Phylogenomic Reconstruction Indicates Mitochondrial Ancestor Was an Energy Parasite

Reconstruction of mitochondrial ancestor has great impact on our understanding of the origin of mitochondria. Previous studies have largely focused on reconstructing the last common ancestor of all contemporary mitochondria (proto-mitochondria), but not on the more informative pre-mitochondria (the last common ancestor of mitochondria and their alphaproteobacterial sister clade). Using a phylogenomic approach and leveraging on the increased taxonomic sampling of alphaproteobacterial and eukaryotic genomes, we reconstructed the metabolisms of both proto-mitochondria and pre-mitochondria. Our reconstruction depicts a more streamlined proto-mitochondrion than these predicted by previous studies, and revealed several novel insights into the mitochondria-derived eukaryotic metabolisms including the lipid metabolism. Most strikingly, pre-mitochondrion was predicted to possess a plastid/parasite type of ATP/ADP translocase that imports ATP from the host, which posits pre-mitochondrion as an energy parasite that directly contrasts with the current role of mitochondria as the cell’s energy producer. In addition, pre-mitochondrion was predicted to encode a large number of flagellar genes and several cytochrome oxidases functioning under low oxygen level, strongly supporting the previous finding that the mitochondrial ancestor was likely motile and capable of oxidative phosphorylation under microoxic condition.     

Germinal proto-mitochondria from rat liver

A number of properties of the smallest (less than 0.2 μm) germinal proto-mitochondria (PRMC) from rat liver have been studied. These PRMC were obtained by filtering the light fraction of hepatic mitochondria (MC) through calibrated millipore membranes. Germinal PRMC contain in general the same proteins as MC. However, they have the reduced content of flavoproteins and zero cytochrome oxidase. Germinal PRMC, in contrast to MC, almost does not contain the “aging pigment” - lipofuscin. They have DNA; the DNA/protein ratio in them is much higher than in MC, i.e. they are poor in protein. The obtained results support the earlier assumption that MC in specialized animal cells can arise from germinal PRMC - particles smaller than 0.2 μm containing DNA. It is assumed that the DNA molecules enter to cytoplasm during degradation of old MC serves as a seed for the formation of PRMC (with the connection of nuclear DNA).     

Effects of Sclerin on Energy-linked Functions and Phospholipase Activity in Mitochondria Isolated from Rat Liver and Some Plants

Sclerin (SCL) not only elevated the respiratory control ratio and ADP/O ratio in mitochondria isolated from rat liver and some plants, but was effective in maintaining the energy-linked functions in these mitochondria during aging. There was a close relationship in the effect of SCL between the liberation of fatty acid and maintenance of the energy-linked functions in mitochondria during aging. The liberation of fatty acid was mainly due to the digestion of mitochondrial phospholipids by endogenous phospholipase. SCL had no effect on the activity of phospholipase and rather raised the level of endogenous phospholipase in mitochondria during aging at 30°C. The activity of phospholipase in mitochondria was inhibited by ATP, but stimulated by DNP. It was supposed that SCL inhibits the activity of phospholipase through ATP or high-energy intermediates which is maintained in mitochondria during aging. SCL had a protective effect on the activity of DNP-activated ATPase in mitochondria stored in the cold, and, at a very low concentration, stimulated the ATP-driven NAD reduction by mitochondria.     

Evolution of Mitochondria Reconstructed from the Energy Metabolism of Living Bacteria

The ancestors of mitochondria, or proto-mitochondria, played a crucial role in the evolution of eukaryotic cells and derived from symbiotic α-proteobacteria which merged with other microorganisms - the basis of the widely accepted endosymbiotic theory. However, the identity and relatives of proto-mitochondria remain elusive. Here we show that methylotrophic α-proteobacteria could be the closest living models for mitochondrial ancestors. We reached this conclusion after reconstructing the possible evolutionary pathways of the bioenergy systems of proto-mitochondria with a genomic survey of extant α-proteobacteria. Results obtained with complementary molecular and genetic analyses of diverse bioenergetic proteins converge in indicating the pathway stemming from methylotrophic bacteria as the most probable route of mitochondrial evolution. Contrary to other α-proteobacteria, methylotrophs show transition forms for the bioenergetic systems analysed. Our approach of focusing on these bioenergetic systems overcomes the phylogenetic impasse that has previously complicated the search for mitochondrial ancestors. Moreover, our results provide a new perspective for experimentally re-evolving mitochondria from extant bacteria and in the future produce synthetic mitochondria.     

