A cytosolic network suppressing mitochondria-mediated proteostatic stress and cell death - PubMed (original) (raw)

. 2015 Aug 27;524(7566):481-4.

doi: 10.1038/nature14859. Epub 2015 Jul 20.

Affiliations

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

Xiaowen Wang et al. Nature. 2015.

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 signalling. 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, respectively. 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.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Extended Data Figure 1

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

(a) Suppression of ρ°-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 37°C.

Extended Data Figure 2

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 four days. (b) HD(25Q) forms aggregates in ρ° 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 Aco1-GFP 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) Means ± s.d. of the four experiments in (c) (P < 0.001, unpaired Student’s t test).

Extended Data Figure 3

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 global protein synthesis rate which is measured by the incorporation of 35S-Methinine after incubating at 25°C for 5 minutes. Data are means ± s.d. of three independent experiments (P < 0.005, unpaired Student’s t test).

Extended Data Figure 4

Extended Data Figure 4. Gene ontology analysis of proteins that are up-regulated in the cytosol of AAC2A128P cells

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

Extended Data Figure 5

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 pulldown 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 Supplemental Information.

Extended Data Figure 6

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 Idh1 precursor instead of its mature form. (d) Frequency of Idh2 peptides identified by mass spectrometry.

Extended Data Figure 7

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 analyzed using an antibody against protein A in the TAP-tag. Full scans of blots are available in Supplemental Information.

Extended Data Figure 8

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 three days.

Extended Data Figure 9

Extended Data Figure 9. Suppression of ρ°-lethality in atp1Δ, mgr2Δ and tom70Δ mutants by over-expression of GIS2 and protein synthesis rate in cells over-expressing 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 over-expressed from the multicopy vector pRS425. (b) In vivo protein synthesis assay. The incorporation of 35S-methionine in the wild-type cells over-expressing GIS2, NOG2 and TMA7 on a multicopy vector was measured at 25°C.

Extended Data Figure 10

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

(a) and (b) Relative steady state levels of Gis2-HA and Nog2-HA in proteasomal mutants. Data are means ± 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) and (d) Half life of Gis2-HA and Nog2-HA in the wild-type (WT) and AAC2A128P (A128P) cells. Data are means ± 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 panel) and ubiquitin (right panel). Note that the IP-purified full length Gis2-HA migrates slower than the protein in the cell lysate, likely due to posttranslational modification during immunoprecipitation. The cryptic modification is unrelated to ubiquitination, based on the lack of reactivity with the anti-ubiquitin antibody.

Fig. 1

Fig. 1. A cytosolic anti-degenerative network that suppresses mitochondria-induced cell death

(a) Schematic of multicopy suppressor screen. (b) Suppression of _AAC2A128P_-induced cell death on galactose (Gal) plus raffinose (Raf) medium. (c) The anti-degenerative network that improves cytosolic protein homeostasis and cell survival in _AAC2A128P_-expressing cells. The symbols * and ’ denote Trm9-skewed and truncated proteins, respectively. (d) Ungrouped anti-degenerative genes.

Fig. 2

Fig. 2. Comparison of cytosolic proteomes from AAC2A128P and wild-type cells

Isobaric Tag for Relative and Absolute Quantitation mass spectrometry (iTRAQ) analysis showing 43 proteins that are upregulated by >2 fold in the cytosol of AAC2A128P cells. * denotes Trm9-skewed non-mitochondrial proteins.

Fig. 3

Fig. 3. Up-regulation of Gis2 and Nog2 in response to mitochondrial damage promotes cell survival

(a) Growth inhibition by AAC2A128P expressed from the GAL10 promoter is suppressed by GIS2, NOG2 and TMA7 on the pRS425 multicopy vector at 33° and 35° but not 30°C. (b) Ribosomal profiles of wild-type (WT) and AAC2A128P (A128P) cells with or without the over-expression of NOG2. (c) Over-accumulation of Gis2-HA and Nog2-HA in response to CCCP treatment. PGK, phosphoglycerate kinase. Full scans of western blots are available in Supplemental Information.

Fig. 4

Fig. 4. Gis2 and Nog2 up-regulation in response to mitochondrial damage provides a feedback loop to suppress mPOS and promote cell survival

Activation of other pathways in the anti-degenerative network as shown in Fig. 1c and 1d may also benefit cell survival.

Comment in

Similar articles

Cited by

References

    1. Wallace DC. A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: a dawn for evolutionary medicine. Annu Rev Genet. 2005;39:359–407. - PMC - PubMed
    1. Veatch JR, McMurray MA, Nelson ZW, Gottschling DE. Mitochondrial dysfunction leads to nuclear genome instability via an iron-sulfur cluster defect. Cell. 2009;137:1247–1258. - PMC - PubMed
    1. Wang X. The expanding role of mitochondria in apoptosis. Genes Dev. 2001;15:2922–2933. - PubMed
    1. Durieux J, Wolff S, Dillin A. The cell-non-autonomous nature of electron transport chain-mediated longevity. Cell. 2011;144:79–91. - PMC - PubMed
    1. Pan Y, Schroeder EA, Ocampo A, Barrientos A, Shadel GS. Regulation of yeast chronological life span by TORC1 via adaptive mitochondrial ROS signaling. Cell Metab. 2011;13:668–678. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources