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The organization and inheritance of the mitochondria genome
Author: Xin Jie Chen,Ronald A. Butow
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"� 2005 Nature Publishing Group Department of Molecular Biology, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, Texas 75390-9148, USA. Correspondence to R.A.B. e-mail: ronald.butow@ utsouthwestern.edu doi:10.1038/nrg1708 Published online 27 September 2005 Mitochondria supply most of the cell?s energy, which is produced as ATP by the oxidative phosphorylation apparatus of the mitochondrial inner membrane. The biogenesis of the ATP-synthesizing machinery requires contributions from two physically separated genomes: one in the nucleus and the other in mitochondria. In humans, more than 80 proteins participate directly in oxidative phosphorylation, of which 13 are encoded by mtDNA. The distribution of oxidative phosphor- ylation components that are encoded by the nuclear and mitochondrial genomes varies between species. However, in all species the essential contribution of the mitochondrial genome demands that mtDNA is faithfully inherited to ensure that respiratory function is maintained during growth and development. Although 4 decades have elapsed since the dis- covery of mtDNA 1,2 , its organization and inheritance remain poorly understood. mtDNA is packaged into protein?DNA complexes that are called, by analogy to the bacterial chromosome, mitochondrial nucleoids (mt-nucleoids). Overall, ~30 proteins from different species have been identified as potential components of mt-nucleoids uniF6AETABLE 1uniF6AF. Some have known func- tions in mtDNA transactions, and might therefore be expected to be present in mt-nucleoids. These proteins include the Abf2 and TFAM (transcription factor A, mitochondrial) family of mtDNA-packaging factors and the mitochondrial ssDNA-binding proteins. However, surprisingly, recent proteomic analyses of yeast 3,4 and Xenopus laevis mt-nucleoids 5 revealed a group of proteins for which the known functions are ostensibly unrelated to mtDNA transactions. Therefore, mitochondria might have evolved unique strategies for the organization and inheritance of their genomes by using proteins that also function in mitochondrial metabolism, potentially coupling these important processes. Another rapidly developing field of mitochondrial biology focuses on understanding the segregation of mt-nucleoids during cell division. In addition to genes that are directly involved in mtDNA recombination, sev- eral other genes have recently been discovered that con- trol mt-nucleoid distribution and stability. Interestingly, these include genes that control mitochondrial fusion, morphogenesis and movement. These findings indicate the existence of a dedicated mt-nucleoid segregation apparatus that coordinates nucleoid segregation with mitochondrial dynamics and cell division 6?9 . THE ORGANIZATION AND INHERITANCE OF THE MITOCHONDRIAL GENOME Xin Jie Chen and Ronald A. Butow Abstract | Mitochondrial DNA (mtDNA) encodes essential components of the cellular energy-producing apparatus, and lesions in mtDNA and mitochondrial dysfunction contribute to numerous human diseases. Understanding mtDNA organization and inheritance is therefore an important goal. Recent studies have revealed that mitochondria use diverse metabolic enzymes to organize and protect mtDNA, drive the segregation of the organellar genome, and couple the inheritance of mtDNA with cellular metabolism. In addition, components of a membrane-associated mtDNA segregation apparatus that might link mtDNA transmission to mitochondrial movements are beginning to be identified. These findings provide new insights into the mechanisms of mtDNA maintenance and inheritance. NATURE REVIEWS | GENETICS ADVANCE ONLINE PUBLICATION | 1 REVIEWS � 2005 Nature Publishing Group Table 1 | mtDNA-associated proteins in yeast, mammals and Xenopus laevis Protein* Primary function mtDNA stability in mutant Yeast Abf2 ?�|| mtDNA packaging ? o or ? ? Aco1 � Citric acid cycle ? o Arg5,6 � Arginine biosynthesis Stable Ald4 � Ethanol metabolism Stable Atp1 � ATP synthesis ? o -lethal Cha1 �� Catabolism of hydroxy amino acids Stable Idh1 � Citric acid cycle Moderate instability Idp1 � Oxidative decarboxylation of isocitrate Stable Ilv5 � Biosynthesis of Val, Ile and Leu Moderate instability Ilv6 � Biosynthesis of Val, Ile and Leu Stable Kgd1 ?� Citric acid cycle Moderate instability Kgd2 ?� Citric acid cycle Moderate instability Lpd1 ?� Citric acid cycle, catabolism of branched-chain amino acids Moderate instability Lsc1 � Citric acid cycle Stable Mgm101 �|| mtDNA maintenance or repair Unstable ? + and ori-lacking ? ? Mip1 �|| mtDNA replication ? o Mnp1 ? Putative mitochondrial ribosomal protein Stable mtHsp60 � Mitochondrial chaperonin Unstable ori-containing ? ? mtHsp10 � Mitochondrial chaperonin Unknown mtHsp70 � Protein import Unknown Pda1 � Oxidation of pyruvate Moderate mtDNA instability Pdb1 � Oxidation of pyruvate Moderate mtDNA instability Rim1 � mtDNA replication ? o Rpo41 � mtDNA transcription ? o or ? ? Sls1 � Coordination of transcription and translation ? o or ? ? Yhm2 � Mitochondrial carrier Stable Mammals TFAM ?# mtDNA transcription and packaging mtDNA depletion Twinkle || mtDNA replication Multiple mtDNA deletions mtSSB ?# mtDNA replication Unknown Polymerase ? # mtDNA replication Multiple mtDNA deletions BRCA1 # Tumour suppressor Unknown PRSS15** Protein degradation Unknown Xenopus laevis mtTFA ? mtDNA transcription and packaging Unknown mtSSB ? mtDNA replication Unknown PDC-E2 ? Oxidation of pyruvate Unknown BCKAD-E2 ? Catabolism of branched-chain amino acids Unknown Prohibitin 2 ? Protein folding Unknown ANT1 ? ADP?ATP exchange on inner membrane Unknown *Mitochondrial DNA (mtDNA)-associated proteins were identified by various procedures as follows: ? density gradient centrifugation of detergent-solubilized mitochondria 5,12,49,111,112 ; � formaldehyde crosslinking followed by density gradient centrifugation 3,4 ; || direct co-localization with mtDNA by GFP tagging 9,13,14,102 ; � protein array-based screens for mtDNA-binding factors 113 ; # immunocytochemistry 64,114 ; and **co-immunoprecipitation with known nucleoid proteins 115 . ANT1, adenine nucleotide translocator 1; BCKAD-E2, branched-chain ?