Mitochondrial Events in the Life and Death of Animal Cells: A Brief Overview

Traditionally, mitochondria have been viewed as the powerhouse of the cell, i.e., the site of theoxidative phosphorylation machinery involved in ATP production. Consequently, much of theresearch conducted on mitochondria over the past 4 decades has focused on elucidating both thosemolecular events involved in ATP synthesis by oxidative phosphorylation and those involved inthe biogenesis of the oxidative phosphorylation machinery. While monumental achievements havebeen made, and continue to be made, in the study of these remarkable but extremely complexprocesses essential for the life of most animal cells, it has been only in recent years that a largebody of biological and biomedical scientists have come to recognize that mitochondria participatein other important processes. Two of these are cell death and aging which, not surprisingly, are relatedprocesses both involving, in part, the oxidative phosphorylation machinery. This new awareness hassparked a new and growing area of mitochondrial research, that has become of great interest to awide variety of scientists ranging from those involved in elucidating the role of mitochondria incell death and aging to those interested in either suppressing or facilitating these processes as itrelates to identifying new therapies or drugs for human disease. It is the purpose of this briefintroductory review to provide an overview of those mitochondrial events involved in the life anddeath of animal cells and to indicate how these events might relate to the human aging process.Much more is known, much remains controversial, and even more remains to be learned as indicatedin the excellent set of minireviews that follow.     

Introduction. Oxidative stress in mitochondria disorders of aging

These special issues of Biological Signals and Receptors are intended to describe mitochondrial DNA damage, oxidative stress and human diseases, including neurodegenerative and neuromuscular diseases, disorders associated with aging, and ischemia-perfusion injury. Traditionally, mitochondria have been viewed as the 'powerhouse' of the cell, i.e., the site of the oxidative phosphorylation machinery involved in adenosine triphosphate (ATP) production. Consequently, much of the research conducted on mitochondria over the past 4 decades has focused on elucidating both those molecular events involved in ATP synthesis by oxidative phosphorylation and those involved in the biogenesis of the oxidative phosphorylation machinery. While monumental achievements have been made, and continue to be made, in the study of these remarkable but extremely complex processes essential for the life of most animal cells, it has been only in recent years that a large body of biological and biomedical scientists have come to recognize that mitochondria participate in other important processes. Two of these are cell death and aging which, not surprisingly, are related processes both involving, in part, the oxidative phosphorylation machinery. This new awareness has sparked a new and growing area of mitochondrial research that has become of great interest to a wide variety of scientists ranging from those involved in elucidating the role of mitochondria in cell death and aging to those interested in either suppressing or facilitating these processes as it relates to identifying new therapies or drugs for human disease.     

Calcium-Dependent Nonspecific Permeability of the Inner Mitochondrial Membrane Is Not Induced in Mitochondria of the Yeast Endomyces magnusii

Mitochondria of the yeast Endomyces magnusii were examined for the presence of a Ca2+- and phosphate-induced permeability of the inner mitochondrial membrane (pore). For this purpose, coupled mitochondria were incubated under conditions known to induce the permeability transition pore in animal mitochondria, i.e., in the presence of high concentrations of Ca2+ and P(i), prooxidants (t-butylhydroperoxide), oxaloacetate, atractyloside (an inhibitor of ADP/ATP translocator), SH-reagents, by depletion of adenine nucleotide pools, and deenergization of the mitochondria. Large amplitude swelling, collapse of the membrane potential, and efflux of the accumulated Ca2+ were used as parameters for demonstrating pore induction. E. magnusii mitochondria were highly resistant to the above-mentioned substances. Deenergization of mitochondria or depletion of adenine nucleotide pools have no effect on low-amplitude swelling or the other parameters. Cyclosporin A, a specific inhibitor of the nonspecific permeability transition in animal mitochondria, did not affect the parameters measured. It is thus evident that E. magnusii mitochondria lack a functional Ca2+-dependent pore, or possess a pore differently regulated as compared to that of mammalian mitochondria.     

