INTRODUCTION

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Mitochondrial DNA (mtDNA) is essential for most eucaryotic (obligate aerobe) species. Evidently, maintenance of the integrity of mtDNA during somatic divisions and sexual reproduction is of extreme importance for their survival. In particular, deletions, duplications, and point mutations in mtDNA have been shown to cause human diseases, either hereditary or sporadic (see references 115, 138, 174, 318, and 337 for reviews). Cases where mutant and wild-type mtDNA molecules are found in the same tissue define a situation termed heteroplasmy. However, the ratio of the two mitochondrial haplotypes often changes over the life of an individual and can be very different between cell types. Despite their fundamental and clinical importance, the factors which act upon the relative distribution of normal and mutant mtDNAs over individual development remain poorly understood. Recently, experimental studies have attempted to address this question (reviewed in reference 22): one of these stresses the roles of the nuclear background (77). In this context, it is noteworthy that several nuclear genes have been implicated in the concomitant accumulation of several classes of deleted mtDNAs in the same individual. Of these, only one has been recently cloned and characterized (219). It encodes thymidine phosphorylase, which is involved in thymidine catabolism and may be essential for mtDNA maintenance.

While very little information is available on nuclear genes which might (directly or indirectly) control mtDNA maintenance in higher eucaryotes, there is an overabundance of data concerning the budding yeast Saccharomyces cerevisiae. In this species, mitochondrial genetics and studies of nuclear control of integrity and transmission of mtDNA are simplified because of two features: (i) this yeast is a facultative aerobe, and (ii) it is very accessible in terms of classical and molecular genetics. However, in spite of its status as a model system, S. cerevisiae exhibits intrinsic weaknesses with respect to these questions: it is a facultative aerobe, it is unicellular and does not stably maintain a heteroplasmic state, and the structure of its mtDNA differs significantly from that of higher eucaryotes. Nonetheless, one can certainly postulate that at least some of the nuclear factors which control mtDNA integrity and transmission have been conserved through evolution. Thus, an understanding of this nucleomitochondrial problem in yeast may shed light on the relevant nuclear genes in higher eucaryotes. Alternate models are rare and have received very little study (see "Conclusions and prospects" below).

This review examines the field of yeast genes involved in mtDNA maintenance. Due to the large number of articles in this area, we may have missed some significant data and apologize for possible omissions. The bibliography was compiled in May 1999.

The symbols used in this review are those currently used by the yeast community: wild-type genes are in capitals and italics, mutant alleles are in lowercase italics, and proteins are in nonitalics and capitalized only on the first letter (e.g., Mip1 or Mip1p).

The cytoplasmic petite mutants (petite colony, vegetative petites, or simply petites) were discovered 50 years ago (84, 85, 278; see reference 87 for a review). Numerous studies then showed that petite mutants lacked a functional mitochondrial (rho⁺) genome but instead showed extensive deletion of mtDNA (rho) or no mtDNA at all (rho⁰) (reviewed in reference 74; see also reference 238). The petite mutations are very pleiotropic because the mutants cannot perform mitochondrial protein synthesis. These mutations differ in this from mit mutations, which are point mutations in mtDNA causing respiratory deficiency through impairment of only one (or a few) specific function (see reference 281 for a definition of mit mutations and the operational differentiation between mit and rho/rho⁰ mutations). The petite mutations are also characterized by their high rate of spontaneous occurrence: in the majority of yeast strains, one finds a small percentage of spontaneously occurring petite clones. However, it should be stressed that the proportion of petite clones with respect to the wild-type clones (often referred to as frequency, percentage, or ratio of petites) is a classical problem of population genetics. The proportion one establishes experimentally results from both the frequency of mutation of rho⁺ cells to rho/rho⁰ cells (measured per cell per generation or per cell per hour) and the ratio of the growth rates of rho⁺ cells to rho/rho⁰ ones. L'Héritier and coworkers (86) have shown that under stationary conditions, when the proportion of petite clones is of the order of 1%, the frequency of mutation is 10 times lower, on the order of 0.1%. Conversely, Ogur and John (223) have shown that the proportion of petite clones, in a population that has multiplied on nonfermentable medium (i.e., ethanol or glycerol) where the petite cells are unable to multiply (but do not die), is equal to the frequency of mutation multiplied by 2. This frequency can be greatly increased (to complete transformation of the cell population into petites) by drugs such as ethidium bromide (280).

In rho mutants, the remaining fragment of rho⁺ mtDNA is regularly repeated along the mtDNA molecules. There are two major types of repeat arrangements: either the sequence is directly repeated with head-to-tail junctions or the arrangement is of the inverted or palindromic type and the repeat unit is an inverted duplication of the retained sequence (91; reviewed in reference 74).

Numerous studies have shown, by crosses between strains with different mitochondrial genotypes, that the heteroplasmic state of the zygotes is very transient. In fact, their vegetative multiplication is coupled with a rapid segregation of mtDNA molecules, leading to homoplasmic clones. After a cross to the wild type, the petite mutants can be classified according to their suppressiveness, i.e., by the relative proportion of petite zygotic clones. Low or moderate suppressiveness probably results from random segregation of mtDNA molecules over cell divisions, while hypersuppressivity implies that rho molecules are preferentially transmitted to the progeny at the expense of the rho⁺ genome. Numerous data have led to the proposal of a replicative advantage for these rho molecules, since they contain several copies of short sequences called ori (rep), which may represent replication origins of mtDNA (reviewed in reference 74). However, more recent data tend to favor the idea of a segregation (transmission) advantage in hypersuppressiveness (see "Nuclear control of biased transmission" below).

This review focuses preferentially on mutations which increase the level of petite production compared to the relevant wild-type background. However, it also concerns the few available mutations which either modify the biased transmission of some rho genomes (see "Nuclear control of biased transmission" below and Table 2) or slow (even abolish) petite production (see "No compromise" below and Tables 10 and 13). It would have been incomplete without a short summary (see "Petite positivity versus petite negativity" below) of recent data concerning the problem of petite positivity versus petite negativity, i.e., the ability or inability of yeast species to produce petite cells. The genes involved in mtDNA maintenance have been roughly classified by their known functions or effects on the mitochondrial genome (see the tables). However, at the beginning of the review, we have focused on the scientific projects which have helped to identify some genes.