-ketoacid dehydrogenase, E2 subunit; BRCA1, breast cancer 1; Hsp, heat-shock protein; mtTFA, mitochondrial transcription factor A; mtSSB, a mitochondrial ssDNA-binding protein; ori, origin of replication; PDC-E2, pyruvate dehydrogenase, E2 subunit; PRSS15, serine protease 15 (also known as LON); TFAM, transcription factor A, mitochondrial; ?�, respiratory-deficient petite cells that lack mtDNA; ? ? , respiratory-deficient cells that contain mtDNA that is deleted for large segments of the wild-type mitochondrial genome. 2 | ADVANCE ONLINE PUBLICATION www.nature.com/reviews/genetics REVIEWS � 2005 Nature Publishing Group HIGH MOBILITY GROUP uniF6AEHMGuniF6AF PROTEINS A family of non-histone proteins that contain DNA-binding HMG-box domains. ATOMIC FORCE MICROSCOPY A method that is used to image materials at the atomic level. PETITE MUTANTS Respiratory-deficient mutants. These either contain a highly repeated, random fragment of the wild-type mitochondrial genome (? ? ) or completely lack mtDNA (? o ). Here we review our current understanding of the organization and inheritance of mitochondrial nucle- oids. Because of its amenability to genetic analysis, the yeast Saccharomyces cerevisiae has provided the most detailed information on these processes, we therefore focus mainly on studies in this species. Findings from other species are discussed where appropriate. Packaging of mtDNA Mitochondrial nucleoids have been visualized using various methods, revealing interspecies differences in the size, morphology and number of nucleoids uniF6AETABLE 2uniF6AF. For example, staining yeast mtDNA with 4?,6 diamidino-2-phenylindole (DAPI) shows nucle- oids as ?strings of beads? that line up with the mitochon- drial reticulum 10?12 (FIG. 1a). Mitochondrial nucleoids can also be detected by visualizing nucleoid-specific proteins that are tagged with GFP (FIG. 1b) 9,13,14 . These structures are the packaged and condensed form of mtDNA. The linear monomer of the ~75-kb mito- chondrial genome in yeast is ~25 �m in length, and is compacted into a globular nucleoid structure with an average diameter of ~0.3 �m in aerobic cells. This contains one to two genome equivalents of mtDNA within a mitochondrial reticulum that has an average radius of ~0.5 �m. In mammals, mt-nucleoids have a diameter of just 0.068 �m; these nucleoids contain sev- eral molecules of the circular mitochondrial genome, which is 16.5-kb long. Abf2 and TFAM ? key players in mtDNA packag- ing. The core packaging elements of mt-nucleoids are mainly non-histone, HIGH MOBILITY GROUP uniF6AEHMGuniF6AF PROTEINS that show homology to the DNA-binding HMG pro- teins of nuclear chromatin 15 . These positively charged mtDNA-binding proteins contain two HMG boxes and are evolutionarily conserved from yeast to humans 16?26 . The best-studied HMG protein in mt-nucleoids is yeast Abf2. This protein exists in solution as an 18.6-kDa monomer and is present at an estimated ratio of 1 mole- cule for every 15?30 bp of mtDNA 26,27 , which is suffi- cient to coat the entire mitochondrial genome. ATOMIC FORCE MICROSCOPY shows that Abf2 introduces sharp bends of ~78� into the DNA backbone, which seems to be sufficient to compact DNA 28 . Abf2 can introduce superhelical turns into relaxed circular DNA 24,26 , but this supercoiling might not be essential for mtDNA packaging 29 . Abf2 generally binds non-specifically to dsDNA, although binding seems to be excluded from simple DNA sequences, such as poly(dA) tracts, which occur in many regulatory and intergenic spacer regions in yeast mtDNA 26,27 . Yeast cells that lack Abf2 have an unstable mito- chondrial genome. When cells are grown on media that contain glucose ? which allows cells to obtain energy through fermentation, avoiding the need for respiration ? respiratory-deficient PETITE MUTANTS are produced 26 . However, mtDNA can be maintained when abf2? mutants are grown on media that contain a non- fermentable carbon source, and in this case mtDNA is hypersensitive to damage from endogenously produced free radicals and from mutagens such as ethidium bro- mide 3,30 . These observations support roles for Abf2 in mtDNA packaging and protection. The mammalian Abf2 homologue, TFAM, is also thought to be involved in packaging the mitochondrial genome. TFAM bends DNA in vitro and can comple- ment the mtDNA-instability phenotype of the yeast abf2? mutation 20,31,32 . TFAM can therefore be consid- ered a non-specific DNA-binding protein. Depending Table 2 | An interspecies comparison of mitochondrial nucleoids Species Cytological appearance Size Number per cell Number of mitochondrial genomes per nucleoid Size of mitochondrial genome Visualization tools References Saccharomyces cerevisiae Globular foci ~0.2?0.4 �m in aerobic and ~0.6?0.9 �m in anaerobic cells (diameter) ~40?60 in aerobic and ~7.6 in anaerobic cells ~1?2 in aerobic and ~20 in anaerobic cells 75?80 kb DAPI, GFP tagging 12,65,102,116 Physarum polycephalum Rod shape Up to ~1.5 �m in length ~15 ~40?80 63 kb DAPI, ethidium and thionine staining, light or electron microscopy 117,118 Crithidia fasciculata Disk shape ~1.0 �m � ~0.35 �m 1 Several thousand mini circles and a few dozen maxi circles 0.5?10 kb for mini circles and 20?40 kb for maxi circles DAPI and ethidium staining, immunofluorescence and GFP tagging, light or electron microscopy 119,120 Humans Globular foci ~0.068 �m (diameter) 466?806 in cell lines ~2?10 16.5 kb Ethidium and PicoGreen staining, immunocytochemical staining with DNA- specific antibodies, bromodeoxyuridine labelling, GFP tagging 14,64,121?125 DAPI, 4?,6-diamidino-2-phenylindole. NATURE REVIEWS | GENETICS ADVANCE ONLINE PUBLICATION | 3 REVIEWS � 2005 Nature Publishing Group a b DIC DIC mt?GFP DAPI Abf2?GFP FOOTPRINT ANALYSIS A technique for identifying sites where proteins bind to DNA at a single-nucleotide resolution. REDUCTIONAL RECOMBINATION Recombination within the highly repeated sequences of ? ? -petite genomes, which produces shorter mtDNA molecules with a reduced number of repeat units. on the cell type, the amount of TFAM that is associated with mtDNA might vary. It is estimated to be present at a ratio of 1 molecule for every 10?20 bp of mtDNA in HeLa cells 33,34 , but estimates of the amount of TFAM that is associated with mtDNA in other cell types are much lower 35,36 . The X. laevis TFAM counterpart, mttfa-A, has also been identified as an abundant com- ponent of purified mt-nucleoids 5 , indicating that it has a similar function. TFAM has other functions in addition to its proposed role in mtDNA packaging. It was initially identified as a transcription factor that facilitates the assembly and promoter recognition of the mitochondrial transcriptional machinery 16,20,31,37 . Through FOOTPRINT ANALYSIS, TFAM has been found at both the light and heavy strand promoter regions (which are origins of asymmetrical replication) of mammalian mtDNA 38,39 . TFAM therefore also shows some DNA-binding specificity that is related to its transcriptional function, as well as non-specific DNA-binding properties. There is also evidence that TFAM might function in repairing mtDNA. Expression of TFAM is elevated in cell lines that are resistant to the DNA-damaging agent cisplatin 40 , and TFAM is reported to bind with increased affinity to mtDNA that has been damaged either oxidatively or by cisplatin 41 . Moderate increases in ABF2 expression (2?2.5 fold) lead to comparable increases in mtDNA copy number in yeast 42 . Abf2 produces this effect either by regulating mtDNA replication or by providing increased protec- tion against degradative turnover, thereby augmenting the copy number. Correlations between TFAM levels and mtDNA copy number have also been reported in mice 43,44 and in a chicken lymphoma cell line 45 . As TFAM is also required for mtDNA transcription 31 , these effects on mtDNA copy number might be a consequence of transcriptional control. However, over- expression of human TFAM in mice in a genetic back- ground that does not alter expression from mtDNA still results in increased mtDNA copy number 33 . Furthermore, the level of expression of the Drosophila melanogaster TFAM homologue is correlated with mtDNA copy number, but not with mtDNA transcrip- tion 46,47 . Finally, the 25-amino-acid, C-terminal tail of TFAM that is required for its transcriptional function is absent from yeast Abf2, and the yeast protein does not stimulate mitochondrial transcription in vitro 48 . These results support the suggestion that the roles of TFAM and its homologues in transcription and mtDNA packaging are separable. Is Abf2 sufficient for mtDNA packaging in yeast? Abf2 shows phased binding to DNA 27 and there is evidence that the physiological concentration of this protein might only allow for a limited compacting capacity 29 . This indicates that mtDNA packaging is more elaborate, requiring other proteins to maintain nucleoid morphology, gene expression and genome maintenance. Indeed, the presence of mt-nucleoids in cells that lack Abf2, although they are altered in morphology 49 , indicates that other packaging factors are present. This suggestion has been strengthened by the identification of genes that interact with ABF2 and have products that are present in mt-nucleoids. Two examples are the metabolic genes ILV5 and ACO1 (aco- nitase 1) (see below). Both of these genes are required for mtDNA maintenance, and their overexpression suppresses the mtDNA instability phenotype of abf2? mutants 3,4,50,51 . Ilv5 ? a prototypical bifunctional mt-nucleoid protein. Ilv5 is a mitochondrial enzyme that functions in branched-chain amino-acid biosynthesis 52 . Direct evidence that Ilv5 is a bifunctional protein came from the identification of two classes of Ilv5 mutants, which independently affect either the biosynthetic activity or the mtDNA maintenance function of the protein 53 . Furthermore, conditions that activate ILV5 expression, such as amino-acid starvation, result in an increase in the number of mtDNA nucleoids without any increase in the overall amount of mtDNA 54 . This ?parsing? of mtDNA into nucleoids is dependent on Ilv5. As revealed from the analysis of ? ? -petite mtDNAs, the parsing of mtDNA into nucleoids is a two-step process. The first step results in an increase in the number of mtDNA molecules, owing to REDUCTIONAL RECOMBINATION across the tandem repeats of ? ? mtDNA or, in the case of ? + mtDNA, to the separation of interlinked genomes. The second step parses the individual mtDNA molecules into smaller nucleoids, increasing nucleoid number without any increase in the amount of mtDNA. This second step requires Ilv5. In its absence, reductional recombination still occurs, but there is no increase in the number of nucleoids. Altogether, these results indicate that Ilv5 functions in assembling mtDNA into mt-nucleoid structures. Figure 1 | Cytological visualization of yeast mitochondrial nucleoids. a | The co-visualization of mitochondria and mitochondrial DNA (mtDNA) in a budding yeast zygote. Cells are visualized by differential interference contrast (DIC) microscopy (left panel). Mitochondria in the same cells were tagged with a GFP construct in which residues 1?52 of citrate synthase (a mitochondrial enzyme) were fused to the amino terminus of GFP (middle panel). mtDNA in these cells was also stained with 4?,6-diamidino-2-phenylindole (DAPI) (right panel). Note the ?string-of-beads? appearance of the mtDNA. b | A visualization of yeast mitochondrial nucleoids by direct GFP tagging of the nucleoid protein, Abf2. As before, the left panel shows DIC imaging and the right panel shows GFP fluorescence. These images are reproduced, with permission, from REF. 9 � (1998) Rockefeller University Press. 4 | ADVANCE ONLINE PUBLICATION www.nature.com/reviews/genetics REVIEWS � 2005 Nature Publishing Group Outer membrane Inner membrane Aco1 Abf2 IIv5 mtDNA Abf2 IIv5 mtDNA Aco1 Abf2 IIv5 mtDNA Aco1 a Glucose repression b Respiration or RTG response c Amino-acid starvation KREBS CYCLE Also known as the tricarboxylic acid (or TCA) cycle. A metabolic pathway in mitochondria that breaks down the products of carbohydrate, fat and protein metabolism into carbon dioxide and water to generate energy. It also provides precursors for other compounds, such as certain amino acids. The bifunctional mitochondrial aconitase, Aco1. The yeast mitochondrial aconitase Aco1 is a KREBSuniF6BACYCLE enzyme that converts citrate into isocitrate. Under all growth conditions tested, Aco1 is essential for mtDNA stability 3,50 , including that of ? ? mtDNAs (X.J.C., X. Wang and R.A.B., unpublished observations). This protein was shown to be bifunctional in an analysis of mutations of three cysteine residues that coordinate the assembly of the Aco1 iron?sulphur cluster 3 , which is essential for catalytic activity 55 . As a result of these mutations, Krebs-cycle activity and the growth of cells are blocked under conditions that require respiration, but mtDNA stability is unaffected 3 . This is reminis- cent of the bifunctionality of some aconitase isoforms in mammals and bacteria. A cytosolic mammalian isoform of aconitase, also known as iron-responsive element-binding protein, binds to 5? or 3? UTRs of specific mRNAs when its iron?sulphur cluster is disassembled in response to iron depletion and oxi- dative stress 56 . This binding modulates the transla- tion and stability of mRNAs that are involved in iron homeostasis. It has also been reported that aconitase from mammalian mitochondria and Bacillus subtilis can bind mRNA 57,58 . Whether a similar ?iron-sulphur switch? controls the conversion between the enzymatic and mtDNA-maintenance forms of Aco1 is unknown. However, as suggested by Shadel 59 , such a regulatory mode could allow Aco1 to function as a stress sensor for the protection of mtDNA. In this model, the dis- assembly of the Aco1 iron?sulphur cluster, induced by free radicals, could result in the reallocation of Aco1 from the Krebs cycle to mt-nucleoids, stabilizing mtDNA under oxidative stress conditions. Wild-type mtDNA is unstable in abf2? cells that are grown on media containing glucose ? which represses mitochondrial function ? but can be stably maintained on media that stimulate respiration 3 . This is mainly due to the robust expression of ACO1 in respiring cells 60 . Ectopic expression of ACO1 from a constitutive promoter to a level which is comparable to that induced by respiratory growth is sufficient to suppress the loss of mtDNA from abf2? cells that are grown in the pres- ence of glucose. Under respiratory conditions, the extra Aco1 is expected to stimulate the Krebs cycle and energy metabolism, but is thought to simultaneously substitute for Abf2 in reorganizing mtDNA. This might also pro- vide metabolically coupled protection of mtDNA against oxidative damage during respiratory growth. A model for the metabolic remodelling of mt-nucleoids. The expression of both ILV5 and ACO1 is metabolically regulated. ILV5 is activated by the general amino-acid control pathway in response to amino-acid starvation 52 . Expression of ACO1 is stimulated by the haem activator protein (HAP) complex and retrograde (RTG) signal- ling pathways 61,62 , which are upregulated by respiratory growth or mitochondrial dysfunction, respectively. Therefore, the genetic interactions of ILV5 and ACO1 with ABF2 indicate that the structural organization of mt-nucleoids could be subject to metabolic remodel- ling (FIG. 2). This is supported by the observation that activation of ILV5 expression by amino-acid starvation increases mtDNA transmission through an increase in the number of nucleoids 54 . Simultaneous inactivation of both the HAP and RTG systems leads to mtDNA instability in an ACO1-dependent manner 3 . Conversely, activation of either HAP or RTG suppresses mtDNA instability in abf2? mutant cells. These findings indicate that the molecular compo- sition of nucleoids might change owing to the fluctuat- ing levels of these bifunctional proteins in response to physiological cues. In glucose-rich media, for example, where the general amino-acid control and HAP path- ways and respiratory activity are repressed, Abf2 might be the predominant factor in packaging mtDNA into a compact structure that has limited accessibility to the mitochondrial transcription apparatus and other mtDNA-transaction systems. This tightly packaged conformation might also be beneficial to the mitochon- drial genome for reducing exposure to adverse micro- environments. In response to increased biosynthetic or respiratory activities, mtDNA might be remodelled into a more active conformation that is favourable for gene expression, which would depend on increased amounts of Ilv5 and Aco1. These two proteins might globally or locally substitute for Abf2 and reorganize mtDNA into a metabolically favourable conforma- tion. They might also specifically protect metabolically active mt-nucleoids so that mtDNA is still protected in the less tightly packed conformation. The observation that overexpression of ILV5 and ACO1 suppresses the hypersensitivity of mtDNA to attack by the mutagen ethidium bromide in an abf2? background might be a reflection of such a protective role (REF. 3; X.J.C., X. Wang and R.A.B., unpublished observations). Comparative analysis of nucleoid composition and structure in cells with different metabolic states will allow the testing of this model. Figure 2 | A model for the metabolic remodelling of mitochondrial nucleoids in yeast. a | In conditions in which respiration is repressed by the presence of glucose, mitochondrial DNA (mtDNA) is tightly packaged by the protein Abf2, limiting mtDNA transactions. b | When cells are shifted to respiratory conditions, or when mitochondrial dysfunction activates the retrograde (RTG) pathway, ACO1 (aconitase 1) expression is increased, resulting in more Aco1 in mitochondrial nucleoids (mt-nucleoids). c | Similarly, in conditions of amino-acid starvation, the expression of ILV5 is activated by the general amino-acid control pathway and Ilv5 accumulates in mt-nucleoids. The increased levels of Aco1 and Ilv5 that are shown in b and c substitute for Abf2 in packaging mtDNA into a metabolically favourable conformation and/or protect mtDNA in the remodelled conformation. The blue oval represents a putative protein, or complex of proteins, that connects mt-nucleoids to the inner mitochondrial membrane. NATURE REVIEWS | GENETICS ADVANCE ONLINE PUBLICATION | 5 REVIEWS � 2005 Nature Publishing Group � mtDNA Zygote Mitochondria ? + ? o Medial bud Mitochondrial fusion 3 hours after fusion CHAPERONIN A protein complex that is required for correct protein folding. DECONVOLUTION MICROSCOPY Microscopy using computer image-processing techniques to reconstruct cross-sectional images from several focal planes, which yields high-resolution images. Mitochondrial nucleoid division The division of mt-nucleoids has been