A yeast mutant temperature-sensitive for mitochondrial assembly is deficient in a mitochondrial protease activity that cleaves imported precursor polypeptides.

We have previously described two yeast mutants which, at elevated temperature, stop growing and accumulate precursors to several imported mitochondrial proteins. We now show that one of these mutants (mas 1) is deficient in a matrix-located protease activity which cleaves the pre-sequences from mitochondrial precursor proteins. Isolated mas 1 mitochondria catalyze oxidative phosphorylation, exhibit respiratory control and import mitochondrial precursor polypeptides, but are defective in removing transient pre-sequences from imported precursors. The phenotype of the mas 1 mutant suggests that the matrix-located processing protease is essential for growth and for mitochondrial assembly.     

Mitochondrial Changes in Ageing Caenorhabditis elegans – What Do We Learn from Superoxide Dismutase Knockouts?

One of the most popular damage accumulation theories of ageing is the mitochondrial free radical theory of ageing (mFRTA). The mFRTA proposes that ageing is due to the accumulation of unrepaired oxidative damage, in particular damage to mitochondrial DNA (mtDNA). Within the mFRTA, the "vicious cycle" theory further proposes that reactive oxygen species (ROS) promote mtDNA mutations, which then lead to a further increase in ROS production. Recently, data have been published on Caenorhabditis elegans mutants deficient in one or both forms of mitochondrial superoxide dismutase (SOD). Surprisingly, even double mutants, lacking both mitochondrial forms of SOD, show no reduction in lifespan. This has been interpreted as evidence against the mFRTA because it is assumed that these mutants suffer from significantly elevated oxidative damage to their mitochondria. Here, using a novel mtDNA damage assay in conjunction with related, well established damage and metabolic markers, we first investigate the age-dependent mitochondrial decline in a cohort of ageing wild-type nematodes, in particular testing the plausibility of the "vicious cycle" theory. We then apply the methods and insights gained from this investigation to a mutant strain for C. elegans that lacks both forms of mitochondrial SOD. While we show a clear age-dependent, linear increase in oxidative damage in WT nematodes, we find no evidence for autocatalytic damage amplification as proposed by the "vicious cycle" theory. Comparing the SOD mutants with wild-type animals, we further show that oxidative damage levels in the mtDNA of SOD mutants are not significantly different from those in wild-type animals, i.e. even the total loss of mitochondrial SOD did not significantly increase oxidative damage to mtDNA. Possible reasons for this unexpected result and some implications for the mFRTA are discussed.     

MAS6 encodes an essential inner membrane component of the yeast mitochondrial protein import pathway

To identify new components that mediate mitochondrial protein import, we analyzed mas6, an import mutant in the yeast Saccharomyces cerevisiae. mas6 mutants are temperature sensitive for viability, and accumulate mitochondrial precursor proteins at the restrictive temperature. We show that mas6 does not correspond to any of the presently identified import mutants, and we find that mitochondria isolated from mas6 mutants are defective at an early stage of the mitochondrial protein import pathway. MAS6 encodes a 23-kD protein that contains several potential membrane spanning domains, and yeast strains disrupted for MAS6 are inviable at all temperatures and on all carbon sources. The Mas6 protein is located in the mitochondrial inner membrane and cannot be extracted from the membrane by alkali treatment. Antibodies to the Mas6 protein inhibit import into isolated mitochondria, but only when the outer membrane has been disrupted by osmotic shock. Mas6p therefore represents an essential import component located in the mitochondrial inner membrane.     

Mitochondria and ageing in Drosophila

Studies in different organisms have revealed that ageing is a complex process involving a tight regulation of gene expression. Among other features, ageing organisms generally display an increased oxidative stress and a decreased mitochondrial function. The increase in oxidative stress can be attributable to reactive oxygen species, which are mainly produced by mitochondria as a by-product of energy metabolism. Consistent with these data, mitochondria have been suggested to play a significant role in lifespan determination. The fruitfly Drosophila melanogaster is a well-suited organism to study ageing as it is relatively short-lived, mainly composed of post-mitotic cells, has sequenced nuclear and mitochondrial genomes, and multiple genetic tools are available. It has been used in genome-wide studies to unveil the molecular signature of ageing, in different feeding and dietary restriction protocols and in overexpression and down-regulation studies to examine the effect of specific compounds or genes/proteins on lifespan. Here we review the various features linking mitochondria and ageing in Drosophila melanogaster.     

Context-Dependent Role of Mitochondrial Fusion-Fission in Clonal Expansion of mtDNA Mutations

The accumulation of mutant mitochondrial DNA (mtDNA) molecules in aged cells has been associated with mitochondrial dysfunction, age-related diseases and the ageing process itself. This accumulation has been shown to often occur clonally, where mutant mtDNA grow in number and overpopulate the wild-type mtDNA. However, the cell possesses quality control (QC) mechanisms that maintain mitochondrial function, in which dysfunctional mitochondria are isolated and removed by selective fusion and mitochondrial autophagy (mitophagy), respectively. The aim of this study is to elucidate the circumstances related to mitochondrial QC that allow the expansion of mutant mtDNA molecules. For the purpose of the study, we have developed a mathematical model of mitochondrial QC process by extending our previous validated model of mitochondrial turnover and fusion-fission. A global sensitivity analysis of the model suggested that the selectivity of mitophagy and fusion is the most critical QC parameter for clearing de novo mutant mtDNA molecules. We further simulated several scenarios involving perturbations of key QC parameters to gain a better understanding of their dynamic and synergistic interactions. Our model simulations showed that a higher frequency of mitochondrial fusion-fission can provide a faster clearance of mutant mtDNA, but only when mutant–rich mitochondria that are transiently created are efficiently prevented from re-fusing with other mitochondria and selectively removed. Otherwise, faster fusion-fission quickens the accumulation of mutant mtDNA. Finally, we used the insights gained from model simulations and analysis to propose a possible circumstance involving deterioration of mitochondrial QC that permits mutant mtDNA to expand with age.     

On the Origin of Mitochondrial Mutants: Evidence for Intracellular Selection of Mitochondria in the Origin of Antibiotic-Resistant Cells in Yeast

In wild-type Saccharomyces cerevisiae, erythromycin and certain other antibacterial antibiotics inhibit the formation of respiratory enzymes in mitochondria by inhibiting translation on mitochondrial ribosomes. This paper is concerned with the origin of mutant cells, resistant to erythromycin by virtue of having a homogeneous population of mutant mitochondrial DNA molecules. Such mutant cells are obtained by plating wild-type (sensitive) cells on a nonfermentable substrate plus the antibiotic. Colonies of mutant cells appear first about four days after the time of appearance of established mutant cells; new colonies continue to appear, often at a constant rate, for many days. Application of the Newcombe respreading experiment demonstrates that most or all of the mutant cells which form the resistant colonies on selective medium arise only after exposure of the population to erythromycin. It is suggested that this result is most probably due to intracellular selection for mitochondrial genomes. Resistant mitochondria arising from spontaneous mutation are postulated to be at a selective disadvantage in the absence of erythromycin; reproducing more slowly than wild-type sensitive mitochondria, they cannot easily accumulate in sufficient numbers in a cell to render it resistant as a whole. In the presence of erythromycin, resistant mitochondria can continue to reproduce while sensitive mitochondria cannot, until there is a sufficient number to make the cell resistant, i.e. to permit normal cell growth. The same phenomenon is seen with respect to chloramphenicol resistance. Intracellular selection is considered more likely than direct induction of mutation by the antibiotic, since mutant cells do not accumulate in the presence of erythromycin if the mitochondrial genome is rendered non-essential by growth on glucose or nontranslatable by chloramphenicol. Intra-cellular selection provides a mechanism for direct adaptation at the cell level, compatible with currently acceptable ideas of spontaneous mutation and selection at the organelle level.     