Throughout this review, as far as possible, we have attempted to identify the type of petites (rho⁰ and/or rho) produced in a given genetic background. We are in fact convinced that this is an important point for the understanding of two main problems: (i) why some genetic backgrounds lead to a complete loss of mtDNA (rho⁰ petites) if the relevant genes are not (directly or indirectly) required for mtDNA replication, and (ii) why some genes are essential for the maintenance of a complete (rho⁺) genome but dispensable for the maintenance of a truncated version (rho) of this genome. Several criteria are available: (i) ensuring that the petite colonies which appear at high levels in a given strain are respiration deficient; (ii) ascertaining a defect in mtDNA; and (iii) distinguishing between rho⁰ and rho petites (see Table 1 footnotes). "Physiological" methods can address only the first goal. Genetic and molecular methods clarify the second. The third problem, however, can be solved by 4',6-diamidino-2-phenylindole (DAPI) staining or extensive genetic tests and/or clear-cut molecular procedures. Unfortunately, in some cases mtDNA stability was clearly not a focus of the authors and in other cases the methods which distinguish between rho⁰ and rho petites were not rigorously applied. These facts have prompted us to be (sometimes) more cautious about conclusions than were the authors. For example, in our opinion, lack of DNA hybridization with a probe containing a limited mtDNA fragment does not justify the conclusion that the relevant clone is of the rho⁰ type: it may very well contain a rho genome with this fragment deleted. The same is true for genetic tests using too few mtDNA markers. It is indeed easier to show that a petite is rho than to demonstrate a rho⁰ state. In addition to these methodological problems, other parameters require that caution be used in drawing definitive conclusions: the overall genetic background and the physiological state of the cells can strikingly modify the amount and nature of petites. As emphasized in some cases, these parameters may explain discrepancies between results obtained by different groups. To underline the influence of the genetic context, it seems that a mitochondrial genome lacking introns is more stable than an intron-containing genome (see below). To place the physiological parameters in a more concrete form, three observations can be reported. First, environmental parameters can strikingly influence petite production in mutant strains. A remarkable example concerns the effects of both temperature and culture medium on the abf2 mutants (see "The ABF2 paradox" below). Second, most rho mutants are not very stable and may evolve to the rho⁰ state (see, as an example, the discussion of overexpression of ADR1 in "Gene overexpression can increase mtDNA instability" below). Thus, a conclusion about the nature of the petites produced by a given mutant may depend on the age of the culture (see, for instance, the discussion of MGM101 in the following section). Third, petite production due to disruption of a nuclear gene can be significantly different depending on the procedures used. Disruption can be achieved in a haploid vegetative cell, and its effect upon petite production can be immediately observed; disruption can be achieved in a diploid cell, and its effect is observed in haploid cells resulting from sporulation of the heterozygous diploids. These two procedures can lead to different observations (as, for instance, in rpo41 mutants; see "Does transcription play a direct role in mtDNA maintenance?" below and Table 3).

Nuclear genes involved in the maintenance of mtDNA can be identified by a systematic search for mutations increasing rho⁰ production at elevated temperature. Screening for heat-sensitive mutations helps to recover mutations in genes essential for cell survival. This procedure was used by Genga et al. (103): among 360 clones able to grow on glycerol at 25°C but not at 35°C, 31 clones were heat sensitive due to massive production of petites, of which 12 completely lacked mtDNA. These 12 mutants belonged to 11 complementation groups. Only one mutant displayed a deficiency in mtDNA synthesis. This identified the MIP1 gene (Table 1), encoding the catalytic subunit of mtDNA polymerase (94). In addition to the initial mutations and deletion, other mip1 mutations impairing replication fidelity have been obtained. Some yield a high level of petites either at 36°C or at both 36 and 23°C. Interestingly, these mutations show a gene dosage effect, since heterozygous diploids are strong producers of petites (139). This effect was also observed (at 36°C) for diploids containing a single copy of the MIP1 gene (177). The data suggest that Mip1p is produced in limited amounts and that the rho⁺ genome cannot be maintained below a particular threshold of that protein.

In 1992, Jones and Fangman (149) used a similar procedure to identify genes required for mitochondrial genome maintenance (MGM genes). This led to the identification of MGM1, encoding a protein whose GTP-binding domain is related to dynamin (117, 149) (see Table 11). Careful examination of DAPI-stained cells of the mgm1-1 mutant (which lost mtDNA after transfer at high temperature) strongly suggests that the defect of the mutant is not due to an asymmetric distribution of mtDNA during cell divisions. Furthermore, deletion of MGM1 causes not only loss of the rho⁺ genome but also that of rho genomes, even those containing a rep (ori) sequence (149). In addition, Guan et al. (117) showed that disruption of MGM1 led to a structural alteration of mitochondria and a defect in mitochondrial Hsp58 protein import. Recently, Shepard and Yaffe (272) have demonstrated that Mgm1 is an integral protein of the outer mitochondrial membrane, which plays a key role in mitochondrial morphology and inheritance (see "Mitochondrial morphology, the cytoskeleton, and mtDNA" below for a further discussion of MGM1).

More recently, Guan (118) described a similar program which led to the recovery of 120 mutants exhibiting temperature-induced loss of mtDNA (rho⁰). These mutants belong to 36 complementation groups. Thirteen mutants contained mutations that could be assigned to six previously described genes: MIP1 (see above and Table 1), CDC8 and CDC21 (see "mtDNA and the cell cycle" below) (Table 1), RPO41 and MTF1 (see "Does transcription play a direct role in mtDNA maintenance" below and Table 3), and MGM1 (see above, "Mitochondrial morphology, the cytoskeleton, and mtDNA" below, and Table 11). This genetic analysis thus offered 30 new genes involved in mtDNA maintenance, of which 2 have been analyzed to date: MGM101 (Table 1) and MGM104 (see Table 9).

The MGM101 gene was sequenced and disrupted. The predicted amino acid sequence showed no similarity to sequences of known proteins. The disrupted gene led to loss of mtDNA (38). Recently, Meeusen et al. (205) found Mgm101 among proteins associated with mitochondrial nucleoids. These authors have also isolated a new heat-sensitive allele of MGM101 (mgm101-2) and attempted, by extensive biochemical and genetic approaches, to determine the precise function of Mgm101p. The data have led to the following conclusions. The protein binds to DNA, which probably explains its specific association with the nucleoid. However, it is not involved in mtDNA packaging, segregation, or partitioning. It is not required for mtDNA replication. Its loss does not cause defects in morphology or transmission of the organelle. In fact, the only clear-cut phenotypic defect of mgm101-2 is the sensitivity of mtDNA to damage induced by UV irradiation and the hypersensitivity of mtDNA to damage induced by gamma rays and H₂O₂. Therefore, the authors propose that MGM101 performs an essential function in the repair of oxidatively damaged mtDNA required for mtDNA maintenance. Interestingly, using the heat sensitivity of mgm101-2, they observed that after 12 h at 37°C, when the cell population is 100% respiration deficient, only 50% of cells are rho⁰, while after a longer exposure to nonpermissive temperature (16 h), the entire population consisted of rho⁰ cells. Thus, at least in this case, this rho⁰ producer exhibits a transition from the rho⁺ to the rho state before undergoing a complete loss of mtDNA (205).

The mgm104-1 mutation causes a greatly increased production of petites at 25 and 37°C, with a stronger effect at high temperature. Attempts to clone the MGM104 gene led to the identification of the TTS1 gene, which encodes the cytosolic tyrosyl-tRNA synthetase (44). TTS1 is able to rescue the mgm104-1 phenotype, even when present on a low-copy-number vector, but is not thought to be the cognate MGM104 gene. This conclusion was drawn since no specific mutations were found in the coding or promotor regions of the TTS1 gene in the mgm104-1 mutant (118). The author suggests two major hypotheses: either Tts1p has a dual function in cytosolic translation and mtDNA maintenance, or it may interact with the mgm104-1-encoded protein and enhance its activity, thus rescuing the phenotypic defect of the mutant. However, one cannot exclude a third hypothesis: a slight overexpression of the tyrosyl-tRNA synthetase results in mischarging of noncognate tRNAs and thus in informational (missense) suppression of the mgm104-1 mutation. The author suggested that additional plasmids able to complement this mutant were required to identify the cognate gene. However, since the TTS1 mRNA is about 60% less abundant in the mgm104-1 strain than in wild type, one cannot exclude the possibility that the mutation lies in the 3' noncoding sequence of TTS1: this would lead to instability of the messenger and cause the mgm104-1 phenotype. In fact, alteration of cytosolic translation may affect mtDNA stability, as shown in the filamentous fungus Podospora anserina (see "Conclusions and prospects" below).

The main advantage of such a wide screening procedure for nuclear mutations leading to complete loss of mtDNA is that it does not assume any mechanism. This is stressed by the few examples cited above: this type of program has permitted the recovery of genes involved in mtDNA replication, genes involved in mtDNA transcription, and genes whose functions are still unknown. Guan's assessment (118) also shows that the screening procedure is far from saturated. This explains why several genes whose mutations lead to rho⁰ production were not identified in his experiments. Among those involved in mtDNA metabolism, one finds, for instance, PIF1, RIM1, ABF2 (discussed below), and PPA2, which encodes a mitochondrial pyrophosphatase (Table 1). Inorganic pyrophosphate (PP_i) is generated in a number of biosynthetic reactions, notably replication and transcription. Hydrolysis of PP_i is thus an essential process which ensures the forward direction of these reactions. Lundin et al. (197) cloned the gene (PPA1) encoding the cytosolic enzyme and used it as a probe for the gene (PPA2) encoding the mitochondrial enzyme. Cells with PPA2 deleted are unable to grow on respiratory carbon sources. DAPI staining showed that these cells have lost their mtDNA (Table 1), which is in accordance with the evident requirement for this enzyme in DNA replication (197).

In addition to genes specifically involved in mtDNA replication discussed in this section, four genes, required for replication of the mitochondrial and the nuclear genomes (CDC8, CDC21, RNR2, and RNR4) were shown to control mtDNA stability (Table 1). Because of their dual cellular function, it seemed to us more convenient to discuss these genes below in "mtDNA and the cell cycle."

As stressed in the Introduction and in "The petite syndrome" (above), mtDNA maintenance is dependent on two basic parameters: the frequency of mtDNA deletions and the management of heteroplasmic states, i.e., the relative transmission of wild-type and altered molecules during cell divisions. In 1991, Zweifel and Fangman (339) found a mutation which disrupted the biased inheritance of hypersuppressive rho genomes (Table 2), thus identifying the first MGT (for "mitochondrial genome transmission") gene. Strikingly, the MGT1 gene was rediscovered by a totally different procedure. Originally, Kleff et al. (158) were interested in a cruciform cutting endonuclease (Cce), which is required during recombination to resolve Holliday junctions. They developed an assay to detect this activity in crude yeast extracts and sought mutants lacking it. Approximately 150 heat-sensitive mutants were screened before a relevant (cce1) mutant was found. Cloning of the CCE1 gene and assays for Cce1p activity yielded evidence that CCE1 was the structural gene for the cruciform cutting endonuclease. It was shown that cce1 mutants did not affect nuclear recombination but that CCE1 and MGT1 were allelic: the mgt1 mutants showed no Cce1p activity and, in turn, the cce1 mutant was resistant to hypersuppressive rho. It was then demonstrated that Cce1p was localized to the mitochondria and associated with the inner membrane (89). Later, Lockshon et al. (194) demonstrated that the lack of Cce1p led to an increased ratio of mtDNA molecules linked by recombination junctions, resulting in aggregation of mtDNA molecules in a smaller number of cytological structures than in wild-type cells. This effect was more pronounced for rho than for rho⁺ genomes. In accordance with these results, overexpression of MGT1/CCE1 reduced the frequency of branched structures and enhanced the transmission of nonsuppressive rho genomes (Table 2). In a recent report (239), it was shown that the mgt1/cce1 background also influences the transmission of respiration-competent mtDNAs lacking intergenic sequences involved in the biased transmission of hypersuppressive rho mutants. Thus, the resolution of recombination junctions plays a key role in the segregation and transmission of yeast mtDNA. However, deletion of MGT1/CCE1 causes only a slight increase in rho production (158). It is noteworthy that this increase is more marked in the presence of a deletion of the NUC1 gene, encoding the major mitochondrial exoendonuclease. This gene was cloned by reverse genetics, using an antibody against the protein. Unexpectedly, neither deletion nor overexpression of the gene resulted in phenotypic effects (334). However, at that time, the authors did not perform extensive analyses of rho production or recombination rates in these two contexts. This should now be undertaken in the light of the interaction between nuc1 and mgt1/cce1 deletions.

View this table: [in this window] [in a new window]   TABLE 2.   Genes which influence biased transmission of rho genomes

Unfortunately, despite its evident interest, very few analyses have been devoted to the problem of biased transmission. However, in addition to the MGT1/CCE1 gene, data concerning the PIF1 gene are available. This gene (described in "The recombination/repair track" below) encodes an mtDNA helicase involved in recombination between rho⁺ and tandemly organized rho genomes. With respect to rho transmission (in crosses with a rho⁺ partner), a high level of suppressiveness was observed when diploids were homozygous for the pif1 mutations. This occurred for all types of rho, regardless of structure (tandem or inverted organization), and was not dependent on the sequence or the size of the repeat (96) (Table 2). This general effect of the pif1 mutants remains unclear. However, when secondary rho clones were obtained from a tandem rho insensitive to the recombinogenic effect of PIF1, those which showed PIF1 sensitivity became hypersuppressive in a pif1 context (98).

Thus, the MGT1/CCE1 and PIF1 data suggest a strong link between mtDNA recombination and transmission at least for rho genomes. With respect to this remarkable point, a systematic analysis of other genes involved in recombination (see "The ABF2 paradox" and "The recombination/repair track" below) should be undertaken.

The presence of a histone-like protein inside mitochondria (HM) was first reported 20 years ago (32). This 20-kDa protein was then rediscovered by Certa et al. (33) in the search for a histone H1 in yeast. It was lastly and serendipitously identified as an ARS binding factor, i.e., a protein binding an autonomously replicating sequence of the nucleus (68). A few years later, it was shown that Abf2 was a mitochondrial protein closely related to DNA-binding proteins of the HMG (high-mobility-group) family (69). These authors showed that abf2 null mutants lose their mtDNA when grown on rich glucose medium (Table 1). However, when tetrads issued from diploids heterozygous for the abf2 null mutation were dissected directly on a nonfermentable substrate, the mutant strains could grow and be maintained indefinitely.

