Our key findings leading to the mPOS hypothesis
Background - More than 20 years ago, we found that mtDNA elimination, but not the disruption of a nuclear-encoded gene essential for oxidative phosphorylation, inhibits the growth of the "petite-negative" aerobic yeast Kluyveromyces lactis (Chen, X.J. and Clark-Walker, G.D.,1993, Genetics 133:517-525). This humble observation invited the hypothesis that a non-bioenergetic factor accounts for the death of cells with severe mitochondrial damage such as mtDNA loss. We subsequently found that gain-of-function mutations in the nuclear MGI (named for Mitochondrial Genome Integrity) and MEX1 (Mgi EXpresion 1) genes can suppress rho-zero-lethality. We showed that the MGI genes encode the alpha-, beta- and gamma-subunits of the mitochondrial F1-ATPase, whereas MEX1 encodes IF1, an intrinsic inhibitor of F1-ATPase (Chen, X.J. and Clark-Walker, G.D,, 1995, EMBO J. 14:3277-3286; Chen, X.J. and Clark-Walker, G.D., 1996, Genetics 144:1445-1454; Clark-Walker, G.D., 2007, FEMS Yeast Res. 7:665-674). Specific mutations in two "gearing rings" of F1-ATPase (see below), and the loss of IF1, convert the ATP-synthesizing molecular machinery into a robust cell-death suppressor, by stimulating the hydrolysis of cytosolically imported ATP in the mitochondrial matrix of rho-zero cells. Thin is turn facilitates the inversed electrogenic ATP(4-)(cytosol)/ADP(3-)(matrix) exchange and the maintenance of membrane potential. Membrane potential is critical to power mitochondrial processes including protein import. These findings raised the possibility that low membrane potential is the key factor that limits the survival of cells with mutated mtDNA. In 1996, we proposed the “two-component” model to explain why mtDNA loss, but not individual disruption of the electron transport chain or the ATP synthase, leads to the collapse of membrane potential and cell death (Clark-Walker, G.D. and Chen, X.J., 1996, Mol. Gen. Genet. 252:746-750). Inspired by these early findings, we started a long journey of investigating how low mitochondrial membrane potential and inner membrane stress cause cell death, and possibly human diseases, in a bioenergetically independent manner.
Specific mutations in two molecular rings in the
the enzyme into
a cell death suppressor
1. Development of an experimental system for "non-bioenergetic" mitochondrial stress: misfolded and clinically relevant Aac2 mutants discovered - The yeast Aac2 protein is homologous to human Ant1, involved in ATP/ADP exchange across the inner mitochondrial membrane (IMM). We found that several clinically relevant variants of Aac2 form large aggregates inside mitochondria. Similar dominant mutations in the human Ant1 protein have been previously shown by the Suomalainen and Zeviani labs to cause autosomal dominant Progressive External Ophthamoplegia (adPEO), myopathy, cardiomyopathy, and neurological symptomes including psychiatric disorder and dementia. We found that the mutant Aac2 affects the biogenesis of respiratory complexes and destabilizes mtDNA. It inhibits cell growth independent of nucleotide transport and bioenergetics. The availability of these experimental tools paved the way for the identification of cellular pathways that suppress cell death induced by primarily non-bioenergetic damage to mitochondria.
Chen, X.J. (2002) Induction of an unregulated channel by mutant nucleotide translocase suggests an explanation for human ophthalmoplegia. Human Molecular Genetics 16: 1835-1843.
Wang, X.W., Salinas, K., Zuo, X.M., Kucejova, B. and Chen, X.J. (2008) Dominant membrane uncoupling by mutant adenine nucleotide translocase in mitochondrial diseases. Hum. Mol. Genet. 17:4036-4044.
Liu, Y., Wang, X. and Chen, X.J. (2015) Misfolding of mutant adenine nucleotide translocase in yeast supports a novel mechanism of Ant1-induced muscle diseases. Molecular Biology of the Cell 26:1985-1994.
2. Proteostatic crosstalk between mitochondria and the cytosol revealed - We found that cell degeneration induced by missense mutations in Aac2 is suppressed by reducing global protein synthesis in the cytosol. This finding unraveled a proteostatic crosstalk between mitochondria and the cytosol. In this study, we also documented that Aac2-induced cellular stress is sufficient to cause the accumulation of unprocessed mitochondrial precursor proteins. Finally, we genetically captured an aging-associated trait that accelerates mitochondria-induced cell degeneration.