Toxicity of familial ALS-linked SOD1 mutants from selective recruitment to spinal mitochondria

One cause of amyotrophic lateral sclerosis (ALS) is mutation in ubiquitously expressed copper/zinc superoxide dismutase (SOD1), but the mechanism of toxicity to motor neurons is unknown. Multiple disease-causing mutants, but not wild-type SOD1, are now demonstrated to be recruited to mitochondria, but only in affected tissues. This is independent of the copper chaperone for SOD1 and dismutase activity. Highly preferential association with spinal cord mitochondria is seen in human ALS for a mutant SOD1 that accumulates only to trace cytoplasmic levels. Despite variable proportions that are successfully imported, nearly constant amounts of SOD1 mutants and covalently damaged adducts of them accumulate as apparent import intermediates and/or are tightly aggregated or crosslinked onto integral membrane components on the cytoplasmic face of those mitochondria. These findings implicate damage from action of spinal cord-specific factors that recruit mutant SOD1 to spinal mitochondria as the basis for their selective toxicity in ALS.     

Biogenesis of mitochondria 23. The biochemical and genetic characteristics of two different oligomycin resistant mutants ofSaccharomyces cerevisiae under the influence of cytoplasmic genetic modification

Two classes ofSaccharomyces cerevisiae mutants resistant to oligomycin, an inhibitor of mitochondrial membrane bound ATPase are described. Biochemical analysis shows thatin vitro the mitochondrial ATPase of both types of mutant are sensitive to oligomycin.In vivo sensitivity of the mutants to oligomycin can be demonstrated following anaerobic growth of the cells, which grossly alters the mitochondrial membrane and renders the ATPase of the mutants sensitive to oligomycin. It is concluded that the mutation to oligomycin resistance in both mutant types is phenotypically expressed as a change in the mitochondrial membrane. The intact mitochondrial membrane in the wild type cell is freely permeable to oligomycin, whereas the resistant mutant is impermeable to oligomycin; alteration of the mitochondrial membrane during isolation of the organelle or physiological modification of the membranes of the mitochondria by anaerobic growth renders the membranes permeable.These mitochondrial membrane mutants differ in their cross-reference patterns and their genetics. One is resistant to oligomycin only, and behaves like previously reported cytoplasmic mutants. The other shows cross-resistance to inhibitors of mitochondrial protein synthesis as well as to oligomycin; although the mutant appears to arise from a single step mutation its genetic properties are complex and show part-nuclear and part-cytoplasmic characteristics. The implications of the observations are discussed.     

Mitochondrial involvement in the ageing process. Facts and controversies

Mitochondria are believed to be involved in human ageing. Whilst it is clear that various mitochondrial DNA mutations do accumulate in human tissues with age, whether or not they interfere with respiratory chain function is uncertain. We question the results of previous studies which have measured respiratory chain function in human skeletal muscle with age. Whilst cytochrome c oxidase deficient fibres are a real finding in skeletal muscle, the contribution of mitochondrial DNA mutations to human ageing is still controversial. Our results show for mitochondria to be involved in ageing then it must be through a more subtle mechanism than a global decline in respiratory chain function. (Mol Cell Biochem 174: 325–328, 1997)     

Measurements of mitochondrial deficiencies in living cells by microspectrofluorometry.

Microspectrofluorometric methods were developed for detection of mitochondrial metabolites and marker molecules in living cells. After excitation in the near UV and blue spectral ranges, respiratory-deficient strains of Saccharomyces cerevisiae showed higher levels of intrinsic fluorescence than corresponding wild types. This may be attributed to an increased emission by NADH and flavin molecules of the mutants. After incubation with the mitochondrial marker rhodamine 123, there was a strong indication that an energy transfer from flavin to rhodamine molecules occurred, which was more pronounced for the respiratory-deficient yeast strains. Skin fibroblasts obtained from patients with mitochondrial diseases showed approximately the same levels of autofluorescence and energy transfer but higher variances than a control cell line. These higher variances may result from a coexistence of intact and defective mitochondria.     

