[International Review of Cytology] Volume 194 || The Petite Mutation in Yeasts: 50 Years On

The Petite Mutation in Yeasts: 50 Years On

Xin Jie Chen and G. Desmond Clark-Walker Molecular and Cellular Genetics Group, Research School of Biological Sciences, The Australian National University, ACT 2601, Australia

Fifty years ago it was reported that baker’s yeast, Saccbaromyces cerevisiae, can form “petite colonie” mutants when treated with the DNA-targeting drug acriflavin. To mark the jubilee of studies on cytoplasmic inheritance, a review of the early work will be presented together with some observations on current developments. The primary emphasis is to address the questions of how loss of mtDNA leads to lethality (#-lethality) in petite- negative yeasts and how s. cerevisiae tolerates elimination of mtDNA. Recent investigations have revealed that #-lethality can be suppressed by specific mutations in the U, p, and y subunits of the mitochondrial F,-ATPase of the petite-negative yeast Kluyveromyces lacfis and by the nuclear pfp alleles in Scbizosaccharomyces pombe. In contrast, inactivation of genes coding for F,-ATPase LY and p subunits and disruption of AAC2, PGSIIPELI, and YMEl genes in S. cerevisiae convert this petite-positive yeast into a petite-negative form. Studies on nuclear genes affecting dependence on mtDNA have provided important insight into the functions provided by the mitochondrial genome and the maintenance of structural and functional integrity of the mitochondria1 inner membrane.

genome integrity, afp mutations, mgi, F,-ATPase. 0 1999 Academic Press. KEY WORDS: Yeast, Petite mutation, mtDNA deletions, $-lethality, Mitochondria1

1. Introduction

In 1949 Boris Ephrussi and colleagues in Paris described the identification and characterization of “petite colonie” mutants in baker’s yeast, Saccharo- myces cerevisiae. A few years later, the salient genetic and biochemical properties of petites were described in two monographs that gave details of

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198 XIN JIE CHEN AND G. DESMOND CLARK-WALKER

a nowMendelian factor needed for respiration (Ephrussi, 1953; Slonimski, 1953). Although these publications marked the beginning of genetic studies on mitochondrial biogenesis, their significance was not appreciated by bio- chemists trying to unravel the secrets of respiration and oxidative phosphorylation in mitochondria from beef heart and rat liver (Schatz, 1993). By 1963, the knowledge that mitochondria in animals, plants, and fungi are capable of incorporating amino acids and contain DNA and RNA (Rabi- nowitz and Swift, 1970) focused attention on the earlier work of cytoplasmic inheritance in yeast. This review, marking the 50th anniversary of the first publications, presents a short historical account of petites and their contribution to the start of studies on mitochondrial biogenesis. The following sections( 111-VI) focus on the recent exciting discoveries concerning factors responsible for differentiating petite-positive from petite-negative yeasts. As will be recounted, unexpected results have moved studies on the petite mutation into new territory. During the intervening years, petite mutants have played an important part in establishing the genetic and physical maps of the mitochondrial genome in baker’s yeast (Linnane and Nagley, 1978; Locker et al., 1979; Dujon, 1981) and continue to be used in the study of mtDNA replication and transmission. In addition, the mechanism of the petite mutation has intrinsic interest, partly because petites appear spontaneously at the high rate of around 1% per generation and can be induced easily by many physical and chemical agents (Ferguson and von Borstel, 1992). The involvement of petite mutants in leading to an understanding of mtDNA recombination, genome rearrangement, replication, and transmission will not be considered in this review but can be found in the citations mentioned previously and other articles (Whittaker, 1979; Wolf and Del Guidice, 1988; Gingold, 1988; Dujon and Belcour, 1989; Clark-Walker, 1992; Piskur, 1994).