An update on the mitochondrial-DNA mutation hypothesis of cell aging.

Our electron microscopic study of aging insects and mammals suggests that metazoan senescence is linked to a gradual process of mitochondrial breakdown (and lipofuscin accumulation) in fixed postmitotic cells. This led us to propose in the early 1980s an oxyradical-mitochondrial DNA damage hypothesis, according to which metazoan aging may be caused by mutation, inactivation or loss of the mitochondrial genome (mtDNA) in irreversibly differentiated cells. This extranuclear somatic gene mutation concept of aging is in agreement with the fact that mtDNA synthesis takes place at the inner mitochondrial membrane near the sites of formation of highly reactive oxygen species and their products. Mitochondrial DNA may be unable to counteract the damage inflicted by those by-products of respiration because, in contrast to the nuclear genome, it lacks excision and recombination repair. Since mtDNA contains the structural genes for 13 hydrophobic proteins of the respiratory chain and ATP synthase as well as mitochondrial rRNAs and tRNAs, damage to this organellar genome will decrease or prevent the 'rejuvenation' of the mitochondria through the process of macromolecular turnover and organelle fission. Thus deprived of the ability to regenerate their mitochondria, the fixed postmitotic cells will sustain a decrease in the number of functional organelles, with resulting decline in ATP production. At higher levels of biological organization, this will lead to a loss in the bioenergetic capacity of cells, with concomitant decreases in ATP dependent protein synthesis and specialized physiological function, thus paving the way for age related degenerative diseases. The above concept is supported by a wealth of recent observations confirming the genomic instability of mitochondria and suggesting that animal and human aging is accompanied by mtDNA deletions and other types of injury to the mitochondrial genome. Our hypothesis of mtDNA damage is integrated with the classic concepts of Weissman and Minot in order to provide a preliminary explanation of the evolutionary roots of aging and reconcile the programed and stochastic views of metazoan senescence.     

Effects of aging and caloric restriction on mitochondrial energy production in gastrocnemius muscle and heart

Mitochondria are chronically exposed to reactive oxygen intermediates. As a result, various tissues, including skeletal muscle and heart, are characterized by an age-associated increase in reactive oxidant-induced mitochondrial DNA (mtDNA) damage. It has been postulated that these alterations may result in a decline in the content and rate of production of ATP, which may affect tissue function, contribute to the aging process, and lead to several disease states. We show that with age, ATP content and production decreased by approximately 50% in isolated rat mitochondria from the gastrocnemius muscle; however, no decline was observed in heart mitochondria. The decline observed in skeletal muscle may be a factor in the process of sarcopenia, which increases in incidence with advancing age. Lifelong caloric restriction, which prolongs maximum life span in animals, did not attenuate the age-related decline in ATP content or rate of production in skeletal muscle and had no effect on the heart. 8-Oxo-7,8-dihydro-2'-deoxyguanosine in skeletal muscle mtDNA was unaffected by aging but decreased 30% with caloric restriction, suggesting that the mechanisms that decrease oxidative stress in these tissues with caloric restriction are independent from ATP availability. The generation of reactive oxygen species, as indicated by H2O2 production in isolated mitochondria, did not change significantly with age in skeletal muscle or in the heart. Caloric restriction tended to reduce the levels of H2O2 production in the muscle but not in the heart. These data are the first to show that an age-associated decline in ATP content and rate of ATP production is tissue specific, in that it occurs in skeletal muscle but not heart, and that mitochondrial ATP production was unaltered by caloric restriction in both tissues.     