Evidence that HM and Abf2 were the same protein was provided by Megraw and Chae (206). In addition to confirming the data of Diffley and Stillman (69), they added two interesting observations. First, disruption of ABF2 results in a heat-sensitive phenotype on respiratory substrates due to mtDNA instability, as shown by the fact that the abf2 mutant is lethal in a op1 context (see "No compromise" below and Table 13 for the op1 mutation and its lethality in a petite background). Second, overexpression of ABF2 leads to a high frequency of rho⁰ petites (Table 1). The same year, three papers dealt with the issue of the evolutionary relationship of Abf2 and DNA-binding proteins involved in DNA maintenance and/or expression. Abf2p is the functional homologue of the bacterial histone-like protein HU: despite the lack of sequence similarity between the two proteins, Abf2 and HU can substitute for each other (206). When directed toward the mitochondria, a yeast nuclear HMG protein is able to complement an abf2 mutant (153). Functional similarities were discovered between a human mitochondrial transcriptional activator (mtTFA or mtTF1) and Abf2p: the human protein partly rescues the phenotypic defects of a abf2 strain (227). However, the two proteins display structural differences. In particular, the tail region of mtTFA, which plays a critical role in its transcriptional activity, is absent in Abf2p (59a). The data as a whole (see also reference 309a) indicate that, unlike its human homologue, Abf2p plays only a small, if any, role in mtDNA transcription, a notion supported by the fact that abf2 mutants are able to maintain and express mtDNA under specific growth conditions (69, 336).

In a recent report, Zelenaya-Troitskaya et al. (335) exemplified the pleiotropic role that ABF2 may play with respect to mtDNA maintenance. These authors show (i) that the distribution and mixing of mtDNA from two abf2 parental cells after zygote formation differ slightly from the patterns observed in wild-type zygotes; (ii) that recombination between mitochondrial markers is reduced five- to sevenfold in the mutant context; (iii) that rho genomes (introduced through cytoduction experiments) are stable in abf2 mutants, in contrast to rho⁺ genomes; (iv) that (in accordance with the data of Megraw and Chae [206]) hyperexpression of ABF2 leads to a complete loss of mtDNA (Table 1); and (v) that a moderate (two- to threefold) increase in the level of the protein raises the content of mtDNA without affecting mtDNA stability. These data have been completed by Okamoto et al. (224a) and by MacAlpine et al. (198). In the first case, the authors showed that in a rho⁺ × rho⁰ zygote, mtDNA (stained by DAPI) is preferentially transmitted to the diploid bud while (GFP-tagged) proteins have equilibrated throughout the zygote and the bud. In homozygous abf2 crosses, mtDNA sorting is delayed and preferential sorting is reduced (224a). In the second case, the authors analyzed the effect of either a null allele or overexpression of ABF2 upon levels of mtDNA recombination intermediates. In rho⁺ genomes this level is reduced when Abf2p is absent and increased when Abf2p is overproduced. In contrast, the absence of Abf2p does not influence the level of recombination intermediates of rho genomes (198).

Three multicopy suppressors of abf2 phenotypes have been isolated to date. Two (SHM1/YMH1 and YMH2) were identified by their ability to restore the growth of abf2 null mutants at 37°C on a respiratory substrate. They could encode mitochondrial carrier proteins. Deletion of SHM1/YMH1 has no effect on mtDNA in a ABF2 context (152). Interestingly, YMH2, whose deletion leads to respiration deficiency, appears to be the first membrane-bound protein associated with the mitochondrial nucleoid in vivo (43). We discuss this important property of YHM2 in "Is there a centromere-like structure for the mitochondrial genome" (below). The third suppressor is ILV5, isolated by its ability to restore mtDNA stability in an abf2 null mutant grown on rich glucose medium. The gene encodes an enzyme of the biosynthesis of branched-chain amino acids (see "The secret life of well-known proteins", below, and Table 8) (336). This discovery and subsequent analyses revealed that mtDNA is highly stable in abf2 mutants grown on glucose medium lacking leucine, isoleucine, and valine, a condition which enhances the expression of ILV5. The inevitable conclusion is therefore that ABF2 is dispensable for mtDNA stability when ILV5 is sufficiently expressed! The effect of ILV5 on petite production by abf2 mutants at high temperature has not yet been tested.

In any case, the data as a whole suggest that Abf2p plays a role in the recombination process. However, they raise more questions than they solve. Why (as observed in other cases) are rho genomes stable in the abf2 mutants while rho⁺ genomes are not? Why do abf2 mutants completely lose their mtDNA even though they are able to maintain rho genomes? Why does hyperexpression of ABF2 lead to loss of rho⁺ and rho genomes? Why are abf2 null mutants unable to maintain a functional mtDNA on respiratory substrates at high temperature? Zelenaya-Troitskaya et al. (335) favor the idea that the main effects of ABF2 result from its role in recombination. This hypothesis is consistent with many data concerning both the properties of HMG proteins and the role of recombination intermediates in mtDNA transmission (reference 335 and references therein) (see also the discussion of MGT1/CCE1, above). However, as pointed out by the authors (335), this hypothesis is difficult to reconcile with the data obtained from the MGT1/CCE1 analysis: in this case, lack of the protein has a very slight effect on mtDNA stability (194). Another interpretation (198) suggests an indirect effect of Abf2p on replication through recombination structures. However, the naive point of view would be that ABF2 is highly pleiotropic: in fact, as a DNA-packaging protein, which plays a role in mtDNA and nucleoid organization (218), Abf2p may be (directly or indirectly) involved in mtDNA replication, recombination, and distribution. The data would therefore be explained by the sum of discrete effects, more or less pronounced depending on the growth conditions and the structure (rho⁺ or rho) of the mitochondrial genome.

Yeast mitochondria are very active in recombination (76), and it has been suggested that mtDNA deletions which give rise to the rho genomes can occur through homeologous recombination between imperfect repeats (102, 279). Although this mechanism does not apply to all petite deletions, mutations affecting recombination may aid in identifying genes involved in mtDNA maintenance.

A direct screen for recessive mutations altering mtDNA recombination is impossible since the experimental test requires the fusion of two cells carrying different mtDNAs: in the resultant diploid, the nuclear mutation cannot be expressed. However, Foury and Kolodynski (96) succeeded by using a genetic ruse, i.e., a mutation which delays nuclear fusion; thus, the daughter cells of the zygote contain a haploid nucleus along with a mixed mtDNA population. By using this procedure, they obtained three allelic mutants which identified the PIF1 (for "petite integration frequency") gene. The pif1 mutations affect the frequency of recombination between rho⁺ and certain rho genomes, which are tandemly organized. The rho genomes with an inverted organization are not sensitive to these mutations. Similarly, recombination between rho⁺ genomes is identical in the wild-type and mutant backgrounds. The pif1 mutations are pleiotropic since, in addition to their specific effects on recombination, they increase the level of rho induced by UV irradiation, cause a total loss of mtDNA at high temperature (Table 1), and increase the suppressiveness of all types of rho genomes (Table 2). PIF1 encodes an mtDNA helicase which is thus involved in recombination, repair, and mtDNA maintenance (169). Amazingly, it was later shown that the protein also acts in the nucleus by inhibiting telomere elongation and de novo telomere formation (268). In fact, PIF1 encodes two forms of the protein through the alternative use of two AUG codons: the longer form goes to the mitochondria, and the shorter form goes to the nucleus.