II. Historical Perspectives

A. “Petite Colonie” Mutants in Baker’s Yeast

Under the heading “Action de l’acriflavine sur les levures,” seven papers were published in 1949 by Boris Ephrussi and collaborators describing the genetic, physiological, and biochemical characteristics of petites (Ephrussi et al., 1949a,b,c; Tavlitzki, 1949; Slonimski, 1949a,b; Slonimski and Ephrussi, 1949). Genetic studies established that a cytoplasmic factor is irreversibly altered or lost (Ephrussi et al., 1949a,b), whereas biochemical and physiological experiments showed that growth is slower (Tavlitzki, 1949), respiration is deficient, and cytochromes a + a3 and b are missing in mutant strains

PETITE MUTATION IN YEASTS 199

(Slonimski, 1949a; Slonimski and Ephrussi, 1949). Subsequently, euflavin, the active ingredient in acriflavin (Marcovich, 1951), was found to induce the petite mutation rather than acting as a selective agent for preexisting petites that occur with a frequency of around 0.2-1.0% in cultures of baker’s yeast (Ephrussi and Hottinguer, 1950, 1951). In regard to spontaneous petites, it was noticed that their frequency is much higher than that expected for mutation of a Mendelian gene and, moreover, it seemed astonishing to Ephrussi and co-workers (1949a) that previous authors had not described the presence of smaller and paler colonies in their plated cultures. However, the occurrence of white colonies in a pink adenine requiring strain, which was attributed to “exhaustion of some gene component easily supplied by outcrossing to any normal stock” (Lindegren and Lindegren, 1947), appears to have been a missed opportunity to discover cytoplasmic inheritance in yeast. Likewise, other observations of S. cerevisiae strains with low levels of respiration obtained after treatment with cyanide or ethylene oxide represent, with hindsight, examples of chemically induced petite mutants (Stier and Castor, 1941; Whelton and Phaff, 1947). The lack of follow-up investigations by the three groups highlights the singular achievement of Boris Ephrussi, Piotr Slonimski, and colleagues in establishing the study of petites on a firm foundation.

Although work from the Paris group provided strong evidence for a cytoplasmic and likely mitochondria1 location of a genetic factor required for respiration (Ephrussi and Slonimski, 1955), a direct demonstration of extranuclear transmission of the element was made by others with transient heterokaryons (Wright and Lederberg, 1957). Using genetically marked haploid wild-type and petite strains, it was demonstrated that parental-type buds from newly fused pairs could have the respiratory phenotype of the opposite partner. In these experiments, use was made of the newly discovered phenomenon of suppressiveness.

Initial studies of crosses between wild-type and petite mutants had given almost 100% respiratory competent diploids. However, a different type of petite mutant was found to give some respiratory deficient diploid progeny in mass mating experiments (Ephrussi et al., 1955). In other words, such strains contain a factor that can suppress the genetic element conferring respiratory ability. Further investigations revealed that suppressive petites show a diversity in their degree of suppressiveness and that this property can be transmitted (Ephrussi and Grandchamp, 1965; Ephrussi et aL, 1966). Current knowledge allows us to interpret suppressive petites as retaining mtDNA with deletions and rearrangements, whereas acriflavin-generated mutants termed neutral petites, which were used in the initial tests, often lack mtDNA (see later). The mechanism of suppressiveness, employing hypersuppressive petites, is still under investigation (Graves et al., 1998; van Dyck and Clayton, 1998) and will not be dealt with in this review.


Serendipity plays an important part in research as illustrated by the discovery of segregational petites. During studies with a diploid strain of baker’s yeast it was noticed that 44% of ascospores were respiratory deficient, suggesting that a Mendelian gene may be responsible. Further analysis confirmed that the diploid strain was heterozygous for a recessive chromosomal gene controlling respiratory ability (Chen et al., 1950). Following this remarkable discovery it was soon recognized that several nuclear genes are required for respiration (Sherman and Ephrussi, 1962; Sherman, 1963; Sherman and Slonimski, 1964). Nomenclature was introduced at this time to differentiate segregational petites, pl , p2, etc., from vegetative petites, p-, with wild types represented by P and p+. Since this early work, more than 200 nuclear genes needed for mitochondrial biogenesis have been isolated and characterized (Attardi and Schatz, 1988; Tzagoloff and Dieck- mann, 1990; de Winde and Grivell, 1993; Poyton and McEwen, 1996). Among the nuclear genes responsible for aspects of mitochondrial metabolism is a category that affects mtDNA replication, recombination, and transmission. However, the influence of these genes on the production of petites is a large topic and will not be considered in this review.