Wang, X.W., Zuo, X.M., Kucejova, B. and Chen, X.J. (2008) Reduced cytosolic protein synthesis suppresses mitochondrial degeneration. Nature Cell Biology 10:1090-1097.
(Recommended by Faculty of 1000 Biology: evaluations for Wang X et al Nat Cell Biol 2008 Aug 10:http://f1000biology.com/article/id/1123372/evaluation)
3. mPOS uncovered in yeast - Our genetic and biochemical (mass spectrometry) studies showed that clinically relevant mitochondrial damage and various mitochondrial stressors (e.g., mtDNA depletion, loss of protein quality control AAA proteases, etc.), with or without directly affecting the core protein import machinery, are sufficient to cause cell death by the toxic accumulation of unimported mitochondrial preproteins in the cytosol. We unambiguously demonstrated that proteostatic stress in the cytosol, rather than the loss of a mitochondria-associated cellular function, is responsible for cell death. The term mPOS (mitochondrial Precursor Overaccumulation Stress) was formally coined to describe a novel mitochondria-induced stress in the cytosol that kills cells independent of bioenergetic defects. We identified a large anti-mPOS proteostatic network in the cytosol that promotes cell survival upon mitochondrial damage.
Wang, X. and Chen, X.J. (2015) A cytosolic network suppressing mitochondria-mediated proteostatic stress and cell death. Nature 524:481-484.
(For commentaries, see News & Views -“Surviving Import Failure”, by Cole Haynes, Nature 524:419-420 (2015); Research Highlights -“Death by Cytoplasmic Accumulation”, by Mirella Bucci, Nature Chemical Biology 11:633 (2015))
4. mPOS and anti-mPOS responses in cultured human cells – We found that overexpression of mitochondrial carrier proteins is sufficient to induce the formation of giant membrane-bound aggresomes in the cytosol of HEK293T cells. The cytosolic aggresomes contain triaged mitochondrial proteins. These data directly demonstrated that mitochondrial protein import is saturable, and that the cytosol has a limited capacity in degrading unimported proteins. We also found that Ant1 overexpression in HEK293T cells induces cytosolic proteostatic adaptations that include the upregulation of EGR1, eEF1A1 and Ubiquitin C. Expression of misfolded Ant1 activates additional stress responses including the upregulation of cytosolic chaperones, the ubuquitin-proteasome system, the integrated stress response, and multiple RNA processing genes such as FUS and SFPQ that are known to be involved in neurodegenerative diseases.
Liu Y.*, Wang, W.*, Coyne, L.P.* (*equal contributions), Yang, Y., Qi, Y., Middleton F.A., and Chen, X.J. (2019) Mitochondrial carrier protein overloading and misfolding induce aggresomes and proteostatic adaptations in the cytosol. Molecular Biology of the Cell 30:1272-1284. (Featured cover and highlighted article)
5. Chronic proteostatic adaptation to mPOS causes progressive muscle atrophy - To model mPOS in vivo, we generated transgenic mice with unbalanced mitochondrial protein loading and import, by moderately overexpressing the nuclear-encoded adenine nucleotide translocase, Ant1. We found that these mice progressively lose skeletal muscle. Interestingly, Ant1 overexpression induces small heat shock proteins and aggresome-like structures in the cytosol, suggesting increased proteostatic burden (mPOS) due to accumulation of unimported mitochondrial preproteins. The transcriptome of Ant1-transgenic muscles is drastically remodeled to counteract proteostatic stress, by repressing protein synthesis and promoting proteasomal function, autophagy, and lysosomal amplification. These proteostatic adaptations collectively reduce protein content thereby reducing myofiber size and muscle mass. Thus, muscle wasting can occur as a trade-off of adaptation to mitochondria-induced proteostatic stress.
This finding could have implications for the understanding of the mechanism of muscle wasting, especially in diseases associated with Ant1 overexpression, including facioscapulohumeral dystrophy.
Muscle atrophy in Ant1-transgenic mice
Lysosomal amplification in Ant1-transgenic muscle
Wang, X., Middleton, F.A., Tawil, R. & Chen, X.J. (2021) Cytosolic Proteostatic Adaptation to Mitochondrial Stress Causes Progressive Muscle Wasting. iScience, Dec 31; 25(1):103715. doi: 10.1016/j.isci.2021.103715. eCollection 2022 Jan 21