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A cytosolic network suppressing mitochondria-mediated proteostatic stress and cell death

Abstract

Mitochondria are multifunctional organelles whose dysfunction leads to neuromuscular degeneration and ageing. The multi-functionality poses a great challenge for understanding the mechanisms by which mitochondrial dysfunction causes specific pathologies. Among the leading mitochondrial mediators of cell death are energy depletion, free radical production, defects in iron–sulfur cluster biosynthesis, the release of pro-apoptotic and non-cell-autonomous signalling molecules, and altered stress signalling1,2,3,4,5. Here we identify a new pathway of mitochondria-mediated cell death in yeast. This pathway was named mitochondrial precursor over-accumulation stress (mPOS), and is characterized by aberrant accumulation of mitochondrial precursors in the cytosol. mPOS can be triggered by clinically relevant mitochondrial damage that is not limited to the core machineries of protein import. We also discover a large network of genes that suppress mPOS, by modulating ribosomal biogenesis, messenger RNA decapping, transcript-specific translation, protein chaperoning and turnover. In response to mPOS, several ribosome-associated proteins were upregulated, including Gis2 and Nog2, which promote cap-independent translation and inhibit the nuclear export of the 60S ribosomal subunit, respectively6,7. Gis2 and Nog2 upregulation promotes cell survival, which may be part of a feedback loop that attenuates mPOS. Our data indicate that mitochondrial dysfunction contributes directly to cytosolic proteostatic stress, and provide an explanation for the association between these two hallmarks of degenerative diseases and ageing. The results are relevant to understanding diseases (for example, spinocerebellar ataxia, amyotrophic lateral sclerosis and myotonic dystrophy) that involve mutations within the anti-degenerative network.

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Figure 1: A cytosolic anti-degenerative network that suppresses mitochondria-induced cell death.
Figure 2: Comparison of cytosolic proteomes from AAC2A128P and wild-type cells.
Figure 3: Upregulation of Gis2 and Nog2 in response to mitochondrial damage promotes cell survival.
Figure 4: Gis2 and Nog2 upregulation in response to mitochondrial damage provides a feedback loop to suppress mPOS and promote cell survival.

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Acknowledgements

We thank M. Mollapour, W. Decatur and the Kane laboratory for help in the ubiquitination and ribosomal analysis experiments, S. Hanes for critical reading of the manuscript, and S. Zhang for processing the iTRAQ data. This work was supported by the National Institutes of Health (NIH) grants R01AG023731 and R21AG047400.

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Authors and Affiliations

Authors

Contributions

X.W. performed most of the experiments described in the study. X.J.C. conceived the project, guided the experiments, constructed yeast strains and performed genetic analysis. X.J.C. and X.W. wrote the manuscript.

Corresponding author

Correspondence to Xin Jie Chen.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Proteostatic crosstalk between mitochondria and the cytosol.

a, Suppression of ρo-lethality in the yme1Δ, atp1Δ, tom70Δ and mgr2Δ mutants by anti-degenerative genes on YPD medium supplemented with ethidium bromide that eliminates mtDNA. b, Schematic depiction of the cytosolic anti-degenerative proteostatic network that suppresses mPOS. c, Synthetic growth defect between yme1Δ and the disruption of anti-degenerative genes (PBP1, RPL40A, SSB1 and POC4) and other genes affecting cytosolic proteostasis (DHH1, BLM10 and RPN4). Cells were grown on YPD medium and incubated at the indicated temperatures.

Extended Data Figure 2 Mitochondrial damage increases protein aggregation in the cytosol.

a, Synthetic lethality between mitochondrial damage and cytosolic protein misfolding. Growth of AAC2A128P but not wild-type cells is inhibited by expression of GAL10-HD(25Q) on galactose medium, whereas growth of both AAC2A128P and wild-type cells is inhibited by expression of GAL10-HD(103Q). Yeast transformants were serially diluted in water and spotted on minimal glucose or galactose medium. The plates were incubated at 25 °C for 4 days. b, HD(25Q) forms aggregates in ρo but not ρ+ cells, whereas HD(103Q) is aggregated in both types of cells. The images are representatives of 200 cells examined for each strain. c, Increased cytosolic accumulation and aggregation of green fluorescent protein (GFP)-tagged Aco1 in AAC2A128P -expressing cells. Representative images are from four independent experiments, with a total number of 2,397 and 1,608 cells examined for the wild-type and AAC2A128P strains, respectively. d, Mean ± s.d. of the four experiments in c (P < 0.001, unpaired Student’s t-test).

Extended Data Figure 3 Growth phenotype and relative protein synthesis rate of yeast cells expressing AAC2A128P .

a, Cells were grown on YPD medium at indicated temperatures for four days. b, Expression of AAC2A128P reduces the global protein synthesis rate, which is measured by the incorporation of [35S]methionine after incubating at 25 °C for 5 min. Data are mean ± s.d. of three independent experiments (P < 0.005, unpaired Student’s t-test).