B. Association of Mitochondria1 DNA with the p Factor

Following the identification of a cytoplasmic factor required for the synthesis of some respiratory enzymes, it seemed reasonable to suppose “that the mutation which results in the formation of vegetative littles consists of the loss of mitochondria” (Ephrussi, 1953). However, an alternative proposal was that loss of the cytoplasmic factor abolished the synthesis of respiratory enzymes but the granules remained unchanged (Slonimski and Ephrussi, 1949). Support for the later view came from the observation that respiratory deficient mutants still contain mitochondria (Yotsuyanagi, 1955,1962). Fur- ther evidence impinging on the nature of the cytoplasmic factor was obtained from a different approach. Based on knowledge that ultraviolet light leads to an increase in petites, it was shown that the action spectrum for mutant production matched the absorption spectrum of nucleic acids, with a peak around 260 nm (Raut and Simpson, 1955). However, the nature of the cytoplasmic factor remained unresolved for some years until it was demonstrated that purified mitochondria from baker’s yeast have an associated DNA (Schatz et al., 1964). Several investigators soon showed that DNA coisolated with mitochondria has a different base composition from nuclear DNA, as evidenced by a lighter buoyant density in CsCl and a lower melting temperature (Tewari et al., 1965; 1966; Corneo et al., 1966; Mounolou et al., 1966; Moustacchi and Williamson, 1966). Later on, electron microscopy studies removed any lingering doubt about the location of the


DNA by showing the presence of DNA fibres in the mitochondria1 matrix (Rabinowitz and Swift, 1970).

The realization that mitochondria contain DNA led investigators to ask if this material has a coding function that could be equated with the p factor. Consequently, in the studies cited earlier, DNA from petite mutants was also examined by buoyant density centrifugation. Results fell into two categories. One set of experiments showed that petites lack mtDNA (Corneo et al., 1966; Moustacchi and Williamson, 1966), whereas other analyses revealed DNA either in reduced amounts (Tewari et al., 1966) or of altered buoyant density (Mounolou et al., 1966). An explanation for the two classes of mutants is that spontaneously arising forms never lose all their mtDNA, whereas ones induced by prolonged acriflavin treatment often do. Indeed, exposure of baker’s yeast to the DNA targeting drug, ethidium bromide (EB), which is more effective than acriflavin for inducing petites (Slonimski et al., 1968), frequently eliminates all mtDNA, rendering cells pa (Goldring et af., 1970; Nagley and Linnane, 1970).

Linkage of suppressiveness to a change in buoyant density of mtDNA provided strong evidence that the p factor is mtDNA (Mounolou et al., 1966). Support for this view came from a number of groups who firmly established the base composition of wild-type mtDNA and its alteration in petite mutants (Mehrotra and Mahler, 1968; Bernardi et af., 1968; Carnev- ali et al., 1969). Shifts in the base composition of mtDNA in petites, generally in the direction of increased adenine and thymine, suggested that deletions rather than point mutations were responsible for loss of respiration. In S. cerevisiae mtDNA, the formation of deletions (Faye et af., 1973) occurs by recombination at regions of sequence homology (Clark-Walker, 1989; Weiller et al., 1991). Likewise, in Kfuyveromyces factis mtDNA, short re- peated sequences are sites for deletions (Hardy et al., 1989; Clark-Walker et al., 1997). Details of these events and the enzymes involved in mtDNA recombination will not be discussed.

