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Structure and function of mitochondria: Their organization and disorders
Structure and Function of Mitochondria: Their Organization and Disorders Takayuki Ozawa, MD, PhD, Masashi Tanaka, MD, PhD, Hiroshi Suzuki, PhD, DMS and Morimitsu Nishikimi, MD, PhD
In this lecture, recent advances in studies on the structure and function of mitochondria were reviewed. In particular, in order to understand the etiology of mitochondrial myopathies, the mechanism of the biogenesis of the mitochondrial structure with proteins synthesized in mitochondria and in the cytoplasm was discussed; namely, how proteins encoded by mitochondriai DNA are biosynthesized, and how nuclealy encoded proteins are targeted into the appropriate compartments inside the mitochondria. Recent advances in mitochondriology have made it possible to isolate and purify the enzyme complexes and their subunits, which are involved in mitochondrial oxidative phosphorylation. Immunochemical analyses using a specific antibody against each complex or subunit enabled us to detect defects in individual subunits in mitochondria isolated from a small amount of biopsied material. Several examples of molecular defects revealed by these methods in patients with mitochondrial myopathies were presented, and the principles of their therapy are discussed on the basis of the pattern of the defect. Specific antibodies are also a powerful tool for the cloning of the human cDNAs for the subunits in the mitochondrial energy-transducing machinery. This approach will hopefUlly facilitate elucidation of the genetic defects underlying these disorders. Key words: Mitochondria, electron-transfer chain, mitochondrial cytopathy, immunochemistry, immunohistochemistry, cytochromes, enzyme deficiency. Ozawa T, Tanaka M, Suzuki H, Nishikimi M. Structure and function of mitochondria: their organization and disorders. Brain Dev 1987; 9: 76-81
Organization of Mitochondria Mitochondria are the organellae which convert the energy derived on oxidation of substrates into the high energy bond of ATP that drives vital cellular· activities. They are equipped with a number of enzyme complexes which are essential for their functions and which are organized in a compact form. Mitochondria are bound on the cytoplasm by their outer membrane. The inner space is divided by an inner membrane into two compartments: the intermembrane space and the matrix space. In order to illustrate how the specialized functions of mitochondria are reflected in their morphological features, we take the example of the oxidation of fatty acids which are selectively utilized by heart muscle. Fatty acids are first converted into acyl-CoA by acyl-CoA synthetase located in the mitochondrial outer membrane.
Then, the acyl group is transferred to carnitine by carnitine acyltransferase I, transported across the inner membrane by carnitine-acylcarnitine translocase, and then transferred again to CoA by carnitine acyltransferase II located on the inner side of the inner membrane. Thus, acyl-Co A is transported to the matrix space, and oxidized to form acetyl-CoA, which is further oxidized to CO 2 via the citric acid cycle. The hydrogen atoms liberated in this process are captured by NAD or flaYins and flow into the respiratory chain which transfers electrons finally to molecular oxygen. The redox energy harvested by the electron-transfer complexes is transmitted to ATP synthetase, which is also localized in the inner membrane. ATP thus formed is transported out, by ADP-ATP translocase, into the cytoplasm where it is utilized. This compartmentalization plays an important role in the harmonious progress of a series of reactions in the cell. Biosynthesis of Mitochondrial Enzymes
From the Department of Biomedical Chemistry, Faculty of Medicine, University of Nagoya, Nagoya. Correspondence address: Takayuki Ozawa, Professor, Department of Biomedjcal Chemistry, Faculty of Medicine, University of Nagoya,65 Tsuruma-cho, Showa-ku, Nagoya 466, Japan.
76 Brain & Development, Vol 9, No 2,1987
Recent advances in biotechnology have allowed the elucidation of the mechanism of localization of mitochondrial enzyme proteins [1]. Some of the mitochondrial proteins are encoded by their own DNAs and synthesized on mitochondrial ribosomes [2]. But most of them are encoded by nuclear DNA, synthesized on cytoplasmic ribosomes as precursors with signal presequences at their
N-terminals, and then transported into mitochondria. The presequences consist of two domains; one contains the information for intracellular targeting, that is, that which directs the precursors to mitochondria, and the other contains the information for intramitochondrial sorting, that is, that which instructs the precursors as to which compartment they should go. In each compartment, the presequences are cleaved off from the precursors by specific proteases to yield mature polypeptides. The proteins of mitochondrial or cytoplasmic origin are assembled into multi-subunit enzyme complexes. The mammalian mitochondrial genome is a circular double-stranded DNA of 16.5 kilobase pairs [2]. The two strands, the heavy (H) chain and the light (L) chain, contain structural genes for 13 subunits in four complexes participating in mitochondrial oxidative phosphorylation, and genes for two ribosomal RNAs and 22 transfer RNAs, which are all essential for the protein synthesis inside the mitochondria. The mitochondrial electron-transfer chain and oxidative phosphorylation system is schematically presented in Fig 1. Complex I (NADH-ubiquinone oxidoreductase), the main entrance to the electron-transfer chain, is composed of 25 different subunits [3] ; seven of which are encoded by the mitochondrial DNA and which form the hydrophobic shell of this enzyme [4]. Complex II (succinateubiquinone oxidoreductase), another en trance to the electron-transfer chain, consists of four different subunits, all of which are of cytoplasmic origin [5]. Complex III (ubiquinol-cytochrome c oxidoreductase), situated in the middle of the respiratory chain, is composed of ten distinct subunits [6]; one of which, the cytochrome b subunit, is of mitochondrial origin [2] . Complex IV (cytochrome c oxidase), at the terminus of the respiratory chain, is composed of seven different subunits [7]. In this complex, subunits 1 and 2, which form the binding
sites for two a-type hemes and two copper atoms [8], and subunit 3, of which the function is unknown, are synthesized in mitochondria [2]. Finally, two subunits, 6 and 8, out of the twelve in Complex V (Fo F 1 -ATPase), which is driven by the electron transfer complexes, are encoded by the mitochondrial DNA [9] . Origin of Mitochondria It is generally accepted that mitochondria originated from ancient prokaryotes, exhibiting oxidative phosphorylation activity, which started symbiotic relationships with anaerobic eukaryotes with only the glycolytic pathway as an energy-producing system. There is wide variation, among animal and plant species, in the distribution of genetic information for the subunits between the mitochondrial DNA and the nuclear DNA [9] . Compared with the mitochondrial genome of lower eukaryotes such as Neurospora [10] or Saccharomyces [11], the genetic organization of the mammalian mitochondrial DNA is compact and concise [12]. It contains genes for only 13 polypeptides, and the genes lack intron structures. It is postulated that the genes for enzymes involved in oxidative phosphorylation possessed by the ancestral prokaryotes were gradually transferred to the nuclei of the host eukaryotes, and that only the genes for the hydrophobic proteins that are inserted into the mitochondrial inner membrane from the matrix side were segregated and organized in a very condensed form. Therefore, the structure of the mitochondria observable in present-day cells has been formed through the interaction between the organellae and cells during the long period of evolution. Characteristic Findings for a Deficiency of Subunits in the Respiratory Enzymes Recently, deficiencies of enzymes in the mitochondrial electron-transfer chain have attracted the attention of a
cytosolic side
I
Matrix side
J[
m
Fig 1 Hypothetical model of mitochondrial electron transport and oxidative phosphorylation system. The dark areas represent the proteins encoded by mitochondrial DNA.
Brain & Development, Vol 9, No 2,1987 77
large number of clinicians as well as basic scientists as a cause of a group of syndromes called mitochondrial cytopathies. We have developed methods for detecting abnormalities in the subunits of respiratory chain enzymes in tissues from patients with mitochondrial cytopathies [13-18]. The characteristic findings on analysis of the patients' mitochondria by these methods can be summarized as follows. 1) A generalized but disproportionate deficiency of subunits. Since the respiratory complexes are multisubunit enzymes, one can expect that a deficiency would be limited to a single subunit of the defective enzyme. In no case, however, has a specific loss of a single subunit been found in our study. The contents of more than one subunit in the complex were decreased in all the patients studied. This is possibly due to the disturbance of the assembly of the whole complex caused by an abnormality in some subunits which serve to anchor the complex to the inner membrane, resulting in a secondary deficiency of other subunits. However, the degrees of the deficiencies of the subunits in the defective complex are not always uniform [14, 15]. In a case of Complex I deficiency [17], we observed that several subunits in the complex were more markedly deficient than the other subunits, and that some of the iron-sulfur clusters in the complex detectable on electron paramagnetic resonance spectroscopy were disproportionately decreased in the submitochondrial particles isolated from the patient's liver (Tanaka and coworkers, in preparation). 2) A frequent deficiency of Complex I and/or Complex IV. We have analyzed mitochondria from more than 20 patients with mitochondrial cytopathies. Most of the patients had a deficiency of Complex I and/or Complex IV [15, 16]. In patients with Complex N deficiency [18] , the deficiency of Complex IV subunits was sometimes accompanied by a partial deficiency of Complex I subunits (Fig 2). Conversely, in patients with Complex I deficiency, a partial deficiency of Complex N subunits
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was observed concomitantly with the deficiency of Complex I subunits [14] . In contrast, a deficiency of subunits of Complex III or Complex V is rare [15, 18]. These results indicate that in mitochondrial cytopathies Complexes I and IV are more susceptible than other enzyme complexes in the inner membrane. Since seven subunits in Complex I and three in Complex IV are encoded by mitochondrial DNA while only one subunit in Complex III and two in Complex V are of mitochondrial origin [9] , this susceptibility might be related to the number of subunits synthesized in the mitochondria. When the protein synthesis in mitochondria is generally suppressed, the complex which contains the larger number of mitochondri ally encoded subunits would be affected more severely. However, the mechanism of the selective deficiency of the two complexes remains to be elucidated. 3) Apparent tissue specificity in the deficiency of subunits of the respiratory chain. In some patients with mitochondrial cytopathies, the enzyme defect is limited to the skeletal muscle [18], and in others the defect is systemic [17]. However, the degree of the enzyme deficiency is not uniform among the tissues even in a patient with a systemic type of mitochondrial cytopathy, and the distribution of the deficiency differs from patient to patient. In a patient with Complex I deficiency, we observed that the deficiency of Complex I subunits was mild in the heart but severe in the skeletal muscle [17]. In this patient, the activity of Complex IV was also decreased, in parallel with the deficiency of the Complex IV subunits , only in the skeletal muscle and the liver. These findings indicate that the deficiency of respiratory complexes in patients with mitochondrial cytopathies exhibits apparent tissue specificity. However, this cannot be explained by a defect in some putative isozymes specific for the respective tissues. The distribution of the enzyme deficiency suggests that the defects in these patients lie in the mitochondrial genes rather than the nuclear genes.
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Fig 2 Immunoblotting of the subunits of the energy-transducing complexes in skeletal muscle mitochondria from a patient with mitochondrial myopathy. A, Complex I; B, Complex Ill; C, Complex IV; D, Complex V. Lane 1, purified complexes; lane 2, beef heart mitochondria; lane 3, normal human skeletal muscle mitochondria; lane 4, patient skeletal muscle mitochondria. Note that not only the Complex IV subunits but also several subunits in Complex I or III are deficient in the patient mitochondria.
that the progressiveness of the clinical, biochemical and histological features in patients with mitochondrial cytopathies might be due to a gradual increase in the ratio of the numbers of defective to normal mitochondria in their muscle fibers and that the proliferation of mitochondria may be strongly enhanced when the ratio exceeds a certain threshold, resulting in the formation of ragged-red fibers.
Immunohistochemical Study on Ragged-Red Fibers In order to clarify the cause of the deficiency of the subunits, we studied the distribution of the enzymes among the skeletal muscle fibers of patients with mitochondrial cytopathies by the immunohistochemical method using specific antibodies against the complexes [16]. In patients with mitochondrial myopathy, encephalopathy, lactic acidosis, stroke-like episodes (MELAS) due to a deficiency of the Complex I subunits, it was demonstrated that a large amount of immunoreactive material of Complex I was accumulated in the ragged-red fibers, which is the pathognomonic finding in this disorder and which is demonstratable by means of modified Gomori trichrome staining (Tanaka and coworkers, in preparation). The results also suggested that the deficiency of Complex I subunits became severer as the number of ragged-red fibers increased and as the immunoreactivity of the nonragged-red fibers decreased. These changes detected with the immunochemical and immunohistochemical methods could be related to the aging of the patients. The increased number of mitochondria in the ragged-red fibers is regarded as a result of enhanced proliferation of mitochondria in response to the decreased energy supply in these cells. Probably because the mitochondria in raggedred fibers are nonfunctional, these fibers are caught in a vicious cycle of proliferation of mitochondria. Actually, we observed in patients with mitochondrial myopathies that, in spite of the abnormal accumulation of immunoreactive material of the Complex IV subunits, the raggedred fibers showed only weak histochemical staining for cytochrome c oxidase activity, suggesting that the enzyme in these fibers was inactive [16]. The decrease in the immunoreactivity of the non-ragged-red fibers indicates that the number of mitochondria is not increased in these fibers, although they are probably also deficient in the enzyme. The reason why the proliferation of mitochondria is enhanced in a progressively increasing number of muscle fibers is still unknown. However, we speculate
Possible Cause of the Defect in the Respiratory Enzymes Since most of the enzyme complexes in the oxidative phosphorylation system are under the dual control of both mitochondrial and nuclear genomes, a deficiency of these complexes can be caused by mutations in structural genes in either of the genomes. Furthermore, the nuclear genes for enzymes or proteins that are directly involved in the transcription or translation of the mitochondrial genes or those for proteins that are responsible for the regulation of the expression of mitochondrial genes should also be considered as possible sites of mutation. Therefore, it is necessary to look at the pathological phenomena in mitochondrial cytopathies in the light of the disturbance of communication between the mitochondria and the nucleus in the expression of each piece of genetic information. Rational Treatment for a Deficiency of the ElectronTransfer Chain Precise diagnosis of the defective segment of the electrontransfer chain in patients with mitochondrial cytopathies by biochemical and immunochemical methods makes it possible to select the rational treatment for the defect. As shown in Fig 3, a defect in any segment of the electrontransfer chain would impair the function of the three energy coupling sites, Complexes I, III and IV, because the electron flow through all of the sites is blocked simultaneously. Administration of succinate is theoretically effective, when the defect is localized in Complex I. Kobayashi et al (in preparation) observed the disappear-
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Brain & Development, Vol 9, No 2,1987 79
Human
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ance of stroke-like episodes after the administration of succinate to a patient with ME LAS due to a deficiency of Complex I. Administration of ubiquinone (CoQ) or menadione is effective, when the defect is limited to Complex III or N. Ubiquinone, which receives electrons from NADH or succinate via Complex I or II, respectively, is bound to ubiquinone-binding proteins (CoQ-N, CoQ-S and CoQ-C). Electrons of ubiquinol are transported through Complex III and cytochrome c to Complex IV, and fmally to molecular oxygen. When ubiquinone is administered, electrons are transferred from the proteinbound ubiquinol to the free ubiquinone. The resultant free ubiquinol transfers the electrons to oxygen to yield the superoxide anion and hydrogen peroxide, which are converted into water and oxygen by scavenging enzymes such as superoxide dismutase, catalase (Cat) and glutathione peroxidase (GPO). Although the rate of the electron flow from ubiquinol to oxygen is much lower than that of the normal activity of Complexes III and IV, respectively the artificial bypass enables Complex I to transfer electrons and to drive the ATP production. Molecular Biological Approaches for Mitochondrial Respiratory Enzymes It is now possible to obtain a human gene for a component in the electron-transfer chain in the test tube. A cDNA clone for cytochrome Cl in Complex III was isolated and sequenced in this laboratory [20]. The amino acid sequence deduced from the nucleotide sequence of the human gene for cytochrome Cl showed 93% homology with that of the bovine protein determined by amino acid sequencing (Fig 4). The localization of the gene among the chromosomes is now in progress. The organization of the genes for respiratory enzymes and the 'regulatory mechanism of their expression would be elucidated using the cloned cDNAs as probes. The combination of the DNA probes for genes of cytoplaSmically synthesized subunits, e.g., cytochrome Cl, and those for the genes of mitochondrially synthesized subuniU, e.g., cytochrome b, would be a useful means of studying the mechanism of coordinated expression of the mitochondrial·and nuclear genes.
80 Brain & Development, Vol 9, No 2,1987
-
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human and bovine cytochrome c \. The amino acid sequence of bovine cytochrome c \ was cited from the results of Wakabayashi et al [J 91. Dashes represent homologous amino acids. Boxes are placed around the residues to which the heme covalently binds.
Prespective Our knowledge of the interaction between the mitochondrial and nuclear genes in their expression is still limited, although we recognize it as a prime determinant of the structure and function of mitochondria. Various lines of basic research, such as molecular cloning of subunits of the electron-transfer enzymes, immunochemical identification of the subunits and in vitro synthesis of the components in the respiratory chain, would facilitate the elucidation of the regulatory mechanisms involved in biosynthesis of the components which make up the structure of the mitochondrial inner membrane. Such progress would elucidate the etiology and the pathophysiology of mitochondrial cytopathies and establish the fundamental diagnosis and treatment for this disorder.
REFERENCES 1. Hurt EC, van Loon APGM. How proteins find mitochondria and intramitochondrial compartments. Trends Biochem Sci 1986;11 :204-7. 2. Anderson S, Bankier AT, Barrell BG, et al. Sequence and organization of the human mitochondrial genome. Nature 1981;290:457-65. 3. Hatefi Y. The mitochondrial electron transport and oxidative phosphorylation system. Ann Rev Biochem 1985;54:101569. 4. Chomyn A, Cleeter MWJ, Ragan CI, Riley M, Doolittle RF, Attardi G. URF6, last unidentified reading frame of human mtDNA, codes for an NADH dehydrogenase subunit. Science 1986;234:614-8. 5. Ohnishi T. Structure of the succinate-ubiquinone oxidoreductase (Complex II). In: Lee CP, ed. Current topics in bioenergetics. New York: Academic Press (in press). 6. Ozawa T, Tanaka M, Shim omura Y. Crystallization of cytochrome bC I complex. Proc Natl Acad Sci 1983;80:921-5. 7. Tanaka M, Ozawa T. Molecular profile of highly purified beef heart cytochrome oxidase. Biochem Int 1982;5 :67-75. 8. Winter DB, Bruyninckx WJ, Foulke FG, Grinich NP, Mason HS. Location of heme a on subunit I and II and copper on subunit II of cytochrome c oxidase. J Bioi Chem 1980;255: 11408-14. 9. Chomyn A, Mariottini P, C1eeter MWJ, et al. Functional assignment of the products of the unidentified reading
10.
11. 12. 13.
14.
frames of human mitochondrial DNA. In: Quagliariello E, Slater Ee, Palmieri F, Saccone C, Kroon AM, eds. Achievements and perspectives of mitochondrial research. Volume II: Biogenesis. Amsterdam·New York·Oxford: Elsevier Science Publishers, 1985 :259-75. Nelson MA, Macino G. Gene organization and expression in Neurospora crassa mitochondria. In: Quagliariello E, Slater EC, Palmieri F, Saccone C, Kroon AM, eds. Achievements and perspectives of mitochondrial research. Volume II: Biogenesis. Amsterdam·New York·Oxford: Elsevier Science Publishers, 1985 :293-304. Tzagoloff A. Mitochondria. New York· London: Plenum Press, 1982:293. Aitardi G. Organization and expression of mammalian mitochondrial genome: a lesson in economy. Trends Biochem Sci 1981 ;6:86-9, 100-3. Tanaka M, Nishikimi M, Suzuki H, Ozawa T, Okino E, Takahashi H. Multiple cytochrome deficiency and deteriorated mitochondrial polypeptide composition in fatal infantile mitochondrial myopathy and renal dysfunction. Biochem BiophysRes Commun 1986;137:911-6. Tanaka M, Nishikimi M, Suzuki H, et aI. Deficiency of subunits in heart mitochondrial NADH-ubiquinone oxidoreductase of a patient with mitochondrial encephalopathy
15.
16.
17.
18.
19. 20.
and cardiomyopathy. Biochem Biophys Res Commun 1986; 140:88-93. Tanaka M, Nishikimj M, Suzuki H, Ozawa T, Koga Y, Nonaka I. Partial deficiency of subunits in Complex I or IV of patients with mitochondrial myopathies. Biochem Int 1987; 14: 525-30. Tanaka M, Nishikimi M, Suzuki H, et al. Deficiency- of subunits of Complex I or IV in mitochondrial myopathies: Immunochemical and immunhistochemical study. J Inher Metab Dis (in press). Tanaka M, Nishikimi M, Suzuki H, et al. Variation in the levels of Complex I subunits among tissues in a patient with mitochondrial encephalopathy and renal dysfunction. Biochem Int 1987 ;14:735-9. Tanaka M, Nishikimi M, Ozawa T, Miyabayashi S, Tada K. Lack of subunit II of cytochrome c oxidase in a patient with mitochondrial myopathy. Ann NY Acad Sci 1987;488: 5034. Wakabayashi S, Matsubara H, Kim CH, King TE. Structural studies of bovine heart cytochrome c l . J Bioi Chem 1982; 257 :9335-44. Nishikimi M, Suzuki H, Ohta S, et al. Isolation of cDNA clone for human cytochrome cI from a Agt11 expression library. Biochem Biophys Res Commun (in press).
Brain & Development, Vol 9, No 2,1987 81
In this lecture, recent advances in studies on the structure and function of mitochondria were reviewed. In particular, in order to understand the etiology of mitochondrial myopathies, the mechanism of the biogenesis of the mitochondrial structure with proteins synthesized in mitochondria and in the cytoplasm was discussed; namely, how proteins encoded by mitochondriai DNA are biosynthesized, and how nuclealy encoded proteins are targeted into the appropriate compartments inside the mitochondria. Recent advances in mitochondriology have made it possible to isolate and purify the enzyme complexes and their subunits, which are involved in mitochondrial oxidative phosphorylation. Immunochemical analyses using a specific antibody against each complex or subunit enabled us to detect defects in individual subunits in mitochondria isolated from a small amount of biopsied material. Several examples of molecular defects revealed by these methods in patients with mitochondrial myopathies were presented, and the principles of their therapy are discussed on the basis of the pattern of the defect. Specific antibodies are also a powerful tool for the cloning of the human cDNAs for the subunits in the mitochondrial energy-transducing machinery. This approach will hopefUlly facilitate elucidation of the genetic defects underlying these disorders. Key words: Mitochondria, electron-transfer chain, mitochondrial cytopathy, immunochemistry, immunohistochemistry, cytochromes, enzyme deficiency. Ozawa T, Tanaka M, Suzuki H, Nishikimi M. Structure and function of mitochondria: their organization and disorders. Brain Dev 1987; 9: 76-81
Organization of Mitochondria Mitochondria are the organellae which convert the energy derived on oxidation of substrates into the high energy bond of ATP that drives vital cellular· activities. They are equipped with a number of enzyme complexes which are essential for their functions and which are organized in a compact form. Mitochondria are bound on the cytoplasm by their outer membrane. The inner space is divided by an inner membrane into two compartments: the intermembrane space and the matrix space. In order to illustrate how the specialized functions of mitochondria are reflected in their morphological features, we take the example of the oxidation of fatty acids which are selectively utilized by heart muscle. Fatty acids are first converted into acyl-CoA by acyl-CoA synthetase located in the mitochondrial outer membrane.
Then, the acyl group is transferred to carnitine by carnitine acyltransferase I, transported across the inner membrane by carnitine-acylcarnitine translocase, and then transferred again to CoA by carnitine acyltransferase II located on the inner side of the inner membrane. Thus, acyl-Co A is transported to the matrix space, and oxidized to form acetyl-CoA, which is further oxidized to CO 2 via the citric acid cycle. The hydrogen atoms liberated in this process are captured by NAD or flaYins and flow into the respiratory chain which transfers electrons finally to molecular oxygen. The redox energy harvested by the electron-transfer complexes is transmitted to ATP synthetase, which is also localized in the inner membrane. ATP thus formed is transported out, by ADP-ATP translocase, into the cytoplasm where it is utilized. This compartmentalization plays an important role in the harmonious progress of a series of reactions in the cell. Biosynthesis of Mitochondrial Enzymes
From the Department of Biomedical Chemistry, Faculty of Medicine, University of Nagoya, Nagoya. Correspondence address: Takayuki Ozawa, Professor, Department of Biomedjcal Chemistry, Faculty of Medicine, University of Nagoya,65 Tsuruma-cho, Showa-ku, Nagoya 466, Japan.
76 Brain & Development, Vol 9, No 2,1987
Recent advances in biotechnology have allowed the elucidation of the mechanism of localization of mitochondrial enzyme proteins [1]. Some of the mitochondrial proteins are encoded by their own DNAs and synthesized on mitochondrial ribosomes [2]. But most of them are encoded by nuclear DNA, synthesized on cytoplasmic ribosomes as precursors with signal presequences at their
N-terminals, and then transported into mitochondria. The presequences consist of two domains; one contains the information for intracellular targeting, that is, that which directs the precursors to mitochondria, and the other contains the information for intramitochondrial sorting, that is, that which instructs the precursors as to which compartment they should go. In each compartment, the presequences are cleaved off from the precursors by specific proteases to yield mature polypeptides. The proteins of mitochondrial or cytoplasmic origin are assembled into multi-subunit enzyme complexes. The mammalian mitochondrial genome is a circular double-stranded DNA of 16.5 kilobase pairs [2]. The two strands, the heavy (H) chain and the light (L) chain, contain structural genes for 13 subunits in four complexes participating in mitochondrial oxidative phosphorylation, and genes for two ribosomal RNAs and 22 transfer RNAs, which are all essential for the protein synthesis inside the mitochondria. The mitochondrial electron-transfer chain and oxidative phosphorylation system is schematically presented in Fig 1. Complex I (NADH-ubiquinone oxidoreductase), the main entrance to the electron-transfer chain, is composed of 25 different subunits [3] ; seven of which are encoded by the mitochondrial DNA and which form the hydrophobic shell of this enzyme [4]. Complex II (succinateubiquinone oxidoreductase), another en trance to the electron-transfer chain, consists of four different subunits, all of which are of cytoplasmic origin [5]. Complex III (ubiquinol-cytochrome c oxidoreductase), situated in the middle of the respiratory chain, is composed of ten distinct subunits [6]; one of which, the cytochrome b subunit, is of mitochondrial origin [2] . Complex IV (cytochrome c oxidase), at the terminus of the respiratory chain, is composed of seven different subunits [7]. In this complex, subunits 1 and 2, which form the binding
sites for two a-type hemes and two copper atoms [8], and subunit 3, of which the function is unknown, are synthesized in mitochondria [2]. Finally, two subunits, 6 and 8, out of the twelve in Complex V (Fo F 1 -ATPase), which is driven by the electron transfer complexes, are encoded by the mitochondrial DNA [9] . Origin of Mitochondria It is generally accepted that mitochondria originated from ancient prokaryotes, exhibiting oxidative phosphorylation activity, which started symbiotic relationships with anaerobic eukaryotes with only the glycolytic pathway as an energy-producing system. There is wide variation, among animal and plant species, in the distribution of genetic information for the subunits between the mitochondrial DNA and the nuclear DNA [9] . Compared with the mitochondrial genome of lower eukaryotes such as Neurospora [10] or Saccharomyces [11], the genetic organization of the mammalian mitochondrial DNA is compact and concise [12]. It contains genes for only 13 polypeptides, and the genes lack intron structures. It is postulated that the genes for enzymes involved in oxidative phosphorylation possessed by the ancestral prokaryotes were gradually transferred to the nuclei of the host eukaryotes, and that only the genes for the hydrophobic proteins that are inserted into the mitochondrial inner membrane from the matrix side were segregated and organized in a very condensed form. Therefore, the structure of the mitochondria observable in present-day cells has been formed through the interaction between the organellae and cells during the long period of evolution. Characteristic Findings for a Deficiency of Subunits in the Respiratory Enzymes Recently, deficiencies of enzymes in the mitochondrial electron-transfer chain have attracted the attention of a
cytosolic side
I
Matrix side
J[
m
Fig 1 Hypothetical model of mitochondrial electron transport and oxidative phosphorylation system. The dark areas represent the proteins encoded by mitochondrial DNA.
Brain & Development, Vol 9, No 2,1987 77
large number of clinicians as well as basic scientists as a cause of a group of syndromes called mitochondrial cytopathies. We have developed methods for detecting abnormalities in the subunits of respiratory chain enzymes in tissues from patients with mitochondrial cytopathies [13-18]. The characteristic findings on analysis of the patients' mitochondria by these methods can be summarized as follows. 1) A generalized but disproportionate deficiency of subunits. Since the respiratory complexes are multisubunit enzymes, one can expect that a deficiency would be limited to a single subunit of the defective enzyme. In no case, however, has a specific loss of a single subunit been found in our study. The contents of more than one subunit in the complex were decreased in all the patients studied. This is possibly due to the disturbance of the assembly of the whole complex caused by an abnormality in some subunits which serve to anchor the complex to the inner membrane, resulting in a secondary deficiency of other subunits. However, the degrees of the deficiencies of the subunits in the defective complex are not always uniform [14, 15]. In a case of Complex I deficiency [17], we observed that several subunits in the complex were more markedly deficient than the other subunits, and that some of the iron-sulfur clusters in the complex detectable on electron paramagnetic resonance spectroscopy were disproportionately decreased in the submitochondrial particles isolated from the patient's liver (Tanaka and coworkers, in preparation). 2) A frequent deficiency of Complex I and/or Complex IV. We have analyzed mitochondria from more than 20 patients with mitochondrial cytopathies. Most of the patients had a deficiency of Complex I and/or Complex IV [15, 16]. In patients with Complex N deficiency [18] , the deficiency of Complex IV subunits was sometimes accompanied by a partial deficiency of Complex I subunits (Fig 2). Conversely, in patients with Complex I deficiency, a partial deficiency of Complex N subunits
A
B
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2
3
4
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was observed concomitantly with the deficiency of Complex I subunits [14] . In contrast, a deficiency of subunits of Complex III or Complex V is rare [15, 18]. These results indicate that in mitochondrial cytopathies Complexes I and IV are more susceptible than other enzyme complexes in the inner membrane. Since seven subunits in Complex I and three in Complex IV are encoded by mitochondrial DNA while only one subunit in Complex III and two in Complex V are of mitochondrial origin [9] , this susceptibility might be related to the number of subunits synthesized in the mitochondria. When the protein synthesis in mitochondria is generally suppressed, the complex which contains the larger number of mitochondri ally encoded subunits would be affected more severely. However, the mechanism of the selective deficiency of the two complexes remains to be elucidated. 3) Apparent tissue specificity in the deficiency of subunits of the respiratory chain. In some patients with mitochondrial cytopathies, the enzyme defect is limited to the skeletal muscle [18], and in others the defect is systemic [17]. However, the degree of the enzyme deficiency is not uniform among the tissues even in a patient with a systemic type of mitochondrial cytopathy, and the distribution of the deficiency differs from patient to patient. In a patient with Complex I deficiency, we observed that the deficiency of Complex I subunits was mild in the heart but severe in the skeletal muscle [17]. In this patient, the activity of Complex IV was also decreased, in parallel with the deficiency of the Complex IV subunits , only in the skeletal muscle and the liver. These findings indicate that the deficiency of respiratory complexes in patients with mitochondrial cytopathies exhibits apparent tissue specificity. However, this cannot be explained by a defect in some putative isozymes specific for the respective tissues. The distribution of the enzyme deficiency suggests that the defects in these patients lie in the mitochondrial genes rather than the nuclear genes.
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78 Brain & Development, Vol 9. No 2.1987
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Fig 2 Immunoblotting of the subunits of the energy-transducing complexes in skeletal muscle mitochondria from a patient with mitochondrial myopathy. A, Complex I; B, Complex Ill; C, Complex IV; D, Complex V. Lane 1, purified complexes; lane 2, beef heart mitochondria; lane 3, normal human skeletal muscle mitochondria; lane 4, patient skeletal muscle mitochondria. Note that not only the Complex IV subunits but also several subunits in Complex I or III are deficient in the patient mitochondria.
that the progressiveness of the clinical, biochemical and histological features in patients with mitochondrial cytopathies might be due to a gradual increase in the ratio of the numbers of defective to normal mitochondria in their muscle fibers and that the proliferation of mitochondria may be strongly enhanced when the ratio exceeds a certain threshold, resulting in the formation of ragged-red fibers.
Immunohistochemical Study on Ragged-Red Fibers In order to clarify the cause of the deficiency of the subunits, we studied the distribution of the enzymes among the skeletal muscle fibers of patients with mitochondrial cytopathies by the immunohistochemical method using specific antibodies against the complexes [16]. In patients with mitochondrial myopathy, encephalopathy, lactic acidosis, stroke-like episodes (MELAS) due to a deficiency of the Complex I subunits, it was demonstrated that a large amount of immunoreactive material of Complex I was accumulated in the ragged-red fibers, which is the pathognomonic finding in this disorder and which is demonstratable by means of modified Gomori trichrome staining (Tanaka and coworkers, in preparation). The results also suggested that the deficiency of Complex I subunits became severer as the number of ragged-red fibers increased and as the immunoreactivity of the nonragged-red fibers decreased. These changes detected with the immunochemical and immunohistochemical methods could be related to the aging of the patients. The increased number of mitochondria in the ragged-red fibers is regarded as a result of enhanced proliferation of mitochondria in response to the decreased energy supply in these cells. Probably because the mitochondria in raggedred fibers are nonfunctional, these fibers are caught in a vicious cycle of proliferation of mitochondria. Actually, we observed in patients with mitochondrial myopathies that, in spite of the abnormal accumulation of immunoreactive material of the Complex IV subunits, the raggedred fibers showed only weak histochemical staining for cytochrome c oxidase activity, suggesting that the enzyme in these fibers was inactive [16]. The decrease in the immunoreactivity of the non-ragged-red fibers indicates that the number of mitochondria is not increased in these fibers, although they are probably also deficient in the enzyme. The reason why the proliferation of mitochondria is enhanced in a progressively increasing number of muscle fibers is still unknown. However, we speculate
Possible Cause of the Defect in the Respiratory Enzymes Since most of the enzyme complexes in the oxidative phosphorylation system are under the dual control of both mitochondrial and nuclear genomes, a deficiency of these complexes can be caused by mutations in structural genes in either of the genomes. Furthermore, the nuclear genes for enzymes or proteins that are directly involved in the transcription or translation of the mitochondrial genes or those for proteins that are responsible for the regulation of the expression of mitochondrial genes should also be considered as possible sites of mutation. Therefore, it is necessary to look at the pathological phenomena in mitochondrial cytopathies in the light of the disturbance of communication between the mitochondria and the nucleus in the expression of each piece of genetic information. Rational Treatment for a Deficiency of the ElectronTransfer Chain Precise diagnosis of the defective segment of the electrontransfer chain in patients with mitochondrial cytopathies by biochemical and immunochemical methods makes it possible to select the rational treatment for the defect. As shown in Fig 3, a defect in any segment of the electrontransfer chain would impair the function of the three energy coupling sites, Complexes I, III and IV, because the electron flow through all of the sites is blocked simultaneously. Administration of succinate is theoretically effective, when the defect is localized in Complex I. Kobayashi et al (in preparation) observed the disappear-
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Fig 3 Schematic representation of the components in the mitochondrial electron-transfer chain and the formation of a bypass on the administration of ubiquinone (CoQ) or menadione in the case of a deficiency of Complex III or IV,
Brain & Development, Vol 9, No 2,1987 79
Human