Method for measuring ATP production in isolated mitochondria: ATP production in brain and liver mitochondria of Fischer-344 rats with age and caloric restriction

The production of ATP is vital for muscle contraction, chemiosmotic homeostasis, and normal cellular function. Many studies have measured ATP content or qualitative changes in ATP production, but few have quantified ATP production in vivo in isolated mitochondria. Because of the importance of understanding the energy capacity of mitochondria in biology, physiology, cellular dysfunction, and ultimately, disease pathologies and normal aging, we modified a commercially available bioluminescent ATP determination assay for quantitatively measuring ATP content and rate of ATP production in isolated mitochondria. The bioluminescence assay is based on the reaction of ATP with recombinant firefly luciferase and its substrate luciferin. The stabilities of the reaction mixture as well as relevant ATP standards were quantified. The luminescent signals of the reaction mixture and a 0.5 microM ATP standard decreased linearly at rates of 2.16 and 1.39% decay/min, respectively. For a 25 microM ATP standard, the luminescent signal underwent a logarithmic decay, due to intrinsic deviations from the Beer-Lambert law. Moreover, to test the functionality of isolated mitochondria, they were incubated with 1 and 5 mM oligomycin, an inhibitor of oxidative phosphorylation. The rate of ATP production in the mitochondria declined by 34 and 83%, respectively. Due to the sensitivity and stability of the assay and methodology, we were able to quantitatively measure in vivo the effects of age and caloric restriction on the ATP content and production in isolated mitochondria from the brain and liver of young and old Fischer-344 rats. In both tissues, neither age nor caloric restriction had any significant effect on the ATP content or the rate of ATP production. This study introduces a highly sensitive, reproducible, and quick methodology for measuring ATP in isolated mitochondria.     

Recent advances in stucture-functional studies of mitochondrial factor B

Since the early studies on the resolution and reconstitution of the oxidative phosphorylation system from animal mitochondria, coupling factor B was recognized as an essential component of the machinery responsible for energy-driven ATP synthesis. At the phenomenological level, factor B was agreed to lie at the interface of energy transfer between the respiratory chain and the ATP synthase complex. However, biochemical characterization of the factor B polypeptide has proved difficult. It was not until 1990 that the N-terminal amino acid sequence of bovine mitochondrial factor B was reported, which followed, a decade later, by the report describing the amino acid sequence of full-length human factor B and its functional characterization. The present review summarizes the recent advances in structure-functional studies of factor B, including its recently determined crystal structure at 0.96?Å resolution. Ectopic expression of human factor B in cultured animal cells has unexpectedly revealed its role in shaping mitochondrial morphology. The supramolecular assembly of ATP synthase as dimer ribbons at highly curved apices of the mitochondrial cristae was recently suggested to optimize ATP synthesis under proton-limited conditions. We propose that the binding of the ATP synthase dimers with factor B tetramers could be a means to enhance the efficiency of the terminal step of oxidative phosphorylation in animal mitochondria.     

Effect of Ca ions on the transmembrane electric potential, synthesis and hydrolysis of ATP in brain mitochondria

The transmembrane potential (delta psi) of rabbit brain mitochondria was measured with the fluorescent dye dis--C2--5. During oxidative phosphorylation a fall in delta psi in the order of 20% was observed. In the presence of inhibitors of ATP synthesis, there was a good correlation between the fall in delta psi and the ADP-stimulated increase in respiration rate. The influence of endogenous calcium on the energetic metabolism of mitochondria was studied by measuring the changes of delta psi. An amount of 12 nmol Ca2+/mg protein cause half-inhibition of the ATP synthesis rate; 50 nmol/mg completely inhibits oxidative phosphorylation. The effect of the Ca2+ load on the ATPase activity of intact mitochondria was studied. It was found that endogenous calcium inhibits in a similar degree synthesis and hydrolysis of ATP. It was shown that both Ca ATP and Mg ATP can serve as a substrate for the mitochondrial ATPase.     