Mitochondrial peptidase IMMP2L mutation causes early onset of age-associated disorders and impairs adult stem cell self-renewal

Mitochondrial reactive oxygen species (ROS) are proposed to play a central role in aging and age-associated disorders, although direct in vivo evidence is lacking. We recently generated a mouse mutant with mutated inner mitochondrial membrane peptidase 2-like (Immp2l) gene, which impairs the signal peptide sequence processing of mitochondrial proteins cytochrome c1 and glycerol phosphate dehydrogenase 2. The mitochondria from mutant mice generate elevated levels of superoxide ion and cause impaired fertility in both sexes. Here, we design experiments to examine the effects of excessive mitochondrial ROS generation on health span. We show that Immp2l mutation increases oxidative stress in multiple organs such as the brain and the kidney, although expression of superoxide dismutases in these tissues of the mutants is also increased. The mutants show multiple aging-associated phenotypes, including wasting, sarcopenia, loss of subcutaneous fat, kyphosis, and ataxia, with female mutants showing earlier onset and more severe age-associated disorders than male mutants. The loss of body weight and fat was unrelated to food intake. Adipose-derived stromal cells (ADSC) from mutant mice showed impaired proliferation capability, formed significantly less and smaller colonies in colony formation assays, although they retained adipogenic differentiation capability in vitro. This functional impairment was accompanied by increased levels of oxidative stress. Our data showed that mitochondrial ROS is the driving force of accelerated aging and suggested that ROS damage to adult stem cells could be one of the mechanisms for age-associated disorders.     

Effect on gluconeogenesis of mutants blocking two mitochondrial transport systems in the yeast Saccharomyces cerevisiae

Two mutants of Saccharomyces cerevisiae, ccr1 and tpy1, have been found to interfere with the transport of small molecules across the inner mitochondrial membrane. Both also have the effect of interfering with the synthesis of a number of cytoplasmically located enzymes involved in gluconeogenesis, even when the cells are released from glucose repression. The ccr1 mutant, defective in the transport of dicarboxylic acids across the inner membrane, represses the synthesis of gluconeogenic enzymes almost totally, but synthesis can be induced on complete medium without a carbon source. This mutant has low levels of intracellular malate under all growth conditions tested. The tpy1 mutant, defective in the transport of pyruvate across the inner membrane, shows repression of gluconeogenesis enzymes under some growth conditions, particularly high levels of ethanol in the medium. These conditions also lead to low levels of malate in the cells. Intracellular levels of malate in these mutants, and in the wild type, are correlated with the levels of gluconeogenic enzymes present. The ability of isolated mutant mitochondria to phosphorylate ADP is shown to be consistent with the interpretation that they are defective in inner membrane transport, although as yet no evidence is available that these defects are the primary lesions in the two mutants. The data are consistent with two general models. In one, the exhaustion of an extramitochondrial corepressor or introduction of a coinducer by mitochondrial activity triggers the induction of gluconeogenic enzyme synthesis. In the second, the mitochondria themselves trigger this induction, but only when the tricarboxylic acid cycle is able to operate at a high level.     

Dynamics of mitochondrial inheritance in the evolution of binary mating types and two sexes

The uniparental inheritance (UPI) of mitochondria is thought to explain the evolution of two mating types or even true sexes with anisogametes. However, the exact role of UPI is not clearly understood. Here, we develop a new model, which considers the spread of UPI mutants within a biparental inheritance (BPI) population. Our model explicitly considers mitochondrial mutation and selection in parallel with the spread of UPI mutants and self-incompatible mating types. In line with earlier work, we find that UPI improves fitness under mitochondrial mutation accumulation, selfish conflict and mitonuclear coadaptation. However, we find that as UPI increases in the population its relative fitness advantage diminishes in a frequency-dependent manner. The fitness benefits of UPI ‘leak’ into the biparentally reproducing part of the population through successive matings, limiting the spread of UPI. Critically, while this process favours some degree of UPI, it neither leads to the establishment of linked mating types nor the collapse of multiple mating types to two. Only when two mating types exist beforehand can associated UPI mutants spread to fixation under the pressure of high mitochondrial mutation rate, large mitochondrial population size and selfish mutants. Variation in these parameters could account for the range of UPI actually observed in nature, from strict UPI in some Chlamydomonas species to BPI in yeast. We conclude that UPI of mitochondria alone is unlikely to have driven the evolution of two mating types in unicellular eukaryotes.     

