The yeast dynamin-like protein, Mgm1p, functions on the mitochondrial outer membrane to mediate mitochondrial inheritance

The mdm17 mutation causes temperature-dependent defects in mitochondrial inheritance, mitochondrial morphology, and the maintenance of mitochondrial DNA in the yeast Saccharomyces cerevisiae. Defects in mitochondrial transmission to daughter buds and changes in mitochondrial morphology were apparent within 30 min after shifting cells to 37 degrees C, while loss of the mitochondrial genome occurred after 4-24 h at the elevated temperature. The mdm17 lesion mapped to MGM1, a gene encoding a dynamin-like GTPase previously implicated in mitochondrial genome maintenance, and the cloned MGM1 gene complements all of the mdm17 mutant phenotypes. Cells with an mgm1-null mutation displayed aberrant mitochondrial inheritance and morphology. A version of mgm1 mutated in a conserved residue in the putative GTP-binding site was unable to complement any of the mutant defects. It also caused aberrant mitochondrial distribution and morphology when expressed at high levels in cells that also contained a wild-type copy of the gene. Mgm1p was localized to the mitochondrial outer membrane and fractionated as a component of a high molecular weight complex. These results indicate that Mgm1p is a mitochondrial inheritance and morphology component that functions on the mitochondrial surface.     

The genome-wide screening of yeast deletion mutants to identify the genes required for tolerance to ethanol and other alcohols

A set of homozygous diploid deletion mutants of the yeast Saccharomyces cerevisiae was screened for the genes required for tolerance to aliphatic alcohols. The screen identified 137, 122 and 48 deletion mutants sensitive to ethanol, 1-propanol and 1-pentanol, respectively. A number of the genes required for ethanol tolerance were those also required for tolerance to other alcohols. Numerous mutants with defective genes encoding for vacuolar H+ -ATPase (V-ATPase) were cosensitive to these alcohols. A global screening approach of yeast deletion library mutants was useful in elucidating the mechanisms of alcohol tolerance based on different lipophilicities.     

Oxidative stress during aging of the yeast in a stationary culture and its attenuation by antioxidants

Oxidative stress during aging of Saccharomyces cerevisiae in stationary culture was documented by demonstration of progressive increase in the formation of superoxide, decrease in the content of acid‐soluble thiols and of acid‐soluble antioxidant capacity of cell extracts, and accumulation of aldehydes and protein carbonyl groups in two yeast strains and decreases in activities of antioxidant enzymes. Cells of a CuZn‐SOD (superoxide dismutase)‐1‐deficient strain showed a higher loss of viability than cells of an isogenic wild‐type strain. Cell survival was augmented, and changes in biochemical parameters were ameliorated, by addition of exogenous antioxidants (ascorbic acid, glutathione and melatonin) in both strains.     

Regeneration of NADH in a bioreactor using yeast cells immobilized in alginate fiber: I. Method and effect of reactor variables

Saccharomyces cerevisiae cells immobilized in a calcium alginate fiber reactor were used as a source of alcohol dehydrogenase for the NAD(+)-to-NADH reaction. The reaction was catalyzed by enzyme in cells on the surface of the fiber. Internal diffusional effects were present. The enzyme cell concentration was optimized by harvesting cells finally grown under anaerobic conditions. The results were expressed as an apparent reaction rate constant that was independent of NAD(+) and excess ethanol concentration, was slightly affected by flow rate above a minimum value, and increased with immobilized cell concentration in the fiber. The reaction was complete after 6 to 7 h under optimal conditions of 36 degrees C and 9.5 pH. The latter was 0.5 pH units above the free enzyme optimum, indicating that microenvironmental effects were in evidence. (c) 1993 John Wiley & Sons, Inc.     

The role of respiration, reactive oxygen species and oxidative stress in mother cell-specific ageing of yeast strains defective in the RAS signalling pathway

We show that the dominant activated allele of the yeast RAS gene, RAS2(ala18,val19), led to redox imbalance in exponential-phase cells and to excretion of almost all of the cellular glutathione into the medium when the cells reached early-stationary phase. The mitochondria of the mutant stained strongly with dihydrorhodamine 123 (DHR) and the cells displayed a very short mother cell-specific lifespan. Adding 1 mM reduced glutathione (GSH) to the medium partly restored the lifespan. The corresponding RAS2(+) rho-zero strain also displayed a short lifespan, excreted nearly all of its GSH, and stained positively with DHR. Adding 1 mM GSH completely restored the lifespan of the RAS2(+) rho-zero strain to that of the wild-type cells. The double mutant RAS2(ala18,val19) rho-zero cells showed the same lifespan as the RAS2(ala18,val19) cells, and the effect of glutathione in restoring the lifespan was the same, indicating that both mutations shorten lifespan through a similar mechanism. In the RAS2(ala18,val19) mutant strain and its rho-zero derivative we observed for the first time a strong electron spin resonance (ESR) signal characteristic of the superoxide radical anion. The mutant cells were, therefore, producing superoxide in the absence of a complete mitochondrial electron transport chain, pointing to the existence of a possible non-mitochondrial source for ROS generation. Our results indicate that oxidative stress resulting from a disturbance of redox balance can play a major role in mother cell-specific lifespan determination of yeast cells.     

Production of reactive oxygen species and loss of viability in yeast mitochondrial mutants: protective effect of Bcl-xL

The capacity of yeast cells to produce reactive oxygen species (ROS), both as a response to manipulation of mitochondrial functions and to growth conditions, was estimated and compared with the viability of the cells. The chronological ageing of yeast cells (growth to late-stationary phase) was accompanied by increased ROS accumulation and a significantly higher loss of viability in the mutants with impaired mitochondrial functions than in the parental strain. Under these conditions, the ectopic expression of mammalian Bcl-x(L), which is an anti-apoptotic protein, allowed cells to survive longer in stationary phase. The protective effect of Bcl-x(L) was more prominent in respiratory-competent cells that contained defects in mitochondrial ADP/ATP translocation, suggesting a model for Bcl-x(L) regulation of chronological ageing at the mitochondria. Yeast can also be triggered into apoptosis-like cell death, at conditions leading to the depletion of the intramitochondrial ATP pool, as a consequence of the parallel inhibition of mitochondrial respiration and ADP/ATP translocation. If respiratory-deficient (rho(0)) cells were used, no correlation between the numbers of ROS-producing cells and the viability loss in the population was observed, indicating that ROS production may be an accompanying event. The protective effect of Bcl-x(L) against death of these cells suggests a mitochondrial mechanism which is different from the antioxidant activity of Bcl-x(L).     

Mitochondrial mutations and aging: random drift is insufficient to explain the accumulation of mitochondrial deletion mutants in short‐lived animals

Mitochondrial DNA deletions accumulate over the life course in post‐mitotic cells of many species and may contribute to aging. Often a single mutant expands clonally and finally replaces the wild‐type population of a whole cell. One proposal to explain the driving force behind this accumulation states that random drift alone, without any selection advantage, is sufficient to explain the clonal accumulation of a single mutant. Existing mathematical models show that such a process might indeed work for humans. However, to be a general explanation for the clonal accumulation of mtDNA mutants, it is important to know whether random drift could also explain the accumulation process in short‐lived species like rodents. To clarify this issue, we modelled this process mathematically and performed extensive computer simulations to study how different mutation rates affect accumulation time and the resulting degree of heteroplasmy. We show that random drift works for lifespans of around 100 years, but for short‐lived animals, the resulting degree of heteroplasmy is incompatible with experimental observations.     

The migration of mitochondrial DNA fragments to the nucleus affects the chronological aging process of Saccharomyces cerevisiae

Migration of fragmented mitochondrial DNA (mtDNA) to the nucleus has been shown to occur in multiple species including yeast, plants, and mammals. Several human diseases, including Pallister–Hall syndrome and mucolipidosis, can be initiated by mtDNA insertion mutagenesis of nuclear DNA. In yeast, we demonstrated that the rate of mtDNA fragments translocating to the nucleus increases during chronological aging. The yeast chronological lifespan (CLS) is determined by the survival of nondividing cell populations. Whereas yeast strains with elevated migration rates of mtDNA fragments to the nucleus showed accelerated chronological aging, strains with decreased mtDNA transfer rates to the nucleus exhibited an extended CLS. Although one of the most popular theories of aging is the free radical theory, migration of mtDNA fragments to the nucleus may also contribute to the chronological aging process by possibly increasing nuclear genomic instability in cells with advanced age.     

Mnsod overexpression extends the yeast chronological (G(0)) life span but acts independently of Sir2p histone deacetylase to shorten the replicative life span of dividing cells

Studies in Drosophila and Caenorhabditis elegans have shown increased longevity with the increased free radical scavenging that accompanies overexpression of oxidant-scavenging enzymes. This study used yeast, another model for aging research, to probe the effects of overexpressing the major activity protecting against superoxide generated by the mitochondrial respiratory chain. Manganese superoxide dismutase (MnSOD) overexpression increased chronological life span (optimized survival of stationary (G₀) yeast over time), showing this is a survival ultimately limited by oxidative stress. In contrast, the same overexpression dramatically reduced the replicative life span of dividing cells (the number of daughter buds produced by each newly born mother cell). This reduction in the generational life span by MnSOD overexpression was greater than that generated by loss of the major redox-responsive regulator of the yeast replicative life span, NAD+-dependent Sir2p histone deacetylase. It was also independent of the latter activity. Expression of a mitochondrially targeted green fluorescent protein in the MnSOD overexpressor revealed that the old mother cells of this overexpressor, which had divided for a few generations, were defective in segregation of the mitochondrion from the mother to daughter. Mitochondrial defects are, therefore, the probable reason that MnSOD overexpression shortens replicative life span.     

A Measurable increase in oxidative damage due to reduction in superoxide detoxification fails to shorten the life span of long-lived mitochondrial mutants of Caenorhabditis elegans

SOD-1 and SOD-2 detoxify superoxide in the cytoplasm and mitochondria. We find that, although several long-lived mutants of Caenorhabditis elegans have increased SOD levels, this phenomenon does not correlate with life span or growth rate. Furthermore, although disruption of sod-1 or -2 expression produces numerous phenotypes, including increased sensitivity to paraquat and increased oxidative damage to proteins (except in daf-2 mutants), this fails to shorten the life span of these long-lived mutants. In fact, sod-1(RNAi) increases the life span of daf-2 mutants and sod-2(RNAi) that of clk-1 mutants. Our results suggest that increased superoxide detoxification and low oxidative damage are not crucial for the longevity of the mutants examined, with the possible exception of daf-2, where our results are inconclusive. These results are surprising because several of the long-lived mutants that we examined specifically affect mitochondrial electron transport, a process whose involvement in life-span determination is believed to be related to superoxide generation. We discuss the significance of our findings in light of the oxidative stress theory of aging.     

The short life span of Saccharomyces cerevisiae sgs1 and srs2 mutants is a composite of normal aging processes and mitotic arrest due to defective recombination

Evidence from many organisms indicates that the conserved RecQ helicases function in the maintenance of genomic stability. Mutation of SGS1 and WRN, which encode RecQ homologues in budding yeast and humans, respectively, results in phenotypes characteristic of premature aging. Mutation of SRS2, another DNA helicase, causes synthetic slow growth in an sgs1 background. In this work, we demonstrate that srs2 mutants have a shortened life span similar to sgs1 mutants. Further dissection of the sgs1 and srs2 survival curves reveals two distinct phenomena. A majority of sgs1 and srs2 cells stops dividing stochastically as large-budded cells. This mitotic cell cycle arrest is age independent and requires the RAD9-dependent DNA damage checkpoint. Late-generation sgs1 and srs2 cells senesce due to apparent premature aging, most likely involving the accumulation of extrachromosomal rDNA circles. Double sgs1 srs2 mutants are viable but have a high stochastic rate of terminal G2/M arrest. This arrest can be suppressed by mutations in RAD51, RAD52, and RAD57, suggesting that the cell cycle defect in sgs1 srs2 mutants results from inappropriate homologous recombination. Finally, mutation of RAD1 or RAD50 exacerbates the growth defect of sgs1 srs2 cells, indicating that sgs1 srs2 mutants may utilize single-strand annealing as an alternative repair pathway.     