Mitochondrial complex I dysfunction in rat heart with aging: critical role of reactive oxygen species and cardiolipin

Reactive oxygen species (ROS) are considered a key factor in the heart aging process. Mitochondrial respiration is an important site of ROS generation and a potential contributor to heart functional changes with aging. We have examined the effects of aging on various parameters related to mitochondrial bioenergetics in rat heart, such as complex I activity, oxygen consumption, membrane potential, ROS production, and cardiolipin content and oxidation. A loss in complex I activity, state 3 respiration, and membrane potential was found in mitochondria with aging. The capacity of mitochondria to produce H(2)O(2) was significantly increased in aged rats. The mitochondrial content of cardiolipin, a phospholipid required for optimal activity of complex I, significantly decreased as a function of aging, whereas there was a significant increase in the level of oxidized cardiolipin. The lower complex I activity in mitochondria from aged rats could be almost completely restored to the level of young heart by exogenously added cardiolipin, but not by other phospholipids nor by peroxidized cardiolipin. It is proposed that aging causes heart mitochondrial complex I deficiency, which can be attributed to ROS-induced cardiolipin peroxidation. These results may prove useful in elucidating the mechanism underlying mitochondrial dysfunction associated with heart aging.     

Entamoeba histolytica as a model for the primitive eukaryotic cell

Entamoeba histolytica is a structurally simple eukaryote lacking mitochondria, peroxisomes and a well-developed Golgi apparatus, also in its biochemistry, it deviates substantially from the more complex eukoryotes. These features have alternatively been interpreted as archaic, ie. the ancestor of Entamoeba branched off before the primitive eukaryotic cell obtained proto-mitochondria, or as regressive, ie. Entamoeba has lost its mitochondria in the course of its adaptation to a parasitic life style. Tilly Bakker-Grunwald and Claudia W?stmann favor the first interpretation and discuss in which respects E. histolytica may serve as a model for the primitive eukaryote.     

Mitochondrial phospholipase A2 activity and mitochondrial aging

The changes in mitochondrial phospholipid metabolism and energy-linked functions have been followed as coupled mitochondria are allowed to age in isotonic sucrose at 18 degrees C. Analysis of the aging process has provided an approach for studying the structure--function relationships within the mitochondrion without adding external agents to perturb the membrane structure. The initial event observed in this process of deterioration is a loss of respiratory control which is paralleled by diminishing levels of ATP. As ATP levels decline, so do the rates of reacylation of monoacyglycerophosphorylethanolamine and fatty acid oxidation. In most cases the previously inactive phospholipase A2 (EC 3.1.1.4, phosphatide-2-acyl-hydrolase) begins rapid hydrolysis of membrane phosphatidylethanolamine as ATP levels approach zero. The final energy-linked phenomenon observed to decline is the anilinonaphthalenesulfonic acid fluorescence response. Evidence is presented which suggests strongly that the activity of the mitochondrial phospholipase A2 on endogenous phospholipids is suppressed in tightly coupled mitochondria. This suppression is temporally linked to ATP levels in the mitochondria. Furthermore, this study demonstrates that mitochondria which are only slightly damaged have the potential to effect membrane repair through reacylation of monoacyl phospholipids.     

Import of an incompletely folded precursor protein into isolated mitochondria requires an energized inner membrane, but no added ATP.

We have studied the post-translational import of incomplete precursor chains into isolated yeast mitochondria. The precursor was a fusion protein containing a mitochondrial presequence attached to mouse dihydrofolate reductase. In vitro-synthesis of the precursor was interrupted by the elongation inhibitor cycloheximide and the arrested nascent chains cosedimenting with ribosomes were released by EDTA. These incomplete chains were efficiently imported by isolated yeast mitochondria; their import resembled that of the complete precursor in requiring an energized inner membrane and a mitochondrial presequence. It differed from that of the completed precursor in its resistance to methotrexate (which only binds to correctly folded dihydrofolate reductase) and its independence of added ATP. The incomplete chains were also more sensitive to proteinase K than the completed precursor. We conclude that the incomplete chains were incompletely folded and suggest that the lack of tight folding caused import into mitochondria to become independent of added ATP. This implies that ATP may participate, directly or indirectly, in the unfolding of the precursor for its transport into mitochondria.     