111. Respiratory Deficient Mutations in Other Yeasts

A. Petite-Positive and Petite-Negative Yeasts

In view of the specific induction by acriflavin of petite mutants in S. cerevisiae, it seems strange that a number of years elapsed before the action of this agent on other yeasts was examined. Perhaps there was a prevalent attitude, similar to that of Alvarez and Mackinnon (1957), who wrote in their paper on a respiratory deficient mutant of Candida albicans that the “hereditary loss of respiratory function may be a frequent phenomenon


among micro-organisms endowed with both aerobic and anaerobic metabolism.” Despite this supposition, the majority of tested ascomyceteous yeasts do not form petite mutants when treated with euflavin as first reported by de Deken (1961) and in more extensive studies by Bulder (1964a,b) and de Deken (1966b). Once the initial observations had been made that yeasts could be separated into petite-positive and petite-negative species (Bulder, 1964a), studies were undertaken to see if any correlation exists between physiological properties and mutational status. Differences in drug uptake were excluded as an explanation because the synthesis of respiratory enzymes is inhibited in both categories of yeasts (Bulder, 1964a; de Deken 1966b). Because euflavin mimics glucose repression by inhibiting cytochrome synthesis, it was proposed that a correlation may occur between the presence of the Crabtree effect (glucose repression of respiration; de Deken, 1966a) and the ability to form petite mutants (de Deken, 1966b). In general, there is a positive relationship, but exceptions exist. For example, Schizosaccharomyces pombe has a threefold inhibition of respiration on shifting from glycerol to glucose yet it is petite negative (Wolf et ul., 1971; Foury and Goffeau, 1972). Furthermore, Brettanomyces anomalus and, to a lesser extent, Kloeckera africana do not show a Crabtree effect but form petites without difficulty (Bulder, 1964a; Clark-Walker and McArthur, 1978; Clark-Walker et al., 1981). A better correlation occurs between the ability to grow anaerobically and petite mutability (Bulder, 1964b; Subik et al., 1974a) but again exceptions are known as K. africana, mentioned earlier does not grow anaerobically under conditions supporting S. cerevisiae and other petite-positive species (G. D. Clark-Walker, unpublished observations).

A further complication for uncovering factors influencing susceptibility to petite mutation is the observation that segregational respiratory deficient mutants can be isolated from the petite-negative species K. lactis (Herman and Griffin, 1968; Del Giudice and Puglisi, 1974; Allmark et al., 1977; Gbelska et al., 1996) and S. pombe (Heslot et al., 1970; Wolf et al., 1971). It had been suggested previously that the petite-negative phenotype of S. rosei could not be explained by insufficient fermentative capacity (Bulder, 1966). Although genetic data indicate that petite-negative species have sufficient fermentative ability to grow in the absence of respiration, a caveat is that the identified chromosomal mutations discussed earlier could be leaky and that residual respiratory capacity may be sufficient to support growth. However, a direct demonstration that K. lactis can grow in the absence of a functional electron transport chain was made by disruption of the unique gene for cytochrome c, CYCl (Chen and Clark-Walker, 1993). Strains containing disrupted CYCl cannot grow on nonfermentable substrates requiring respiration for their metabolism but do grow on glucose. In other words, K. lactis has sufficient fermentative ability to support


growth but it cannot form cytoplasmic petite mutants. An implication from these results is that the mitochondrial genome in K. lactis is vital and that it codes for one or more essential genes. Consequently, questions are raised as to what mitochondrial genes are vital in petite-negative yeasts and what differences are present in petite-positive species that allow them to survive loss of mtDNA? An experimental approach to answering these questions will be considered in Sections IV, V, and VI.