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ance of stroke-like episodes after the administration of succinate to a patient with ME LAS due to a deficiency of Complex I. Administration of ubiquinone (CoQ) or menadione is effective, when the defect is limited to Complex III or N. Ubiquinone, which receives electrons from NADH or succinate via Complex I or II, respectively, is bound to ubiquinone-binding proteins (CoQ-N, CoQ-S and CoQ-C). Electrons of ubiquinol are transported through Complex III and cytochrome c to Complex IV, and fmally to molecular oxygen. When ubiquinone is administered, electrons are transferred from the proteinbound ubiquinol to the free ubiquinone. The resultant free ubiquinol transfers the electrons to oxygen to yield the superoxide anion and hydrogen peroxide, which are converted into water and oxygen by scavenging enzymes such as superoxide dismutase, catalase (Cat) and glutathione peroxidase (GPO). Although the rate of the electron flow from ubiquinol to oxygen is much lower than that of the normal activity of Complexes III and IV, respectively the artificial bypass enables Complex I to transfer electrons and to drive the ATP production. Molecular Biological Approaches for Mitochondrial Respiratory Enzymes It is now possible to obtain a human gene for a component in the electron-transfer chain in the test tube. A cDNA clone for cytochrome Cl in Complex III was isolated and sequenced in this laboratory [20]. The amino acid sequence deduced from the nucleotide sequence of the human gene for cytochrome Cl showed 93% homology with that of the bovine protein determined by amino acid sequencing (Fig 4). The localization of the gene among the chromosomes is now in progress. The organization of the genes for respiratory enzymes and the 'regulatory mechanism of their expression would be elucidated using the cloned cDNAs as probes. The combination of the DNA probes for genes of cytoplaSmically synthesized subunits, e.g., cytochrome Cl, and those for the genes of mitochondrially synthesized subuniU, e.g., cytochrome b, would be a useful means of studying the mechanism of coordinated expression of the mitochondrial·and nuclear genes.
80 Brain & Development, Vol 9, No 2,1987
-
Tyr
-
human and bovine cytochrome c \. The amino acid sequence of bovine cytochrome c \ was cited from the results of Wakabayashi et al [J 91. Dashes represent homologous amino acids. Boxes are placed around the residues to which the heme covalently binds.
Prespective Our knowledge of the interaction between the mitochondrial and nuclear genes in their expression is still limited, although we recognize it as a prime determinant of the structure and function of mitochondria. Various lines of basic research, such as molecular cloning of subunits of the electron-transfer enzymes, immunochemical identification of the subunits and in vitro synthesis of the components in the respiratory chain, would facilitate the elucidation of the regulatory mechanisms involved in biosynthesis of the components which make up the structure of the mitochondrial inner membrane. Such progress would elucidate the etiology and the pathophysiology of mitochondrial cytopathies and establish the fundamental diagnosis and treatment for this disorder.
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frames of human mitochondrial DNA. In: Quagliariello E, Slater Ee, Palmieri F, Saccone C, Kroon AM, eds. Achievements and perspectives of mitochondrial research. Volume II: Biogenesis. Amsterdam·New York·Oxford: Elsevier Science Publishers, 1985 :259-75. Nelson MA, Macino G. Gene organization and expression in Neurospora crassa mitochondria. In: Quagliariello E, Slater EC, Palmieri F, Saccone C, Kroon AM, eds. Achievements and perspectives of mitochondrial research. Volume II: Biogenesis. Amsterdam·New York·Oxford: Elsevier Science Publishers, 1985 :293-304. Tzagoloff A. Mitochondria. New York· London: Plenum Press, 1982:293. Aitardi G. Organization and expression of mammalian mitochondrial genome: a lesson in economy. Trends Biochem Sci 1981 ;6:86-9, 100-3. Tanaka M, Nishikimi M, Suzuki H, Ozawa T, Okino E, Takahashi H. Multiple cytochrome deficiency and deteriorated mitochondrial polypeptide composition in fatal infantile mitochondrial myopathy and renal dysfunction. Biochem BiophysRes Commun 1986;137:911-6. Tanaka M, Nishikimi M, Suzuki H, et aI. Deficiency of subunits in heart mitochondrial NADH-ubiquinone oxidoreductase of a patient with mitochondrial encephalopathy
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and cardiomyopathy. Biochem Biophys Res Commun 1986; 140:88-93. Tanaka M, Nishikimj M, Suzuki H, Ozawa T, Koga Y, Nonaka I. Partial deficiency of subunits in Complex I or IV of patients with mitochondrial myopathies. Biochem Int 1987; 14: 525-30. Tanaka M, Nishikimi M, Suzuki H, et al. Deficiency- of subunits of Complex I or IV in mitochondrial myopathies: Immunochemical and immunhistochemical study. J Inher Metab Dis (in press). Tanaka M, Nishikimi M, Suzuki H, et al. Variation in the levels of Complex I subunits among tissues in a patient with mitochondrial encephalopathy and renal dysfunction. Biochem Int 1987 ;14:735-9. Tanaka M, Nishikimi M, Ozawa T, Miyabayashi S, Tada K. Lack of subunit II of cytochrome c oxidase in a patient with mitochondrial myopathy. Ann NY Acad Sci 1987;488: 5034. Wakabayashi S, Matsubara H, Kim CH, King TE. Structural studies of bovine heart cytochrome c l . J Bioi Chem 1982; 257 :9335-44. Nishikimi M, Suzuki H, Ohta S, et al. Isolation of cDNA clone for human cytochrome cI from a Agt11 expression library. Biochem Biophys Res Commun (in press).
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