On the relevance of mitochondrial fusions for the accumulation of mitochondrial deletion mutants: a modelling study

The molecular mechanisms underlying the aging process are still unclear, but the clonal accumulation of mitochondrial deletion mutants is one of the prime candidates. An important question for the mitochondrial theory of aging is to discover how defective organelles might be selected at the expense of wild-type mitochondria. We propose that mitochondrial fission and fusion events are of critical importance for resolving this apparent contradiction. We show that the occurrence of fusions removes the problems associated with the idea that smaller DNA molecules accumulate because they replicate in a shorter time--the survival of the tiny (SOT) hypothesis. Furthermore, stochastic simulations of mitochondrial replication, mutation and degradation show that two important experimental findings, namely the overall low mosaic pattern of oxidative phosphorylation (OXPHOS) impaired cells in old organisms and the distribution of deletion sizes, can be reproduced and explained by this hypothesis. Finally, we make predictions that can be tested experimentally to further verify our explanation for the age-related accumulation of mitochondrial deletion mutants.     

Thread-grain transition of mitochondrial reticulum as a step of mitoptosis and apoptosis

Association of mitochondrial population to a mitochondrial reticulum is typical of many types of the healthy cells. This allows the cell to organize a united intracellular power-transmitting system. However, such an association can create some difficulties for the cell when a part of the reticulum is damaged or when mitochondria should migrate from one cell region to another. It is shown that in these cases decomposition of extended mitochondria to small roundish organelles takes place (the thread-grain transition). As an intermediate step of this process, formation of beads-like mitochondria occurs when several swollen parts of the mitochondrial filament are interconnected with thin thread-like mitochondrial structures. A hypothesis is put forward that the thread-grain transition is used as a mechanism to isolate a damaged part of the mitochondrial system from its intact parts. If the injury is not repaired, spherical mitochondrion originated from the damaged part of the reticulum is assumed to convert to a small ultracondensed and presumably dead mitochondrion (this process is called 'mitoptosis'). Then the dead mitochondrion is engulfed by an autophagosome. Sometimes, an ultracondensed mitoplast co-exists with a normal mitoplast, both of them being surrounded by a common outer mitochondrial membrane. During apoptosis, massive thread-grain transition is observed which, according to Youle et al. (S. Frank et al., Dev Cell 1: 515, 2002), is mediated by a dynamin-related protein and represents an obligatory step of the mitochondria-mediated apoptosis. We found that there is a lag phase between addition of an apoptogenic agent and the thread-grain transition. When started, the transition occurs very fast. It is also found that this event precedes complete de-energization of mitochondria and cytochrome c release to cytosol. When formed, small mitochondria migrate to (and in certain rare cases even into) the nucleus. It is suggested that small mitochondria may serve as a transportable form of organelles ('cargo boats' transporting some apoptotic proteins to their nuclear targets).     

Division versus fusion: Dnm1p and Fzo1p antagonistically regulate mitochondrial shape

In yeast, mitochondrial division and fusion are highly regulated during growth, mating and sporulation, yet the mechanisms controlling these activities are unknown. Using a novel screen, we isolated mutants in which mitochondria lose their normal structure, and instead form a large network of interconnected tubules. These mutants, which appear defective in mitochondrial division, all carried mutations in DNM1, a dynamin-related protein that localizes to mitochondria. We also isolated mutants containing numerous mitochondrial fragments. These mutants were defective in FZO1, a gene previously shown to be required for mitochondrial fusion. Surprisingly, we found that in dnm1 fzo1 double mutants, normal mitochondrial shape is restored. Induction of Dnm1p expression in dnm1 fzo1 cells caused rapid fragmentation of mitochondria. We propose that dnm1 mutants are defective in the mitochondrial division, an activity antagonistic to fusion. Our results thus suggest that mitochondrial shape is normally controlled by a balance between division and fusion which requires Dnm1p and Fzo1p, respectively.     