observed in the slime mould Physarum polycephalum and in human cell lines. In P. p o l y c e p h a l u m , division is syn- chronized with mitochondrial fission 63 , whereas in human cells it occurs in the absence of mitochondrial division 64 . The molecular mechanisms that drive the division of mt-nucleoids are unclear, but recent studies indicate that heat shock protein 60 (Hsp60), another bifunctional protein, might be involved 65 . Hsp60 is an essential CHAPERONIN that is required for the correct folding of many proteins that are imported into mitochondria 66 . It is functionally and structurally homologous to the bacterial chaperonin GroEL and is organized into torroidal structures that are composed of two seven-member rings. Hsp60 also binds with high specificity in vitro to mtDNA ori sequences 4 , which are putative origins of mtDNA replication, and this binding is preferential for the template strand of ori mtDNA. mtDNAs that contain intact ori sequences are unstable in hsp60 temperature-sensitive (hsp60 ts ) mutant backgrounds, in which the chaperonin activity of Hsp60 is close to that of the wild type. However, mtDNAs that lack ori sequences or have mutations in these sequences are as stable in the hsp60 ts background as in wild-type cells 65 . The mtDNA instability in hsp60 ts mutants might arise because ori-dependent segrega- tion complexes fail to assemble correctly owing to a compromised Hsp60 function at ori sequences. How does Hsp60 function in stabilizing mtDNA? Using high-resolution DECONVOLUTION MICROSCOPY, Kaufman et al. 65 found that nucleoids in hsp60 ts mutant cells are fewer in number than in wild-type cells and have an abnormal appearance, often having elongated, barbell-like or serpentine structures. This indicates that nucleoid division is delayed after mtDNA replication, which would impair nucleoid transmission in these mutants. The interaction between Hsp60 and mtDNA ori sequences could therefore be an important determi- nant of nucleoid division. Although further studies will be necessary to understand the relationship between Hsp60, ori sequences, nucleoid division and nucleoid transmission, these findings indicate that nucleoid divi- sion is a regulated process that is important for mtDNA transmission. Mitochondrial nucleoid segregation At the G 1 ?S transition of the yeast cell cycle, a subset of mitochondrial structures are selected and actively moved into the bud that will form the new daughter cell 67,68 . Although the segregation of mt-nucleoids into daughter cells is dependent on the movement of mitochondrial structures, these two events are not necessarily coupled. Much of our current understanding of the mecha- nism of mt-nucleoid segregation has benefited from the use of the yeast zygote system. In yeast the mito- chondrial genome is inherited from both parents after the formation of zygotes from two strains of opposite mating type. As the zygotes have defined positions for bud emergence during the first post-zygotic cell division, the relative movements of mitochondria and mt-nucleoids can therefore be followed cytologically with reference to this unique topographical landmark. In addition, within a relatively few divisions, diploid buds that are formed from heteroplasmic zygotes become homoplasmic. These unique features pro- vide a robust experimental system for following how individual parental mtDNAs are segregated into the progeny. This allows easy tracking of the drift from the heteroplasmic to homoplasmic states of mtDNAs during cell division. Early genetic studies in yeast indicated that most parental mtDNAs do not mix in zygotes 69,70 , and that only a small fraction of the total mtDNA pool is trans- ferred post-zygotically to diploid buds 71 . Together with more recent investigations 6,8 , these results provide us Figure 3 | Understanding the mechanism of mitochondrial nucleoid segregation. An experimental system that can be used to track the sorting of mitochondrial protein and DNA (mtDNA) after mating between two strains of the yeast Saccharomyces cerevisiae. The two strains are ? + (which carry normal mtDNA) and ?� (which lack mtDNA). Haploid ? + cells are transformed with a vector from which a GFP fusion protein is expressed. This protein is targeted to the mitochondrial matrix or to the inner or outer mitochondrial membranes, and is expressed under the control of the inducible galactosidase (GAL) promoter so that it is induced in cells grown on galactose. After growth of the transformed ? + cells on a galactose medium, these cells are mated in a glucose-containing medium to non-transformed ?� cells. Zygotes are examined by differential interference contrast and fluorescence microscopy at various time points after mating, and mtDNA is stained with 4?,6-diamidino-2-phenylindole (DAPI). The mitochondrial GFP fusion protein (indicated by the green colour) can be seen to spread evenly throughout the zygote and into the medial diploid bud within ~3 hours of mating. However, in most zygotes (>80%) mtDNA is preferentially segregated to the medial diploid bud, indicating that mtDNA movements are controlled independently of mitochondrial proteins. These experiments also indicate the existence of an mtDNA segregation apparatus. 6 | ADVANCE ONLINE PUBLICATION www.nature.com/reviews/genetics REVIEWS � 2005 Nature Publishing Group ROLLINGuniF6BACIRCLE REPLICATION A form of DNA replication in which a circular DNA molecule produces linear daughter molecules. HOMOPLASMY The state of the mitochondrial genetic system in which all copies of the mitochondrial genome within a cell are identical. HETEROPLASMY The state of the mitochondrial genetic system in which a cell contains mitochondrial genomes that are genetically different. FLUORESCENCE IN SITU HYBRIDIZATION A microscopic technique that uses fluorescently tagged DNA probes to detect the cytological localization of specific DNAs by in situ hybridization. with important information on the relative sorting of mitochondrial proteins and mt-nucleoids. Soon after mating parental mitochondria fuse, which allows the mixing of mitochondrial constituents within a reticu- lum that extends throughout the zygote and into the diploid buds. Surprisingly, in crosses in which only one of the two haploid parents contained mtDNA, mt-nucleoids in most