6 . Evolution of the Petite-Positive Trait

Even though there does not appear to be a determining physiological property shared by all petite-positive yeasts, it is possible that extraneous elements may mask a common attribute. For instance, the failure of the petite-positive species K. africana to grow anaerobically, even when supple- mented with ergosterol and unsaturated fatty acids that are required for S. cerevisiae (Andreasen and Stier, 1953, 1954), may be due to lack of a factor that requires oxygen for its synthesis or some compound not supplied in the medium. In its natural habitat, K. africana may be able to grow in a microaerobic environment due to the presence of such a compound. Hence it is still possible that the capacity to grow in low oxygen could be a shared trait of petite-positive species and that a collateral, but incidental, phenotype is an ability to survive loss of mtDNA.

Until now it has not been feasible to determine the phylogenetic distribu- tion of petite mutability because of the unsatisfactory state of yeast taxonomy. The application of DNA sequence comparisons to yeast phylogeny has revealed some curious examples of misclassification that must be due to convergent evolution of the physiological characteristics used previously (see the position of Saccharomyces kluyveri, a petite-negative yeast: Kurtz- man and Robnett, 1998). When sequence-based phylogenetic trees are examined for the occurrence of petite-positive species, it is apparent that a susceptibility to mutation has arisen on at least two occasions and, in one instance, the ability to form petites appears to have been lost (Hoeben et al., 1993).

There are two well-separated clades of petite-positive yeasts. Most of the species in the genus Saccharomyces, which includes close relatives and more distant forms, are petite positive (Nagai et al., 1961; Bulder, 1964a; de Deken, 1966b; Clark-Walker et al., 1981). Likewise, all except one species, Brettanomyces custersianus, are petite positive in the Dekkera Brettano- myces genus (Bulder, 1964a; Subik et al., 1974a; Hoeben et al., 1993). Loca- tion of these two petite-positive groups on trees constructed by sequence comparisons shows that they are widely separated by petite-negative yeasts (Cai et al., 1996; Kurtzman and Robnett, 1998). Positioned between these


groups are other petite-positive species, such as Candida glabrata, Kluyvero- myces yarrowii, Kloeckera africana, and Hanseniaspora vinea, that further studies may show are allied to the Saccharomyces clade (Clark-Walker et al., 1981 and unpublished observations). Nevertheless, wide separation between the Saccharomyces and Dekkera clades indicates that the ability to form petite mutants has arisen on two separate occasions. As only a small proportion of the more than 500 ascomyceteous yeasts (Kurtzman and Fell, 1997) have been examined for mutational status, it would be informative to undertake a comprehensive analysis of this trait now that yeast taxonomy is on a firmer footing. Such studies, together with a more detailed appraisal of physiological properties, may reveal the evolutionary niche occupied by petite-positive yeasts. It is the ability to exploit a particu- lar environment, perhaps a microaerobic one, that has selected a physiological trait shared by petite-positive yeasts. Perhaps this trait is the ability to maintain the functional integrity of mitochondria in the absence of an electron transport chain. Loss of mtDNA from such cells, leading to a physiological condition similar to anaerobiosis, would no longer be lethal.

C. Naturally Occurring Respiratory Deficient Yeasts

In his studies on petite induction, Bulder (1964a) listed Schizosaccharo- myces versatalis, Torulopsis lactis-condensi, T. pintolopesii, and Candida slooffii as being respiratory deficient (obligatory fermentative) strains that were isolated outside the laboratory. Others have subsequently confirmed the absence of respiration in C. sloofii, T. pintolopesii (Watson et al., 1980), and S. versatilis (Subik et al., 1974a). The question posed by the isolation of respiratory deficient strains, often from anaerobic environments such as animal intestinal tracts (Mendonca-Hagler and Phaff, 1975), is whether they are similar to vegetative petites or have arisen from chromosomal gene mutations. Studies with three different isolates of C. sloofii have shown that each contains a circular DNA of low buoyant density, resembling mtDNA from petite mutants of baker’s yeast, and that the size profile of the circles differs in each case (Arthur et al., 1978). The similarity of these strains to petite mutants is further supported by the observation that euflavin could eliminate the light buoyant density DNA (Arthur et al., 1978). Subsequent studies with T. pintolopesii indicated that this isolate also resem- bles a petite mutant (Watson et al., 1980). Earlier observations from DNA buoyant density analysis and hybridization experiments suggested that the respiratory competent parents of C. sloofii and T. pintolopesii could be Saccharomyces telluris andlor Torulopsis bovina and that the four yeasts are representatives of the same taxon (Mendonca-Hagler and Phaff, 1975). It seems likely that C. sloofii and T. pintolopesii are petite mutants that have