Although its role in mtDNA repair and recombination remains to be elucidated, we must mention here the RIM1 (for "replication in mitochondria") gene. RIM1 was discovered as a partial suppressor of the heat-sensitive phenotype of the pif1 null mutants (310). Interestingly, the suppressor effect was observed with a few additional copies of the RIM1 gene (i.e., when carried on a centromeric vector). Unfortunately, it has not been reported if strong RIM1 overexpression could lead to a complete suppression of pif1 mutants. RIM1 encodes a mitochondrial single-stranded DNA-binding (SSB) protein required for mtDNA maintenance; deletion of this gene causes complete mtDNA loss (310) (Table 1). Since the heat sensitivity of the pif1 disruptants remains unclear, it is difficult to speculate on the precise role of Rim1p in mtDNA stability. However, SSB proteins and DNA helicases often perform complementary functions in DNA metabolism, involving unwinding of duplex DNA. Thus, Rim1p might be involved not only in mtDNA replication but also in mtDNA recombination, repair, and transcription (310). As suggested by the authors (310), it would be interesting to test the ability of RIM1 to suppress the defects of the pif1 mutants in mtDNA repair and recombination. It could also be informative to know the effect of strong overexpression of RIM1 in a PIF1 (wild-type) background.

A genetic procedure linking mtDNA repair and recombination was used by Ling et al. (183). Of two mutants displaying an elevated UV induction of rho mutants, one also exhibited a deficiency in gene conversion. The MHR1 (for "mitochondrial homologous recombination") gene is also involved in mtDNA maintenance at high temperature (Table 1). Genetic localization showed that MHR1 was a previously unknown gene.

A direct search for genes involved in DNA repair was undertaken by Reenan and Kolodner (245) to identify the yeast homologue of the mutS gene, which encodes a component of the bacterial MutHLS system. This was performed by reverse genetics, using degenerate oligonucleotide primers and PCR. Two genes were identified, one of which encoded a protein targeted to the mitochondria. Disruption of the MSH1 (for "MutS homologue") gene leads to a strikingly rapid accumulation of rho mutations, mostly hypersuppressive (246) (Table 1). DAPI staining of the mutant cells revealed large fluorescent patches instead of the small dispersed patches observed in wild-type cells. This phenotype might be the result of either altered mtDNA distribution or abnormal morphology and distribution of mitochondria (246). In addition, Chi and Kolodner (42) noted that strains heterozygous for a MSH1 deletion accumulated mtDNA point mutations (mit) faster than wild-type strains did. The authors propose a role for Msh1p in the suppression of homeologous recombination, as supported by the in vitro binding specificity of the protein for DNA with multiple mismatches and loops of unpaired nucleotides. They also suggest that Msh1 could interact with other mitochondrial proteins, especially Pif1 and Rim1 (see above) (Table 1), which are, respectively, a DNA helicase and an SSB protein: they have similarities to components of the Escherichia coli MutHLS system, and both are required for mtDNA maintenance. This assumption could be tested by using the two-hybrid system in yeast or, even more clearly, by performing coimmunoprecipitation experiments, since the three proteins, Pif1, Rim1, and Msh1, have been purified.

Thus, the recombination/repair approach (which has been difficult to perform) appears very promising with respect to mtDNA maintenance, even though most of the genes required for these processes in mitochondria remain to be identified. Furthermore, as described in "Nuclear control of biased transmission" (above), this specific screen also identified the CCE1 gene (allelic to MGT1 [Table 2]), encoding a mitochondrial cruciform cutting endonuclease (158). Although discovered by other methods, ABF2 (see "The ABF2 paradox" above) must be added to the list of the genes involved in recombination. In fact, among the genes required for mtDNA maintenance, four are clearly involved in recombination processes: MGT1/CCE1, PIF1, MHR1, and ABF2. However, in the first three cases, recombination between markers in rho⁺ × rho⁺ crosses is unaffected or only slightly affected in the relevant mutant backgrounds (96, 183, 339). Thus, ABF2 is the only gene among these four to be implicated in intergenic recombination between rho⁺ genomes (335). There is another striking point concerning ABF2: it stands apart from MGT1/CCE1 with respect to recombination intermediates. In fact, their level is reduced in the absence of ABF2 and increased in the absence of MGT1/CCE1; moreover, these effects are seen only on rho⁺ genomes in the first case and preferentially on rho genomes in the second case (194, 198). Clearly, the field of recombination has been underinvestigated.

It has long been known that mitochondrial protein synthesis is required for maintenance of rho⁺ genomes (reference 213 and references therein). This conclusion has been confirmed over time: all nuclear or mitochondrial mutations which completely block mitochondrial translation inescapably lead to rho genome production (see below). In accordance with this constraint, mutations which impair mtDNA transcription should lead to rho genome production. The transcriptional apparatus has also been thought to be directly involved in mtDNA replication. This assumption is based on indirect (mostly in vitro) data, suggesting that (at least in vertebrates) mtRNA polymerases are primases for mtDNA replication (reviewed in reference 49). Thus, transcription might play a dual role in mtDNA maintenance: directly through initiation of mtDNA synthesis and indirectly through translation. However, evidence for a direct role is still questionable.

The RPO41 gene encoding the catalytic subunit of mtRNA polymerase in yeast was cloned by reverse genetics (155). Disruption experiments led to the conclusion that this enzyme was required for mtDNA maintenance (112) (Table 3). Since the authors provided evidence for a complete loss of mtDNA in a rpo41 mutant background, it seemed of utmost importance to determine if Rpo41p was also required for maintenance of rho genomes. This experiment was performed by Fangman et al. (90). They used two types of rho strains. The first was hypersuppressive, containing an (amplified) mtDNA fragment with an ori/rep sequence, which is assumed to offer a replicative advantage in matings with rho⁺ strains. The second was neutral, lacking such sequences and consisting solely of AT base pairs. Such rho genomes replicate by an unknown process and are most often lost after mating with a rho⁺ strain. Both rho genomes were maintained in haploid strains carrying a disrupted rpo41 gene. Thus, Rpo41p did not seem to be required for rho mtDNA replication. This observation prompted the authors to reexamine the effects of RPO41 disruption on rho⁺ genomes. In contrast to the results of Greenleaf et al. (112), they observed that the rpo41 mutation did not cause a complete loss of mtDNA but led mainly to the accumulation of rho genomes (90) (Table 3). As emphasized by the latter authors, this discrepancy is probably due to the experimental procedures used in the two laboratories. In one case (112), the observations were performed on cultures obtained from haploid spores which inherited the rpo41 mutation through meiosis, while in the other case (90), the observations were made on vegetative cells after inactivation of the RPO41 gene. When these authors repeated the experiment by sporulating a diploid strain heterozygous for the rpo41 mutation, they observed a lower recovery of mtDNA than in their previous experiment with vegetative cells. These data as a whole lead to three clear-cut conclusions. First, inactivation of RPO41 does not cause a complete loss of mtDNA. Second, even if the mutant strain produces a large amount of rho⁰ petites, cytoduction experiments provide evidence that it is able to maintain rho genomes. This interesting feature has also been noticed for the abf2 mutants (see "The ABF2 paradox" above). Third, conclusions about the effect of a nuclear mutation upon the production of rho⁰ versus rho petites must be drawn cautiously, since they may depend on the experimental procedures used in different laboratories. In particular, this could explain why rpo41 mutations were recovered by Guan (118) among rho⁰ producers (see "Wide-scale screening for rho⁰ production" above).

Another very interesting way to test the putative role of transcription for replication was to analyze the effects of a rpo41 deletion on biased transmission of hypersuppressive rho, assumed to result from their replicative advantage. This experiment was also performed in Fangman's laboratory (196). Since rho⁺ genomes are unstable in a rpo41 background while rho genomes are stable, the authors examined diploid clones issued from matings between hypersuppressive and neutral petites. The hypersuppressive rho genomes were always preferentially transmitted regardless of the nuclear background, RPO41 or rpo41 (deletion). The authors proposed two hypotheses based on all available data. First, hypersuppressive petites indeed have a replication advantage, but transcription is not required for replication initiation (at least for rho genomes). Second, the preferential transmission of rho genomes containing a ori/rep sequence is not at the level of replication but at the level of segregation. This hypothesis is in agreement with the deletion and overexpression of MGT1/CCE1 (see "Nuclear control of biased transmission" above and Table 2), which suggest that the resolution of recombination junctions could play a key role in the segregation and transmission of mtDNA. A similar hypothesis has also been proposed for ABF2 (see "The ABF2 paradox" above).

The catalytic (Rpo41) subunit of mtRNA polymerase requires an additional factor for promoter recognition. This factor (Mtf1 or sc-mtTfb) was initially characterized biochemically (264), while the gene was first identified as a high-copy suppressor of an rpo41 heat-sensitive mutant (188) (Table 3). The relationship between the protein and its structural gene was then established by Jang and Jaehning (144) using reverse genetics. The geneticists (188) claimed that disruption of MTF1 led to a complete loss of mtDNA. However, since their experimental evidence was limited to a lack of hybridization with a single probe (21S rDNA), we must be more cautious (Table 3). Directed mutageneses of the MTF1/mtTFB gene were performed by Shadel and Clayton (271). The mutants showing respiration deficiency were not analyzed at the mtDNA level. This would have been especially interesting with respect to a small deletion (of 5 amino acids) resulting in a mutant phenotype, while the protein showed significant levels of transcription in vitro. This was the only mutant of the series exhibiting a contradictory (in vitro/in vivo) phenotype. This led the authors to propose that the Mtf1/mtTfb protein could have a mitochondrial function other than transcription.

Thus, to date, the data neither prove nor disprove a role of transcription in replication initiation of the rho⁺ genome. However, they lead to two conclusions concerning petite mutants: (i) rho genomes (either hypersuppressive or neutral) do not require transcription for their replication, and (ii) preferential transmission of hypersuppressive rho genomes is maintained in the absence of transcription.

THE TRANSLATION-ATP SYNTHASE CONNECTION

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Background

The idea that mitochondrial translation is required for mtDNA stability was first suggested by experiments demonstrating that growth of yeast in the presence of inhibitors of mitochondrial protein synthesis caused a high frequency of rho genomes (references in reference 213). This idea has been reinforced by these authors, who showed that the inactivation of four genes involved in mitochondrial translation led to loss of wild-type mtDNA (213). They also showed that induction of rho was observed only if the mutations were stringent and not if the relevant nuclear genes carried leaky mutations. Finally, they provided evidence that rho genomes could be maintained in these contexts, which thus appeared deleterious only for the wild-type mitochondrial genome. In these experiments (213) (Table 3), the genes studied encoded two aminoacyl-tRNA synthetases, one elongation factor, and a putative ribosomal protein later shown to be a bona fide r-protein (60). To date, there has been no exception to the rule: rho genomes are produced at high frequency as long as a stringent mutation occurs in a mitochondrial or nuclear gene encoding an essential component of the mitochondrial translational apparatus. It would be redundant to quote all reports on the subject published over the last 14 years.

One must keep in mind that the effect of a mutation may be indirect, if the product of the relevant gene acts upstream of translation. Among many examples, two illustrate this fact. The first case concerns the RPM2 gene, encoding the protein component of a ribonucleoprotein required for 5' maturation of mitochondrial tRNAs (154, 210). A less obvious example concerns the NAM1 gene, first identified as a multicopy suppressor of mitochondrial splicing deficiencies caused by mitochondrial mutations (73). The effects of NAM1 inactivation were carefully analyzed in two mitochondrial genetic backgrounds, i.e., in strains carrying either an intron-containing or an intronless mitochondrial genome. The data led to the conclusion that Nam1p was required for the processing and/or stability of cytb and cox1 intron-containing pre-mRNAs and of the atp6 transcript (which does not contain introns) (116). Interestingly, NAM1 inactivation leads to 20 to 50% petite production in a strain with an intron-containing mitochondrial genome (16) while there is no increase in petite production (with respect to wild type) when the mitochondrial genome does not contain introns (116). According to the authors, the high level of petites observed in the first context may be explained by a defect in mitochondrial translation due to a starvation for tRNA^Glu, which is cotranscribed with the cytb gene, the intron-containing cytb transcript being unstable in the absence of Nam1p. Although this attractive hypothesis has yet to be experimentally tested, it strikingly illustrates the chain of events which can indirectly link a mutation with mitochondrial translation and hence with petite production.

In 1985, Myers et al. (213) proposed two hypotheses concerning the role of mitochondrial translation in maintenance of the rho⁺ genome. First, mtDNA would encode a protein required for replication or repair. Second, perturbation of the inner membrane (due to the lack of a component encoded by mtDNA) might specifically prevent the import of proteins required for replication or repair of the rho⁺ genome. Fourteen years later, the question remains open. However, many data have meanwhile focused on the mitochondrial ATP synthase.

The yeast mitochondrial ATP synthase (ATPase) contains at least 14 subunits, of which 5 (, , , , and ) are found in the catalytic F₁ sector (responsible for both ATP synthesis and hydrolysis) while the rest participate in the proton membrane channel F₀ sector (see reference 232 for a general review on ATPases and Table 4 for yeast mtATPase). Three of the F₀ subunits (Atp6, Atp8, and Atp9) are encoded by the mitochondrial genome, and the remainder of the complex is specified by nuclear genes (Table 4).

Two of the three mitochondrial genes, those encoding Atp9 (54) and Atp6 (254), were initially identified through mutations conferring resistance to oligomycin, an inhibitor of mitochondrial ATPase (OLI1 and OLI2, respectively) (10). Later, mit mutations leading to the loss of ATPase activity were obtained, defining two genes, PHO2 and PHO1, which mapped near the OLI1 and OLI2 loci, respectively (54, 97). These loss-of-function mutations produced high levels of petites (Table 4). To simplify the nomenclature, the genes are now called ATP9 and ATP6. A final group of mit mutations helped to identify the third gene, AAP1 (ATP8), encoding Atp8 (199). Again, the mutants were high producers of petites (214) (Table 4). At the time, it seemed clear that mutations in the ATPase mitochondrial genes so far identified were inherently unstable with respect to the mitochondrial genome. This conclusion was strengthened by the observation that very few mit mutations were found in these three genes compared to the mitochondrial genes encoding cytochrome oxidase subunits and cytochrome b. For instance, it was reported that mutants with deficiencies in cytochrome b and in cytochrome oxidase were, respectively, 50 and 300 times more frequent than mutations in the ATP9 gene (54, 163, 307). As discussed by these authors, the rare occurrence of the ATP9 mutants could have two explanations. First, one must take into account the relative sizes of the targets: ATP9 is a small gene (this is also the case for ATP6 and even more so for ATP8, which encodes a 48-amino-acid protein). Second, all loss-of-function mutations in the ATP9 gene (and in ATP6 and ATP8 as well) are unstable. In fact, one can presume that all ATP9 (and ATP6 and ATP8) mutations able to produce nearly 100% petites could not have been recovered as mit mutations. This hypothesis implies that most mit mutations in the three relevant genes are leaky. Although frameshift and nonsense mutations have been obtained in these genes, this does not imply stringent mutations: phenotypic suppression occurs in mitochondria, due to its error-prone translational apparatus (see reference 284 for an example of a leaky frameshift mutation in the ATP6 gene).