zygotes were preferentially sorted to the first medial bud 9 (FIG. 3). These studies indicated that mt-nucleoid segregation is a directed process that is driven by a putative nucleoid-segregation apparatus. Before discussing the molecular nature of this appara- tus, we first consider how mtDNA structure itself can influence mt-nucleoid organization and inheritance. mtDNA recombination and segregation. mtDNA structure can influence both the number and size of nucleoids. The wide variation among species in mtDNA size, shape, replication mechanisms, unique sequence elements and the presence or absence of a highly active recombination system indicates that the details of nucleoid assembly and dynamics will also vary. In S. cerevisiae, a fraction of mtDNA is present as concatemers 72 , which can arise by ROLLINGuniF6BACIRCLE REPLICATION or homologous recombination. Fangman and co-workers 73 showed that the level of Mgt1 (also known as the mitochondrial cruciform-cutting endo- nuclease, Cce1), which cuts cruciform structures such as recombination junctions, has an important effect on how mtDNAs segregate both mitotically and in crosses. Their studies focused on mtDNA in ? ? -petite mutants, which generally have fewer mt-nucleoids than ? + cells, despite having about the same amount of mtDNA. As expected, overexpression or deletion of MGT1 affects the number of recombination junctions in mtDNA. Interestingly, manipulation of MGT1 also affects mt-nucleoid number, the mitotic stability of mtDNA and its transmission properties in crosses. In mgt1? cells, more mtDNA is connected by recombi- nation junctions, fewer nucleoids are observed and the efficiency of mitotic and zygotic propagation of mtDNA from parent cells is reduced; overexpression of MGT1 had the opposite effect. Therefore, the rate of resolution of recombination junctions determines the number of, and consequently, the transmission of mt-nucleoids. A further relationship between mtDNA structure, recombination and inheritance has been indicated from the analysis of another yeast gene, MHR1. This gene encodes a mitochondrial matrix protein that promotes the ATP-independent pairing of homologous DNAs in vitro 74 . Shibata and co-workers 74,75 observed a high rate of formation of petites in a temperature- sensitive mhr1 mutant, and showed that this mtDNA instability is exacerbated when the mhr1 mutation is combined with an MGT1 deletion. Furthermore, in mating experiments, the rate at which post-zygotic HOMOPLASMY was achieved was reduced approximately 2-fold from mhr1/mhr1 zygotes and accelerated 2.5-fold from zygotes overexpressing MHR1 uniF6AEREF. 76uniF6AF. These effects were not observed from similar manipulations of the expression of either MGT1 or ABF2. MHR1 therefore seems to be the primary determi- nant of homoplasmy in vegetatively growing cells. The generation of homoplasmic cells from heteroplasmic ones is usually an efficient process, regardless of how the HETEROPLASMY originated ? whether from a muta- tion in vegetatively growing cells or from matings between haploid cells of different mtDNA genotypes. Based on the apparent enrichment of genome-length, monomeric mtDNA molecules in buds versus the prevalence of concatemers in mother cells, Ling and Shibata 76 suggested that Mhr1 has a role in mtDNA concatemer formation by promoting a type of rolling- circle replication that was first proposed by Clark- Walker and co-workers 77 . The model proposed by Ling and Shibata 76 states that homoplasmy could be achieved in relatively few divisions by the transmission of a small number of randomly selected concatemers, which are converted by unknown mechanisms to genome-length monomers during the partitioning of mtDNA from the mother to the daughter cells. Direct measurements of the rates of segrega- tion of mitochondrial genomes have been made by McAlpine et al. 78 using FLUORESCENCE IN SITU HYBRIDIZATION (FISH). Heteroplasmic zygotes were created in which one of the two parental mitochondrial genomes was known from genetic studies to have a marked segrega- tional or replicative advantage over the other in crosses. Although these experiments focused on biased rather than purely random outputs of mtDNAs, about eight post-zygotic divisions were nevertheless required for the appearance of near-homoplasmic diploid cells. A further interesting finding was that few if any nucleoids contained both parental mitochondrial genomes. As it is known that genotypically distinct mitochondrial genomes interact in such mating experiments (for example, through recombination), questions arise about genome selection in mt-nucleoids and the proc- esses that allow genotypically distinct mtDNAs in separate nucleoids to interact. Links with mitochondrial dynamics and morphology. Mitochondria are dynamic structures: they can read- ily fuse and divide, and undergo morphological transi- tions, which are regulated by various nuclear genes 68,79,80 . Intriguing links between mitochondrial dynamics and mtDNA inheritance come from the observation that interference with fusion, but not fission, compromises mt-nucleoid stability 81 . Among the yeast genes that function in mitochon- drial fusion are those that encode the outer membrane proteins Fzo1, which is a GTPase, and Ugo1, which contains a proposed single-membrane-spanning domain 82 . The D. melanogaster homologue of Fzo1, fuzzy onion 83 , is known to be required for mitochon- drial fusion in spermatogenesis. Fzo1 and Ugo1 form a complex with Mgm1, a dynamin-related GTPase that is located in the intermembrane space. This complex, which is a key component of the mito- chondrial fusion apparatus 84?86 , is in contact with the inner mitochondrial membrane. The process of fusion requires the coordinated fusion of the outer and inner NATURE REVIEWS | GENETICS ADVANCE ONLINE PUBLICATION | 7 REVIEWS � 2005 Nature Publishing Group PREuniF6BAPROTEIN TRANSLOCASE A complex of proteins that function in the import of nuclear-encoded mitochondrial proteins that were synthesized on cytoplasmic ribosomes. mitochondrial membranes. Current models propose that remodelling these membranes during the fusion process is achieved through contacts between outer and inner membranes that are mediated by the linking of the GTPases Fzo1 and Mgm1 by Ugo1 uniF6AEREFS 84,86uniF6AF. Mutations in the components of this apparatus not only lead to aberrant mitochondrial morphology, but also to a loss of mtDNA stability. How does disruption of mitochondrial fusion affect mtDNA stability? When fusion is compro- mised, the continuing fission process gives rise to fragmented mitochondrial structures, some of which might no longer contain mtDNA. Inheritance of those mitochondria would lead to the production of ?