found a growth niche where they can compete with parental forms. However, the origin of S. versatilis is far less clear. As noted, this yeast is respiratory deficient; however, in contrast to vegetative petites, it appears to lack cytochrome c (Subik etal., 1974a). Phylogenetic analysis based on sequence comparisons does not reveal a close relative of S. versatilis that could be a respiratory competent parent (Naehring etal., 1995; Kurtzman and Robnett, 1998). Until further data are available, it appears that the status of S. versafitis as a chromosomal or cytoplasmic mutant must remain undecided.

IV. What Is the Vital Role of mtDNA in Petite-Negative Yeasts?

A. Nuclear Genes Coding for Electron Transport or ATP-Synthase Are Not Vital

As mentioned previously, it has been possible to isolate chromosomal respiratory deficient mutants in both K. Zactis and S. pombe. At least for K. lactis, the use of a Rag+ strain in this research was fortuitous for the recovery of mutants. It is now known that K. lactis strains vary in their ability to grow fermentatively when mitochondria1 respiration is inhibited. Fermentative growth depends on the allelic status of RAG1 and RAG2 genes (Goffrini etal., 1989) that encode a glucose transporter (WCsolowski- Louvel et al., 1992) and a phosphoglucose isomerase, respectively (Goffrini et al., 1991). Some alleles of these genes do not permit growth on glucose in the presence of antimycin, which inhibits respiration. An implication from these observations is that some petite-negative species may behave like Rag- mutants of K. Zactis. Indeed, lack of sufficient fermentative capacity was one of the first explanations advanced for the occurrence of petite- negative yeasts. Nevertheless, if a Rag+ strain of K. lactis is used, it is possible to disrupt genes coding for components of both the electron transport chain and oxidative phosphorylation (ATP synthase).

As described previously, it has been found that K. lactis disrupted in the unique cytochrome c gene (CYCl) is viable on fermentable carbon sources, indicating that electron transport is not essential (Chen and Clark-Walker, 1993). The same conclusion can be drawn when viable null mutants were constructed by disrupting the K. Zactis QCR8 gene, encoding subunit VIII of the bcl complex (Mulder et al., 1994), and the K. lactis COX18 homolog, required for assembly of a functional cytochrome c oxidase (Hikkel et al., 1997). Moreover, successful disruptions of genes encoding subunits of the FIFo-ATP synthase have also been reported. These genes include A TPl, ATP2, ATP3, ATP6, and ATPE, encoding the a, p, y, 6, and E subunits of


the F1 complex (Chen and Clark-Walker, 1995; 1996; Hansbro et al., 1997; X. J. Chen, unpublished data), and ATP4, ATP5, and ATP7, the three nuclear encoded genes for subunits b, OSCP, and d of the membrane FO sector (Chen et al., 1998). One can thus conclude that neither electron transport nor ATP synthesis is essential for K. lactis. However, K. lactis respiratory deficient mutants do not grow anaerobically (G. D. Clark- Walker, unpublished data). As discussed earlier, the synthesis of some cellular components probably requires oxygen in this species.