Role of Mitochondria in Human Aging

Mitochondria are the major intracellular source and target sites of reactive oxygen species (ROS) that are continually generated as by-products of aerobic metabolism in animal and human cells. It has been demonstrated that mitochondrial respiratory function declines with age in various human tissues and that a defective respiratory chain results in enhanced production of ROS and free radicals in mitochondria. On the other hand, accumulating evidence now indicates that lipid peroxidation, protein modification and mitochondrial DNA (mtDNA) muutation are concurrently increased during aging. On the basis of these observations and the fact that the rate of cellular production of superoxide anions and hydrogen peroxide increases with age, it has recently been postulated that oxidative stress is a major contributory factor in the aging process. A causal relationship between oxidative modification and mutation of mtDNA, mitochondrial dysfunction and aging has emerged, although some details have remained unsolved. In this article, the role of mitochondria in the human aging process is reviewed on the basis of recent findings gathered from our and other laboratories.     

Adrenal glucocorticoids, adenine nucleotide translocation, and mitochondrial calcium accumulation.

The administration of dexamethasone to rats markedly diminished the initial rate and maximal extent of substrate-dependent calcium uptake in subsequently isolated liver mitochondria, and enhanced the release of calcium. The apparent Km for calcium transport was not altered by dexamethasone treatment and it ranged from 50 to 80 muM when an EDTA/Ca buffer system was used in the presence of magnesium, and 20 muM when an NTA/Ca buffer system without magnesium was employed. In contrast, when ATP was employed as the energy source, there was no significant difference in initial rate, Km, or the extent of calcium accumulation between mitochondria from control and dexamethasone-treated animals. Although mitochondria from dexamethasone-treated animal showed 15% less cytochrome c oxidase activity/mg of protein, overall respiratory capacity and ATP production from ADP were the same as in control mitochondria. However, mitochondria from dexamethasone-treated animals translocated ATP from inside to outside faster than those from control animals. When the ATP in the medium was depleted by glucose and hexokinase, both types of mitochondria retained essentially all the preloaded calcium until total ATP reached a critical level (7 approximately 5 mumol of ATP/mg of protein). When ATP content fell below this critical level, mitochondria released all the calcium quickly. Dexamethasone treatment increased the susceptibility of mitochondria to the depletion of ATP. These data indicate that the dexamethasone-induced decrease in maximal calcium transport and in calcium retention carrier system per se, but o an altered ability of the mitochondria to regulate intramitochondrial ATP content.     

Energy-independent protection of the oxidative phosphorylation capacity of mitochondria against anoxic damage by ATP and its non-metabolizable analogs

Preservation of the oxidative phosphorylation capacity of mitochondria by addition of ATP under anaerobic conditions was analyzed by use of non-metabolizable adenine nucleotide analogs. The capacity was well preserved in the presence of ATP and did not require the hydrolysis of ATP, since ATP analogs, such as beta, gamma-methylene adenosine triphosphate (AMPPCP), alpha, beta-methylene adenosine triphosphate (AMPCPP), and adenylyl imidodiphosphate (AMPPNP), were as effective as ATP. These analogs were incorporated into mitochondria through ATP/ADP translocase to maintain the original level of total adenine nucleotides in the mitochondria. ADP apparently had the same effect as ATP, but its effect was shown to be due to ATP generated from it by adenylate kinase in mitochondria. An analog of ADP, alpha, beta-methylene adenosine diphosphate (AMPCP), which was found to be a substrate of the translocase but not of adenylate kinase, could not replace ADP or ATP. From these results, it was concluded that the oxidative phosphorylation capacity of mitochondria was maintained by ATP, but not ADP, through a process not requiring energy.