� petites. Indeed, in cultured Nicotiana tabaccum cells, mito- chondrial fission products often lack mt-nucleoids 87 . Frequent fusions are probably required to overcome the heterogeneous distribution of mtDNA nucleoids and the eventual segregation into progeny cells of those mitochondria that lack mtDNA. Alternatively, defects in the assembly of a functional mtDNA seg- regation apparatus (see below) could underlie the compromised mt-nucleoid inheritance in mitochon- drial fusion mutants. If this is the case, other genes that are involved in mitochondrial morphogenesis, such as those that have roles in general membrane remodelling and mitochondrial volume control, might also affect the stability and inheritance of mt-nucleoids. This might explain the correlation between increased mtDNA instability and mitochon- drial morphology in the mdm38 mutant, which is defective for an inner membrane K + /H + antiporter that is necessary for mitochondrial volume control 88 , and in cells treated with nigericin, which alters mitochondrial ion homeostasis 89 . Membrane tethering and the mt-nucleoid segrega- tion apparatus. What is the molecular mechanism that drives the segregation of mt-nucleoids during cell division? Early biochemical data support the view that, similar to the bacterial chromosome, mtDNA is membrane-associated, therefore provid- ing a platform for mtDNA segregation 90 . The molec- ular constituents that might tether mt-nucleoids to the membrane are now being revealed through the genetic analysis of several yeast mitochondrial membrane proteins that affect mitochondrial struc- ture and motility, as well as mt-nucleoid organization and stability 91?94 . The mitochondrial membrane proteins Mmm1, Mdm10 and Mdm12 are present in a complex that connects mitochondria to the actin cytoskeleton 7,95 . Mutations of the encoded genes not only result in aberrant mitochondrial morphology and alterations in mitochondrial motility, but also lead to changes in nucleoid structure and loss of mtDNA 96,97 . Mdm10 is also involved in the assembly of the PREuniF6BAPROTEIN TRANSLOCASE of the outer mitochondrial membrane 98 , highlighting the possibility that this protein has many functions, any of which could lead to mitochondrial morphology defects when absent. An important insight into the link between the struc- ture of mitochondria, their distribution within cells and nucleoid stability came from a study of Mmm1 by Jensen and co-workers 91,93 , who showed that this protein forms punctate structures, a subset of which were localized in apposition to mt-nucleoids. In contrast to Mdm10 and Mdm12, which are located in the outer membrane, Mmm1 is thought to span both the outer and inner mitochondrial membranes, although the N-terminal region that extends to the matrix is not essential for mtDNA maintenance 99 . Interactions of Mmm1 with other proteins might link mt-nucleoids to the outer membrane and cytoskeleton, which together would provide a way to tether mt-nucleoids to components that control mitochondrial distribution and morphology. Dimmer et al. 100 recently showed that two inner mito- chondrial membrane proteins, Mdm31 and Mdm32, also have dual roles in nucleoid stability and mito chondrial morphology. Importantly, the absence of either pro- tein is lethal in combination with a deletion of any one of MMM1, MDM10 and MDM12, as well as with a deletion of MMM2, which encodes another mitochon- drial distribution protein that is located in the outer mitochondrial membrane 101 . No synthetic lethality was observed among these four genes. Although direct bio- chemical evidence is lacking, these findings raise the possibility that the connection between nucleoids and the Mdm10?Mdm12?Mmm1 complex is mediated through Mdm31 and Mdm32. Further support for this comes from the finding that, in the absence of either Mdm31 or Mdm32, mtDNA-nucleoid morphology is disordered and apposition of Mmm1 to mt-nucleoids mostly disappears, whereas the abundance of Mdm10, Mdm12, Mmm1 and Mmm2 is unchanged. Because these proteins do not seem to be evolutionarily con- served, and the mitochondrial movements are not linked to the actin cytoskeleton in higher eukaryotes, other mechanisms might operate in these organisms to ensure the coordinated transmission of mt-nucleoids with mitochondrial inheritance. The significance of the apposition of mt-nucleoids to the outer membrane proteins remains to be clari- fied, as does the biochemical nature of this associa- tion. Remarkably, some mt-nucleoid proteins, such as Mgm101 uniF6AEREF. 102uniF6AF and certain mutant forms of Ilv5, seem to occupy the same sites as mt-nucleoids in cells that lack mtDNA (J. Batman and R.A.B., unpublished observations). These findings support the existence of a structure that is stably maintained regardless of the respiratory state of the cell. Identifying the deter- minants that specify the localization of these proteins might provide important clues to how nucleoids associate with the inner mitochondrial membrane. FIGURE 4 summarizes the current state of knowledge about the yeast mt-nucleoid segregation apparatus. According to this general model, contacts between the inner and outer mitochondrial membrane might have to be maintained to preserve the association of nucleoids with the segregation apparatus and with mitochondrial tethering devices. So, the latter would not only contrib- ute to mitochondrial movements, but also to nucleoid 8 | ADVANCE ONLINE PUBLICATION www.nature.com/reviews/genetics REVIEWS � 2005 Nature Publishing Group Outer membrane Inner membrane Aco1 Abf2 IIv5 mtDNA Mdm31 Mdm32 Mmm2Mdm10 Mitochondrial nucleoids Actin Cytoplasm Mitochondrial matrix Mdm12 Mmm1 REPLISOME A DNA-replicating structure that is located at the replication fork, which consists of DNA-replication enzymes and associated proteins. movements and therefore to mtDNA inheritance. The