This high instability of the rho⁺ genome, observed when the F₀ sector of ATPase is deficient, is not limited to the mitochondrial genes. In fact, mutations in nuclear genes encoding the other F₀ subunits also display this property (Table 4). Three F₀ subunits (e, g, and k) have been omitted in Table 4 since they are not required for the formation of enzymatically active ATP synthase (6).

In 1973, Ebner and Schatz (79) showed that a nuclear mutant (pet936) lacking F₁ produced a large number of petites. However, this observation was forgotten, and before 1992 it was believed that the F₁ ATPase sector was not involved in mtDNA stability (see, for instance, reference 2). In fact, at that time two pet mutations, later shown to lie in the structural genes for  and  subunits (260, 297), appeared genetically stable with respect to mtDNA (Table 4). In any case, the situation was strikingly modified when it appeared that deletions of the genes encoding the three other F₁ subunits (, , and ) led to a more or less pronounced production of petites (Table 4). Very recently, Lai-Zhang et al. (170) showed that strains carrying a deletion of either ATP1 (subunit ) or ATP2 (subunit ) produce less than 1% petites. The authors did not discuss the fact that deletions of either one of these two genes give a petite level 10-fold below what they observed in the wild-type strain from which the deletions were obtained (Table 4). Our hypothesis is that these mutants cannot survive in a petite context (see "No compromise," below, for a more extensive discussion of nuclear mutants killed by mtDNA instability).

In addition to these structural genes, several nuclear genes whose products are involved either in ATPase subunit synthesis or in assembly of the complex have been identified. Three concern the expression of the mitochondrial ATP9 gene: AEP1 (for "ATPase expression protein"), AEP2 (ATP13), and NCA1 (for "nuclear control of ATPase"). The first is necessary for ATP9 translation (231). Disruption of the AEP1 gene leads to a high frequency of petites (230). The second gene, isolated by two research groups, is required for ATP9 transcript maturation or stabilization. The original pet mutant, which helped identify the ATP13 gene (1), showed a low level (8%) of petite production (306). This can be explained by some leakiness of the mutant, since disruption of AEP2 (ATP13) clearly shows an instability of mtDNA (93). The third gene (NCA1) involved in ATP9 expression is required for transcript stability, but there are no data concerning the effects of either the original mutation or the NCA1 disruption upon petite production (338).

Three other genes are required for stabilization (NAM1) or normal maturation (NCA2 and NCA3) of the ATP8-ATP6 cotranscripts. As mentioned above, inactivation of the NAM1 gene leads to a high production of petites in a strain bearing an intron-containing mitochondrial genome (probably due to an indirect effect on translation) whereas there is only a basal level of petites when the mitochondrial genome contains no introns. In fact, in the latter condition, the only defect of the nam1 mutant is a diminution of the ATP6 transcripts. However, this is a leaky phenotype, as shown by the fact that the nam1 strain is able to grow (although only slowly) on glycerol-containing medium at 28°C: this probably explains the mtDNA stability. It would have been interesting to observe petite production by the mutant at 36°C, a temperature at which it displays a complete respiratory growth defect (116). The two other genes so far identified, which affect the synthesis of ATP6 and ATP8 subunits, are part of a complex procedure that is not yet understood. It was demonstrated that the simultaneous presence of mutations in two genes resulted in a cold-sensitive phenotype (on a nonfermentable carbon source), associated with a small amount of one of the ATP8-ATP6 cotranscripts (234). Isolation of the NCA2 (29) and NCA3 (233) genes shed no light on their functions. The petite levels were not reported, either for the original double mutant or for the double-disruption strain.

Three genes (ATP10, ATP11, and ATP12) specifically involved in ATPase assembly have so far been described. The Atp11 and Atp12 proteins are required for normal assembly of the F₁ sector. The atp11 and atp12 mutants display 10% of wild-type ATPase activity and seem to be defective in a late step of F₁ assembly, the incorporation of  and  subunits into an active oligomer (3). The two mutants display a basal (1 to 5%) level of petite production (2). The genes were cloned (2, 23), but the effects of null alleles on the fate of mtDNA have been reported only for ATP11 by Lai-Zhang et al. (170): a strain carrying a deletion of this gene produces 1% petites. With respect to ATP10, biochemical data suggest that the mutants have an abnormal F₀ structure, which would impair the coupling of F₁ to F₀. Biochemical criteria, in accordance with growth phenotypes, also suggest that the mutants (the original pet as well as the disruptant) are leaky. One of the pet mutants has been analyzed with respect to mtDNA stability. According to the authors, it is genetically stable since it accumulates only 10 to 15% petites after long-term culture (4).

The final class of nuclear genes reported here are those whose products are required to facilitate the assembly of ATPase and other complexes in the mitochondrial inner membrane as well, namely, cytochrome oxidase. They are the AFG3 (YTA10), RCA1 (YTA12), and OXA1 genes (reference 248 and references therein). Again, all the mutants (even the null mutants) display a leaky phenotype with respect to the ATPase complex. For instance, as reported by Rep et al. (249), afg3 rca1 double mutants contain 56% of fully assembled ATPase relative to wild-type. The single- and double-mutant strains are genetically stable with respect to mtDNA (5). The same situation is encountered with the oxa1 mutants (G. Dujardin, personal communication).

These data as a whole lead to the following conclusions and comments. First, null alleles of all genes encoding essential subunits of the F₀ sector cause a strong increase in petite production. Second, deletion (disruption) of the genes encoding subunits , , and  of the F₁ sector also increase mtDNA instability. Third, the status of the  and  subunits (which are the catalytic subunits of ATP synthase) remains ambiguous. Ebner and Schatz (79) showed that absence of ATP synthase activity (due to an uncharacterized pet mutation) increases petite production. However, the petite colonies could be detected only after 6 to 10 days (instead of 2 to 3 days in the usual procedures). This raises a fundamental question concerning the conclusions of Lai-Zhang et al. (170). In fact, these authors observed that deletion of ATP1 ( subunit) or ATP2 ( subunit) resulted in less than 1% petites (compared to 9% in their reference strain). However, they did not state whether they found any petites and whether they examined the plates after long-term culturing. The last conclusion concerns the regulatory proteins. Clearly, some of them facilitate only ATPase assembly, since, in their absence, significant amounts of ATPase are assembled. In these cases, the mutant strains do not accumulate petites at high rates. In contrast, deletions of AEP1 or AEP2, required for ATP9 expression, increase mtDNA instability.

We must now reformulate the problem of mitochondrial translation. If the lack of one (or several) protein encoded by mtDNA is responsible for the high rates of petites observed when translation is blocked, one can easily exclude cytochrome b and subunits I, II, and III of cytochrome oxidase: stringent mutations in the four genes do not increase the levels of petites. However, in direct contrast, each time a mutation abolishes the synthesis (or drastically alters the structure) of one of the three ATPase subunits encoded by mtDNA, an extreme level of petite production is observed (see above and Table 4). The question must be posed: what is the link between Atp6, Atp8, Atp9, and mtDNA stability?

To answer this question, one must keep in mind that mutations which abrogate the synthesis of any of the ATPase subunits, even those which are nuclearly encoded (with the possible exception of  and ), cause the same phenotype, i.e., high production of petites. A careful analysis of the data leads one to favor a special role for Atp6 (see references in Table 4 for most of the relevant data). First, Atp6 assembly depends on Atp9 and Atp8 (reviewed in reference 214). Second, the lack of either of the nuclearly encoded F₀ subunits is always associated with the absence of Atp6. Third, in a strain lacking the F₁ subunit , the concentrations of the F₀ subunits Atp6, Atp8, and Atp9 are reduced by more than 70% (although petite production is only 20% in the culture analyzed) (228). Fourth, when the F₁ subunit  is absent, Atp6, Atp8, and Atp9 are present but there is a proton leak, suggesting a conformational change of F₀ (119). The relevant observations cannot be performed with subunit , since deletion of the gene leads to 100% petites. Finally, disruption of ATP2 (subunit ) blocks the functional assembly of F₀ (298). Thus, the data as a whole show (i) that the F₀ sector is altered when one of the F₀ or F₁ subunits is absent and (ii) that Atp6 is the ultimate and most sensitive target of F₀ assembly.

These data do not argue against the proposals of Myers et al. (213) but suggest the following considerations: in the absence of mitochondrial translation, either the import of proteins required for replication/repair of the rho⁺ genome would be specifically prevented by alteration of the F₀ sector or the putative mtDNA-encoded protein required for replication/repair of the rho⁺ genome would be Atp6. In our opinion, these hypotheses now appear quite unlikely and we favor a structural and more direct role of Atp6 in the maintenance of the rho⁺ genome: for instance, Atp6 might be involved in physical attachment of the rho⁺ molecule to a centromere-like structure required for its faithful transmission (see "Is there a centromere-like structure for the mitochondrial genome?" below).

In any case, a critical test with respect to the translation-ATPase connection would be to supply the mitochondria with Atp6, Atp8, and Atp9 encoded by artificial nuclear genes specifying imported forms of the proteins (already performed with Atp8 [214]) and then examine the effect of a mitochondrial translational block on petite production. If in this context the level of petites remains stable, one may conclude that at least one of these subunits is indeed the target of mitochondrial translation with respect to rho⁺ maintenance. However, if a high production of petites is observed, one must assume that either another (uncharacterized) protein encoded by the mitochondrial genome or translation per se of the relevant subunit is involved in the rho⁺ maintenance.

The problem of ATPase does not end here: we will find it again as a possible key in the old problem of petite positivity versus petite negativity (see "No compromise" and "Petite positivity versus petite negativity" below).

POSTTRANSLATIONAL EVENTS

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Expected and Surprising Roles for Proteases (Peptidases) in mtDNA Stability

As reviewed in reference 247, mitochondrial proteases can be divided into two classes: processing of precursor proteins and involvement in protein degradation. Most mitochondrial proteins encoded by nuclear genes are translated as precursors whose N-terminal presequences are required for mitochondrial targeting. These presequences are eliminated by proteolytic cleavage during their transport across the mitochondrial membranes. This is achieved by the mitochondrial processing peptidase (Mpp). One may assume that Mpp is indirectly required for mtDNA stability through processing of numerous proteins directly (or indirectly) involved in this process. However, this prediction cannot be tested, because the genes encoding the two subunits of Mpp are essential for cell viability (147, 329). This is not surprising if Mpp is a global mitochondrial processing enzyme: mitochondria are indeed required for many functions besides respiration (reviewed in reference 305). A few proteins, encoded by nuclear genes and delivered to the intermembrane space or encoded by the mitochondrial genome and devoted to the inner membrane, are processed by Imp, a inner membrane peptidase (reference 221 and references therein). This protein is beyond the scope of this review, since mutations of the two genes encoding its catalytic subunits (though leading to respiration deficiency) do not cause mtDNA instability.

A subset of nuclearly encoded proteins are processed to the mature form in two steps. The precursors are first cleaved by Mpp and then cleaved by the mitochondrial intermediate peptidase "Mip" (we put this acronym in quotation marks to avoid confusion with the acronym used above for the mitochondrial DNA polymerase [Table 1]). The yeast "MIP1" gene (Table 5) was cloned by hybridization with the cDNA encoding this enzyme in the rat. Disruption of the gene leads to respiration deficiency (142). Among the precursors which require "Mip" in order to be processed in the mature form, one r-protein, one translational elongation factor, and the Rim1 precursor were found (24). Because mitochondrial translation (see the previous section) (Table 3) and Rim1p (see "The recombination/repair track" above) (Table 1) are involved in mtDNA stability, the petite production observed when "MIP1" is inactivated is an indirect but expected effect (24).

The problem of the Pim1/Lon protease is much more puzzling. It belongs to the second group of mitochondrial proteases involved in protein degradation (247). The gene encoding this ATP-dependent protease was cloned independently by two groups. In one case (313), it was discovered during the systematic sequencing project of chromosome II and identified due to similarities of the deduced protein to bacterial Lon proteases. In the other case (295), the gene was obtained through PCR experiments, using the sequence conservation between these bacterial proteases and their human homologues. Evidence for proteolytic activity of the protein in vivo was obtained. The data also showed its role both in selective protein turnover (295) and in degradation of misfolded proteins (316). The PIM1/LON gene was rediscovered as a multicopy suppressor of mutations in two nuclear genes encoding proteins of the mitochondrial inner membrane, AGF3/YTA10 and RCA1/YTA12 (248). Mutations in these two genes lead to defects in degradation of mitochondrially synthesized proteins and in assembly of inner-membrane complexes. Several arguments led to the conclusion that these proteins may play a dual role, with both degradation and chaperone activities (reviewed in reference 247). Interestingly, overproduction of the Pim1/Lon protein suppressed respiration-dependent growth defects and protein assembly defects of the agf3/yta10 and rca1/yta12 mutants but not defects in protein degradation. Furthermore, suppression was maintained and even enhanced when the proteolytic activity of the Pim1/Lon protein was destroyed (249). This suggests that this protein (at least when overproduced) also displays a chaperone-like function in the assembly of mitochondrial complexes.

Disruption of the PIM1/LON gene is not lethal but leads to respiration deficiency and rho genome production (295, 313) (Table 5). The stability of mtDNA depends on the proteolytic activity of the Pim1/Lon protein. Two types of data lead to this conclusion. First, a missense mutation which impairs this activity results in mtDNA aberrations. Second, a strain carrying a deletion of the PIM1 gene is complemented (at 30 but not 37°C)