The ldb1 mutant of Saccharomyces cerevisiae is defective in Pmr1p,the yeast secretory pathway/Golgi Ca(2+)/Mn(2+)-ATPase

The LDB1 gene of Saccharomyces cerevisiae was identified by complementation of the ldb1 mutant phenotype with a genomic library. We found that the ldb1 defect is complemented by PMR1 which codes for the yeast secretory pathway/Golgi Ca(2+)/Mn(2+)-ATPase. Besides that, the analysis of a null mutation of the PMR1 gene revealed a phenotype identical to that of ldb1 mutant. Thus, LDB1 must be considered a synonym of PMR1.     

The bulk of UCP3 expressed in yeast cells is incompetent for a nucleotide regulated H+ transport

The impact of uncoupling protein (UCP) 1, UCP3 and UCP3s expressed in yeast on oxidative phosphorylation, membrane potential and H⁺ transport is determined. Intracellular ATP synthesis is inhibited by UCP3, much more than by UCP1, while similar levels of UCP3 and UCP1 exist in the mitochondrial fractions. Measurements of membrane potential and H⁺ efflux in isolated mitochondria show that, different from UCP1, with UCP3 and UCP3s there is a priori a preponderant uncoupling not inhibited by GDP. The results are interpreted to show that UCP3 and UCP3s in yeast mitochondria are in a deranged state causing uncontrolled uncoupling, which does not represent their physiological function.     

The isolation and characterisation of a Saccharomyces cerevisiae gene (LIP2) involved in the attachment of lipoic acid groups to mitochondrial enzymes

Lipoic acid is an essential cofactor for a variety of mitochondrial enzymes. We have characterised a gene from Saccharomyces cerevisiae which appears to encode a protein involved in the attachment of lipoic acid groups to the pyruvate dehydrogenase and glycine decarboxylase complexes. The predicted protein product of this gene has significant identity to the lipoyl ligase B of both Escherichia coli and Kluyveromyces lactis. A strain harbouring a null allele of this S. cerevisiae gene is respiratory deficient due to inactive pyruvate dehydrogenase, and is unable to utilise glycine as a sole nitrogen source.     

The role of loop ZA and Pro371 in the function of yeast Gcn5p bromodomain revealed through molecular dynamics and experiment

Biological experiments were combined with molecular dynamics simulations to understand the importance of amino acidic residues present in the bromodomain of the yeast histone acetyltransferase Gcn5p. It was found that residue Pro371 plays an important role in the molecular recognition of the acetylated histone H4 tail by Gcn5p bromodomain. Crystallographic analysis of the complex showed that this residue does not directly interact with the histone substrate. It has been demonstrated that a double mutation Pro371Thr and Met372Ala in the Gcn5p bromodomain impairs chromatin remodeling activity. It is demonstrated here that, in this double mutant and in the fully deleted bromodomain strain, there is lower growth under amino acid deprivation conditions. By in vitro surface plasmon resonance (Biacore) experiments it is shown that the binding affinity of the double mutation to acetyl lysine 16 histone H4 peptide decreases. Molecular dynamics simulations were used to explain this loss in acetyl lysine-Gcn5p bromodomain affinity, in the double mutant. By comparing nanosecond molecular dynamics trajectories of the native as well as the single and doubly mutated bromodomain, it is concluded that the presence of Pro371 is important to the functionality of the Gcn5p bromodomain. In the simulation a point mutation involving this highly conserved residue induced an increase in the flexibility of the ZA loop, which in turn modulated the exposure of the binding pocket to the acetyl lysine. The combined double mutations (Pro371Thr-Met372Ala) not only markedly perturb the motion of the ZA loop but also destabilize the entire structure of the bromodomain.     

The effect of donor age on the in vitro life span of cultured human arterial smooth-muscle cells

Summary The number of population doublings of cultured human arterial smooth-muscle cells decreased as a function of donor age (0.5 to 82 years). Cells from older donors als showed longer latent periods for outgrowth from explants. These results extend other comprable observations with human skin fibroblasts to another cell type, and may have relevance to the pathogenesis of atherosclerosis with aging in vivo. This study was supported by a reserach program project grant (AG00299) from the National Institutes of Health.