Reduced cytosolic protein synthesis suppresses mitochondrial degeneration - PubMed (original) (raw)

Reduced cytosolic protein synthesis suppresses mitochondrial degeneration

Xiaowen Wang et al. Nat Cell Biol. 2008 Sep.

Abstract

Mitochondrial function degenerates with ageing and in ageing-related neuromuscular degenerative diseases, causing physiological decline of the cell. Factors that can delay the degenerative process are actively sought after. Here, we show that reduced cytosolic protein synthesis is a robust cellular strategy that suppresses ageing-related mitochondrial degeneration. We modelled autosomal dominant progressive external ophthalmoplegia (adPEO), an adult- or later-onset degenerative disease, by introducing the A128P mutation into the adenine nucleotide translocase Aac2p of Saccharomyces cerevisiae. The aac2(A128P) allele dominantly induces ageing-dependent mitochondrial degeneration and phenotypically tractable degenerative cell death, independently of its ADP/ATP exchange activity. Mitochondrial degeneration was suppressed by lifespan-extending nutritional interventions and by eight longevity mutations, which are all known to reduce cytosolic protein synthesis. These longevity interventions also independently suppressed ageing-related mitochondrial degeneration in the pro-ageing prohibitin mutants. The aac2(A128P) mutant has reduced mitochondrial membrane potential (delta psi(m)) and is synthetically lethal to low delta psi(m) conditions, including the loss of prohibitin. Mitochondrial degeneration was accelerated by defects in protein turnover on the inner membrane and was suppressed by cycloheximide, a specific inhibitor of cytosolic ribosomes. Reduced cytosolic protein synthesis suppressed membrane depolarization and defects in mitochondrial gene expression in aac(A128P) cells. Our finding thus establishes a link between protein homeostasis (proteostasis), cellular bioenergetics and mitochondrial maintenance during ageing.

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Conflict of interest statement

Author Information: The authors declare no competing financial interests. X.W., X.Z., B.K. and X.J.C. performed experiments; X.J.C. designed experiments, analyzed data and wrote the manuscript. All authors discussed the results and commented on the manuscript.

Figures

Figure 1

Figure 1. The aac2A128P alleles induces defect in mitochondrial biogenesis independent of ADP/ATP exchange activity or mtDNA instability

a, Projected localization of Ala128 (red, yAla128, equivalent to Ala114 in human Ant1) and Arg252, 253 and 254 (green) in yeast Aac2p on the crystal structure of bovine Ant1 in the cytosolic conformation bound by carboxyatractyloside (magenta). M, matrix; IMS, inter-membrane space. b, aac2A128P dominant-negatively inhibits cell growth on YPD medium at 25°C in a manner independent of nucleotide exchange activity. c, Cold-induced defects in Hsp60 processing and Cox2 biogenesis in _aac2A128P_-expressing cells. Pgk1, 3-phosphoglycerate kinase; Hsp60, large subunit of mitochondrial chaperonine; Hsp60-p, precursor of Hsp60; Cox2, cytochrome c oxidase subunit 2. d, mtDNA profile in cells co-expressing aac2A128P and the wild type AAC2 incubated at 30°C and 25°C. i.c., internal control for sample loading.

Figure 2

Figure 2. Aging accelerates mitochondrial degeneration and degenerative cell death

a, Flow cytometry analysis showing mitochondrial depolarization induced by increasing copy number of aac2A128P in diploid cells. The mean fluorescent intensities of the subpopulations are indicated in parentheses. b, aac2A128P induces the formation of degenerative micro-colonies (circles) on YPD medium in AAC2/aac2A128P heterozygous diploid cells. Totally 270 individual cells were analysed and representative micro-colonies from the AAC2/aac2A128P cells are circled. No micro-colony is detectable in the wild type control. c, Representative pedigree demonstrating irreversible commitment of mother cells co-expressing AAC2 and aac2A128P (boxed) in the production of daughter cells that form degenerative micro-colonies (circled) on YPD medium. Top, a full non-degenerative pedigree with 19 daughters (n=38); Bottom, a full degenerative pedigree with 18 daughters (n-38). The daughters are orderly placed from left to right and top to bottom. d, The fraction of mother cells committed to produce their first degenerative daughters at a given age. A total of 59 pedigrees were analysed, among which 29 are degenerative. e, Frequency of degenerative micro-colony formation on YPD, in function of the replicative age of wild type (WT) and mutant cells co-expressing AAC2 and aac2A128P (A128P).