Extended Data Figure 4 Gene Ontology analysis of proteins that are upregulated in the cytosol of AAC2A128P cells.

Coloured in red are those involved in ribosomal biogenesis/translation.

Extended Data Figure 5 Increased cytosolic retention and Ssb1 association of mitochondrial precursors in AAC2A128P cells.

a, Western blot showing increased retention of representative mitochondrial proteins (Aco1, Ssc1, Idh1 and Aac2) in the cytosol of AAC2A128P cells. Pgk1, the cytosolic 3-phosphoglycerate kinase, is used as a control. b, SDS–PAGE (left) and western blot (right) showing Coomassie-stained Ssb1–TAP (tandem affinity purification) pull-down products and increased association of Ssb1–TAP with the mitochondrial Aco1 and Abf2 in AAC2A128P cells, respectively. Full scans of blots and gels are available in the Supplementary Information.

Extended Data Figure 6 iTRAQ analysis showing the presence of Idh1 and Idh2 precursors in the cytosol of AAC2A128P -expressing cells.

a, Underlined are Idh1 peptides detected by mass spectrometry. The presequence of Idh1 is shown in red. The cleavage site of mitochondrial peptidase for Idh1 maturation is indicated by the red arrow. Boxed is the peptide 1 detected by mass spectrometry after trypsinization. Peptide 1 encompasses the last threonine residue of the presequence, suggesting that it is derived from the Idh1 precursor instead of its mature form. b, Frequency of Idh1 peptides identified by mass spectrometry. c, Underlined are Idh2 peptides detected by mass spectrometry. The presequences of Idh2 are shown in red. The cleavage sites of mitochondrial peptidase for Idh2 maturation are indicated by the red arrows. Boxed is the peptide 1 detected by mass spectrometry after trypsinization. Peptide 1 encompasses the last 1–2 residues of the presequence, suggesting that it is derived from the Idh2 precursor instead of its mature form. d, Frequency of Idh2 peptides identified by mass spectrometry.

Extended Data Figure 7 Western blot showing the expression levels of TAP-tagged Ssa1, Ssa2, Sse1, Sse2, Ssb1 and Ssb2 in wild-type and AAC2A128P cells.

Equal amounts of lysates from cells grown at 25 °C were analysed using an antibody against protein A in the TAP tag. Full scans of blots are available in the Supplementary Information.

Extended Data Figure 8 The heat sensitivity of the yme1Δ mutant is suppressed by one and two extra copies of SSB1 integrated into the genome.

Cells were diluted in water, spotted on YPD plates and incubated at the indicated temperatures for 3 days.

Extended Data Figure 9 Suppression of ρo-lethality in atp1Δ, mgr2Δ and tom70Δ mutants by overexpression of GIS2 and protein synthesis rate in cells overexpressing GIS2, NOG2 and TMA7.

a, Ethidium bromide (EB) sensitivity test. Yeast transformants were diluted in water and spotted on YPD with or without ethidium bromide. The plates were incubated at 30 °C for four days. GIS2 was overexpressed from the multicopy vector pRS425. b, In vivo protein synthesis assay. The incorporation of [35S]methionine in the wild-type cells overexpressing GIS2, NOG2 and TMA7 on a multicopy vector was measured at 25 °C.

Extended Data Figure 10 Stability of Gis2–HA and Nog2–HA.

a, b, Relative steady-state levels of Gis2–HA and Nog2–HA in proteasomal mutants. Data are mean ± s.d. of 3 and 5 independent experiments for Gis2–HA and Nog2–HA, respectively (*P < 0.05; **P < 0.01; unpaired Student’s t-test). c, d, Half-life of Gis2–HA and Nog2–HA in the wild-type and AAC2A128P (A128P) cells. Data are mean ± s.d. of three experiments (P < 0.05 for Gis2–HA and P = 0.15 for Nog2–HA, unpaired Student’s t-test). e, Western blot analysis showing no evidence of Gis2–HA ubiquitination in the poc4Δ, ump1Δ and rpn4Δ cells. The ump1Δ and rpn4Δ cells are temperature-sensitive because of defective proteasomal function. Cells were grown at the non-permissive temperature (37 °C) before being lysed for protein extraction and SDS–PAGE. f, Gis2–HA was immunoprecipitated from the wild-type (WT) and AAC2A128P (A128P) cells and analysed by western blot using antibodies against HA (left) and ubiquitin (right). Note that the immunoprecipitation-purified full-length Gis2–HA migrates slower than the protein in the cell lysate, probably due to posttranslational modification during immunoprecipitation. The cryptic modification is unrelated to ubiquitination, based on the lack of reactivity with the anti-ubiquitin antibody.

Supplementary information

Supplementary Information

This file contains Supplementary Tables, 1-4, a Supplementary Note, additional references and full scans for Extended Data Figures 3c, 5a, 5b and 7. (PDF 8338 kb)

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Wang, X., Chen, X. A cytosolic network suppressing mitochondria-mediated proteostatic stress and cell death. Nature 524, 481–484 (2015). https://doi.org/10.1038/nature14859

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