The mitochondrial COB region in yeast codes for apocytochrome b and is mosaic.

Mitochondrial mutants of Saccharomyces cerevisiae defective in cytochrome b were analyzed genetically and biochemically in order to elucidate the role of the mitochondrial genetic system in the biosynthesis of this cytochrome. The mutants mapped between OLI1 and OLI2 on mitochondrial DNA in a region called COB. A fine structure map of the COB region was constructed by rho- deletion mapping and recombination analysis. The combined genetic and biochemical data indicate that the COB region is mosaic and contains at least five distinct clusters of mutants, A-E, with A being closest to OLI2 and E being closest to OLI1. Clusters A, C and E are probably coding regions for apocytochrome b, whereas clusters B and D seem to be involved in as yet unknown functions. These conclusions rest on the following evidence. 1. Most mutants in clusters A, C and E have specifically lost cytochrome b. Many of them accumulate smaller mitochondrial translation products; some of these were identified as fragments of apocytochrome b by proteolytic fingerprinting. The molecular weight of these fragments depends on the map position of the mutant, increasing in the direction OLI2 leads to OLI1. The mutant closest to OLI1 accumulates an apocytochrome b which is slightly larger than that of wild type. 2. A mutant in cluster C exhibits a spectral absorption band of cytochrome b that is shifted 1.5 nm to the red. 3. Mutants in clusters B and D are pleiotropic. A majority of them are conditional and lack the absorption bands of both cytochrome b and cytochrome aa3; these mutants also fail to accumulate apocytochrome b and subunit I of cytochrome c oxidase and instead form a large number of abnormal translation products whose nature is unknown. 4. Zygotic complementation tests reveal at least two complementation groups: The first group includes all mutants in cluster B and the second group includes mutants in clusters (A + C + D + E).     

Frataxin and mitochondrial carrier proteins, Mrs3p and Mrs4p, cooperate in providing iron for heme synthesis

Frataxin is a conserved mitochondrial protein implicated in cellular iron metabolism. Deletion of the yeast frataxin homolog (YFH1) was combined with deletions of MRS3 and MRS4, mitochondrial carrier proteins implicated in iron homeostasis. As previously reported, the Deltayfh1 mutant accumulated iron in mitochondria, whereas the triple mutant (DeltaDeltaDelta) did not. When wild-type, Deltamrs3/4, Deltayfh1, and DeltaDeltaDelta strains were incubated anaerobically, all strains were devoid of heme and protected from iron and oxygen toxicity. The cultures were then shifted to air for a short time (4-5 h) or a longer time (15 h), and the evolving mutant phenotypes were analyzed (heme-dependent growth, total heme, cytochromes, heme proteins, and iron levels). A picture emerges from these data of defective heme formation in the mutants, with a markedly more severe defect in the DeltaDeltaDelta than in the individual Deltamrs3/4 or Deltayfh1 mutants (a "synthetic" defect in the genetic sense). The defect(s) in heme formation could be traced to lack of iron. Using a real time assay of heme biosynthesis, porphyrin precursor and iron were presented to permeabilized cells, and the appearance and disappearance of fluorescent porphyrins were followed. The Mrs3/4p carriers were required for rapid iron transport into mitochondria for heme synthesis, whereas there was also evidence for an alternative slower system. A different role for Yfh1p was observed under conditions of low mitochondrial iron and aerobic growth (revealed in the DeltaDeltaDelta), acting to protect bioavailable iron within mitochondria and to facilitate its use for heme synthesis.