MPSR1 degradation (arbitrary units) is plotted versus control MBP levels. The data shown are the averages of three replicates ±SD (n = 3). (E) Transiently expressed Δ2GFP stabilizes MPSR1. GFP or Δ2GFP were expressed in protoplasts from two 35S:MPSR1-OE transgenic seedlings and their expression levels were determined with an α-GFP antibody. MPSR1 levels were determined with an α-MPSR1 antibody. The cartoon shows the structure of the GFP and Δ2GFP expression vectors. (F) Chemically induced Δ2GFP stabilizes endogenous MPSR1. The endogenous MPSR1 levels in WT and XVE:Δ2GFP transgenic plantlets treated with β-estradiol (5 and 20 µM) for a day are shown. MPSR1 levels were determined with an α-MPSR1 antibody. Induced Δ2GFP was determined with an α-GFP antibody. Equal loading of samples was determined with an α-actin antibody. (G) The relative amounts of MPSR1 were calculated by image analysis and plotted (arbitrary units) versus the level of the initial sample (F). The data shown are average values of four immunoblot analyses with ±SD (n = 4). (H) The self-ubiquitination of MPSR1 is hindered by Δ2GFP. (I) The self-ubiquitination of MPSR1 is not hindered by GFP. Ubiquitinated MPSR1 was determined with an α-GST antibody. The red square brackets indicate ubiquitinated proteins.
Fig. 4.
Fig. 4.
MPSR1 sustains the integrity of the 26S proteasome complex under proteotoxic stress. (A) In vitro proteasome activity assay using the cytosolic fraction of WT, 35S:MPSR1-OE (OE), 35S:MPSR1-KD (KD), and 35S:MPSR1-DN (DN), and short peptide Suc-LLVY-AMC as substrate. Log2 values of OE/WT, KD/WT, or DN/WT activity ratio are shown on the graph. The data shown are the averages of three independent assays ±SD (*P < 0.05, **P < 0.01 Student’s t test, n = 12). (B) The expression levels of 26S proteasomal subunits in wild-type and two transgenic plants. The 20S core complex was detected with an α-20S alpha antibody. The 19S lid and base complexes were detected with an α-RPN12 antibody and an α-RPT5 antibody, respectively. Equal loading of samples was determined with an α-actin antibody. (C) Size-fractionation analysis of the 26S proteasome using the cytosolic fractions of WT, 35S: MPSR1-OE (OE), and 35S:MPSR1-KD (KD) under nonstress and stress condition. The size-fractionation profile of the 19S lid, the base, and 20S core complex in each plant was determined with an α-RPN12, α-RPT5, and α-20S alpha antibody, respectively. (D and E) Native in-gel analysis of WT, 35S:MPSR1-OE, and 35S:MPSR1-KD to reveal the latent activity of the proteasome complexes. Proteasome activity was visualized using Suc-LLVY-AMC as substrate. The protein levels of proteasome complexes were determined with an α-RPN12 antibody. Equal loading of samples was confirmed with the levels of Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) using SDS/PAGE. (F) In vivo degradation assay of Δ2GFP. The levels of Δ2GFP in WT/XVE-Δ2GFP and 35S:MPSR1-KD/XVE:Δ2GFP transgenic plants that were treated with β-estradiol (5 µM) for the indicated times are shown. The induced levels of Δ2GFP were monitored with an α-GFP antibody. (G) Deficiency of MPSR1 results in the accumulation of insoluble pUb-catalase in vivo. AZC (5 mM)-treated WT and 35S:MPSR1-KD plantlets were fractionated into soluble extracts and insoluble pellets. The amount of catalase in each fraction was determined with an α-catalase antibody. In all tests, equal loading of samples was determined with an α-actin antibody. Red square bracket indicates unmodified CAT proteins.
Fig. 5.
Fig. 5.
Misfolded protein-mediated association between MPSR1 and the 26S proteasome. (A) Subcellular colocalization of MPSR1 and the 26S proteasome in 35S:MPSR1-OE seedlings determined by immunohistochemistry using α-flag for MPSR1 and α-RPN12 for the 26S proteasome. After AZC treatment, green fluorescence from MPSR1 and red fluorescence from RPN12 colocalized as speckles around the nucleus. The nucleus was detected with DAPI staining. (B) Subcellular colocalization of MPSR1 and RPT5 in 35S:MPSR1-OE seedlings determined by immunohistochemistry using α-flag for MPSR1 and α-RPT5 for the 26S proteasome. Under the AZC treatment only, green fluorescence from MPSR1 and red fluorescence from RPT5 granulated as speckles. White squares show the zoomed-in area. White dotted circles indicate the nucleus. The nucleus was detected with DAPI staining. (C) In vivo coimmunoprecipitation analysis of flag-MPSR1 and the 26S proteasome using 35S:MPSR1-OE and wild-type seedlings treated with AZC (5 mM). flag-MPSR1 and associated proteins were precipitated with an α-flag antibody and the presence of 26S proteasome was determined with α-RPN12, α-RPT5, and α-20S alpha antibodies. (D) In vitro pull-down assay of GST-MPSR1 and purified 26S proteasome complex. GST-MPSR1 was used as the bait and the 26S proteasome as the prey in the presence or absence of His-Δ2GFP. Retrieved proteins were determined with an α-RPT1 antibody. (E) In vitro pull-down assay of GST-MPSR1 and purified 26S proteasome complex. GST-MPSR1 was used as the bait and the 26S proteasome as the prey in the presence or absence of His-GFP. Retrieved proteins were determined with an α-RPT1 antibody. (F) In vitro pulldown assay of GST-MPSR1 and His-RPT1-3HA. GST-MPSR1 was used as the bait and His-RPT1-3HA as the prey in the presence or absence of His-Δ2GFP. Retrieved proteins were determined with an α-HA antibody.

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References

    1. Wickner S, Maurizi MR, Gottesman S. Posttranslational quality control: Folding, refolding, and degrading proteins. Science. 1999;286:1888–1893. - PubMed
    1. Goldberg AL. Protein degradation and protection against misfolded or damaged proteins. Nature. 2003;426:895–899. - PubMed
    1. McClellan AJ, Tam S, Kaganovich D, Frydman J. Protein quality control: Chaperones culling corrupt conformations. Nat Cell Biol. 2005;7:736–741. - PubMed
    1. Kim YE, Hipp MS, Bracher A, Hayer-Hartl M, Hartl FU. Molecular chaperone functions in protein folding and proteostasis. Annu Rev Biochem. 2013;82:323–355. - PubMed
    1. Rock KL, et al. Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules. Cell. 1994;78:761–771. - PubMed

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