Figure 3

Figure 3. Longevity interventions suppress mitochondrial degeneration

a, The cold-induced defect of _aac2A128P_-expressing cells to form viable colonies is suppressed by gpr1Δ, tor1Δ, sch9Δ, rei1Δ, rpl6BΔ, tma19Δ, rpd3Δ and rpl31AΔ, but not by fob1Δ or the introduction of an extra copy of SIR2 (2XSIR2). Three independent experiments were carried out and error bars represent standard deviations. Unpaired Student's t test: *, P<0,05; **, P<0.005; ***, P<0.0001. b, Growth defect of nascent meiotic segregants co-expressing AAC2 and aac2A128P is suppressed by rpl6BΔ at 30°C. Four representative tetrads are shown. The _aac2A128P_-expressing segregants are circled and those that co-segregated with rpl6BΔ are boxed. The plates were incubated for four days before being photographed. c, The formation of degenerative micro-colonies at 25°C by nascent meiotic segregants co-expressing AAC2 and aac2A128P (circled) is suppressed by caloric restriction (0.5% D) and by growth on minimal medium supplemented with adenine, leucine and uracil. Four representative tetrads are shown for each growth condition. d, Longevity interventions suppress the aging-dependent formation of degenerative micro-colonies. e, The short replicative lifespan of haploid cells co-expressing AAC2 and aac2A128P is suppressed by rpl6BΔ, rei1Δ and sch9Δ respectively. Wild type (wt), rpl6BΔ, rei1Δ and sch9Δ single mutants are analysed in parallel as controls. Median lifespans are indicated in parentheses.

Figure 4

Figure 4. Longevity interventions suppress aging-dependent mitochondrial degeneration in prohibitin mutants

a, Growth phenotype of four representative tetrads. Meiotic spores co-segregating aac2A128P with cyt1Δ, cox4Δ, phb1Δ and phb2Δ, but not with atp1Δ, form degenerative micro-colonies on YPD at 30°C (boxed). b, Growth phenotype of representative tetrads showing that synthetic lethality between aac2A128P and phb1Δ among the meiotic segregants is suppressed by lifespan-extending mutations. Box, aac2A128P phb1Δ double mutant. Triangle, triple mutants with gpr1Δ, tor1Δ, sch9Δ, rpl6BΔ or rei1Δ. c, Suppression of synthetic lethality between aac2A128P and phb1Δ among the meiotic segregants by lifespan-extending nutritional conditions. Boxed are aac2A128P phb1Δ double mutants. d, The sensitivity of phb1Δ cells to ethidium bromide (EB)-induced mtDNA elimination is suppressed by longevity mutations. e, Aging-dependent formation of degenerative micro-colonies in phb1Δ cells is suppressed by longevity mutations.

Figure 5

Figure 5. Modulation of _aac2A128P_-induced cell death by mitochondrial protein loading

a, (A) In vivo protein synthesis assay showing the incorporation of 35S-methionine in longevity mutants (15 min, 30°C). The standard deviations between three independent samples are shown. Unpaired Student's t test: **, P<0,05; ***, P<0.005. CYH, cycloheximide. b, Growth phenotype of representative tetrads showing the synthetic lethality between aac2A128P and yme1Δ among the meiotic segregants and the suppression of the synthetic lethality by lifespan-extending mutations and by cycloheximide (CYH, 100 ng/ml). Box, aac2A128P yme1Δ double mutant. Triangle, triple mutants with rpl6BΔ, rei1Δ or sch9Δ. c, The formation of degenerative micro-colonies at 25°C by nascent meiotic segregants co-expressing AAC2 and aac2A128P (circled) is suppressed by cycloheximide on YPD medium. d, Western-blot analysis of crude mitochondrial preparations showing that the loss of Cox2p in _aac2A128P_-expressing cells is suppressed by rpl6BΔ, sch9Δ, rei1Δ, and cycloheximide (25 ng/ml). The level of the matrix Ilv5p is an internal control for sample loading.

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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. Spinazzola A, Zeviani M. Disorders of nuclear-mitochondrial intergenomic signaling. Gene. 2005;354:162–168. - PubMed
    1. Suomalainen A, Kaukonen J. Diseases caused by nuclear genes affecting mtDNA stability. Am J Med Genet. 2001;106:53–61. - PubMed
    1. Kaukonen J, et al. Role of adenine nucleotide translocator 1 in mtDNA maintenance. Science. 2000;289:782–785. - PubMed
    1. Esposito LA, Melov S, Panov A, Cottrell BA, Wallace DC. Mitochondrial disease in mouse results in increased oxidative stress. Proc Natl Acad Sci USA. 1999;96:4820–4825. - PMC - PubMed

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