underlying model is that mt-nucleoid interaction with the segregation apparatus ? perhaps in the form of the mitochondrial REPLISOME 102 ? is a crucial feature in nucle- oid organization and maintenance. Disruption of the connection between the inner and outer mitochondrial membranes would also disrupt the link to nucleoids, leading to their disorganization and instability. Concluding remarks Cytological, biochemical and genetic studies have pro- vided insights into the organization and composition of mt-nucleoids. One concept that is emerging from these investigations is that mitochondria have recruited various proteins of mitochondrial metabolism and biogenesis to mt-nucleoids. This would allow mtDNA packaging, nucleoid division and nucleoid inherit- ance to be coupled to these processes. Much remains to be learned about how these bifunctional proteins, together with the core nucleoid components such as Abf2 or TFAM, participate in mtDNA organization and inheritance. The role of many other metabolic pro- teins that are associated with mtDNA uniF6AETABLE 1uniF6AF remains to be determined. The full physiological significance of the organizational remodelling of mt-nucleoids with respect to mtDNA expression and the oxidative phosphorylation capacity of the cell also await further exploration. Another challenge will be to develop a molecular-level understanding of the mt-nucleoid segregation apparatus and how it functions in mtDNA transmission. This will not only require a detailed view of the composition and organization of mt-nucleoids, but also insights into how these structures functionally interact with mitochondria, and possibly with other cellular components. Many advances in understanding the organiza- tion and segregation of mt-nucleoids have benefited from the amenability of the genetic system in yeast. It will be interesting to determine what aspects of the molecular mechanisms that underlie these fundamen- tal processes are conserved in humans. Furthermore, understanding how mt-nucleoids are organized and segregated could also be of clinical importance. Mutations in mtDNA are responsible for a range of neuromuscular disorders 103,104 and are suspected to underlie cellular ageing 105 . Recent studies have demonstrated that increased mtDNA mutations can directly cause premature ageing in mouse models 106 . How these mtDNA mutations arise is a pressing issue in mitochondrial disease and ageing. Mutations in several mt-nucleoid proteins ? including the Twinkle helicase, PolgA and the Ant1 isoform of the adenine nucleotide translocase ? cause inherited forms of adult-onset neuromuscular diseases 14,107,108 . This indicates that functional loss of other mt-nucleoid proteins, especially those vulnerable to oxidative damage, such as aconitase, might contribute to sporadic or ageing-related mtDNA mutations that are due to compromised mtDNA metabolism and organization. Perturbation of mt-nucleoid segregation might also directly contribute to the pathogenesis of mito- chondrial diseases. In many cases the severity of mitochondrial disorders is determined by the ratio of wild-type to mutant mtDNA molecules in the cells of the affected tissue 103,109 . The ratio of mtDNAs of different genotypes within heteroplasmic cells is determined by how these mtDNAs segregate during development. Bottlenecks in mtDNA segregation during development can lead to dramatic shifts in the extent of heteroplasmy in specific tissues when a fetus inherits mixed mtDNAs from its mother 110 . Defects in the organization and inheritance of mt-nucleoids could directly alter these bottlenecks. Elucidating the molecular mechanisms that underlie mt-nucleoid segregation should help to understand the pathogenesis of these diseases. Figure 4 | A model of the yeast mitochondrial nucleoid segregation apparatus. The core components of the segregation apparatus consist of the Mdm10?Mdm12? Mmm1 (mitochondrial membrane proteins) complex, which might also interact with Mmm2. This core complex forms the contact site between the inner and outer mitochondrial membranes. It is located primarily in the outer membrane, with the possible extension of Mmm1 into the inner membrane and its possible protrusion into the mitochondrial matrix. The inner membrane proteins, Mdm31 and Mdm32, are also proposed to be part of this double membrane-spanning complex, on the basis of genetic interactions with Mdm10, Mdm12, Mmm1 and Mmm2 uniF6AEREF. 100uniF6AF. This supramolecular complex mediates the attachment of mitochondria to actin cables in the cytosol and tethers mitochondrial nucleoids (mt-nucleoids) on the matrix side through as yet unknown proteins. This provides a mechanism for linking mt-nucleoids to the cytoskeleton so that mt-nucleoids move with mitochondria into the progeny cell during cell division 7 . How the Mdm10?Mdm12?Mmm1 complex, Mmm2, and the inner membrane proteins Mdm31 and Mdm32 interact has yet to be clarified. NATURE REVIEWS | GENETICS ADVANCE ONLINE PUBLICATION | 9 REVIEWS � 2005 Nature Publishing Group 1. Nass, M. M. K. & Nass, S. 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Cell cycle dependent morphology changes and associated mitochondrial DNA redistribution in mitochondria of human cell lines. Mitochondrion 1, 425?435 (2002). Acknowledgements X.J.C. is supported by the US National Institutes of Health (NIH) and American Heart Association and R.A.B is supported by the NIH and The Robert A. Welch Foundation. We are grateful to our colleagues for their helpful discussions and a critical reading of the manuscript. Competing interests statement The authors declare no competing financial interests. Online links DATABASES The following terms in this article are linked online to: Entrez: http://www.ncbi.nlm.nih.gov/entrez/query. fcgi?db=gene Abf2 | ACO1 | Cce1 | Fzo1 | ILV5 | MHR1 | Mdm10 | Mdm12 | Mdm31 | Mdm32 | Mgm101 | Mmm1 | mttfa-A | Ugo1 MedlinePlus: http://www.nlm.nih.gov/medlineplus/ medlineplus.html cisplatin Swiss-Prot: http://cn.expasy.org/sprot TFAM FURTHER INFORMATION Ronald Butow?s homepage: http://www.utsouthwestern.edu/ findfac/professional/0,2356,11009,00.html Access to this interactive links box is free online. NATURE REVIEWS | GENETICS ADVANCE ONLINE PUBLICATION | 11 REVIEWS "
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