Another yeast that is widely studied as a model petite-negative species is Schizosaccharomyces pombe. Heslot and co-workers (1970) first characterized segregational respiratory deficient mutants of S. pombe that are deficient in cytochrome a + a3 and respire at a low rate. Segregational mutants lacking cytochrome c oxidase and succinate-cytochrome c reduc- tase activities were also isolated by Wolf and collaborators (1971). Boutry and Goffeau (1982) identified S. pombe mutants specifically altered in the (Y or p subunits of F1-ATPase. In a more recent study, Bonnefoy et al., (1996) described the disruption of the S. pombe ABCl gene that is required for the correct functioning of the bcl complex of the mitochondria1 respiratory chain. Likewise, targeted disruption of the genes encoding the F1- ATPase (Y and p subunits in S. pombe has yielded viable cells (D. I. O’Connor, X. J. Chen, and G. D. Clark-Walker, unpublished data). These observations are in agreement with the findings with K. lactis that neither electron transport chain nor ATP synthase is essential for survival of these petite-negative species.

A number of other petite-negative yeasts have been investigated for the production of respiratory deficient mutants after chemical mutagenesis, ultraviolet irradiation, or high temperature treatment. These species include Saccharomycodes ludwigii (Nagai et al., 1976), Candida albicans (Aoki and Ito-Kuwa, 1987; Roth-Ben Arie et al., 1998), Schwanniomyces castellii (Claisse et al., 1991), Zygosaccharomyces rouxii (Yagi et al., 1992), Candida apicola, and Candida bombicola (Hommel et al., 1994). Although these mutants are deficient in cytochromes or in cytochrome c oxidase, it is uncertain whether the mutations are leaky with the isolates retaining a residual capacity for respiration.

B. Petite-Negative Cells Are Refractory to mit- Mutations

One peculiar observation in the study of respiratory deficient mutants from petite-negative yeast is that nearly all mutants recovered are chromosomal in origin. Because both electron transport and ATP synthesis are dispens- able for viability, one would at least expect lesions in genes such as those encoding cytochrome b, cytochrome c oxidase subunits, or the ATP syn-


thase subunits 6,8, and 9 to be identified as cytoplasmic respiratory deficient mutants. However, isolation of mit- mutants has proved to be a rare event.

Two special cases can be mentioned. In S. pombe, although attempts to induce m i - mutants by acriflavin mutagenesis proved unsuccessful, such mutants can be recovered in specific mitochondrial mutator strains (Seitz- Mayr and Wolf, 1982). In these strains, respiratory deficient mutants can rise at a rate of 2-20% and roughly 20% of them carry mtDNA deletions of 50-1500 bp (Ahne et aZ., 1984,1988). The mutator strains carry mutations in the urfa sequence (unassigned reading frame; Zimmer et al., 1991) that has significant homology to a mitochondrial ribosomal protein encoded by varl in S. cerevisiae and other species (Neu et aZ., 1998). How mutations in urfa induce the formation of point mutations and small deletions remains unknown.

In K. Zactis, only one cytoplasmic mutant has been reported. It has been found that the respiratory deficient mutant Gly-3.9 has a rearranged mitochondrial genome that leads to a deletion of 22 amino acids from the carboxyl terminus of the 75 amino acid ATP synthase subunit 9 protein (Clark-Walker et al., 1997). This mutant, induced by heavy EB mutagenesis, lacks Atp9p. However, using a hybrid strain between K. Zactis and S. cerevisiae, respiratory deficient mtDNA deletion mutants have been isolated from the petite-negative KF4 that has a chromosomal composition and mtDNA profile essentially like K. Zactis (Hardy et aZ., 1989; Maleszka and Clark- Walker, 1990).

In the light of the persistent failure in isolating mit- mutants from wild- type S. pombe and K. Zactis, it appears that the mitochondrial genomes in these yeasts are refractory to mutations or deletions under the mutagenic procedures used so far. The recovery of mit- mutants at high frequency in the mutator strains of S. pombe and in the atp (mgi) mutants of K. Zactis (see Section V) excludes the possibility that these yeasts lack specific mtDNA sequences or a DNA recombination machinery that would enable deletions to occur as suggested earlier (Clark-Walker and Miklos, 1974). As genetic analysis of the K. Zactis atp9 mutant (see earlier) failed to identify any possible nuclear mutation that predisposes this isolate to the formation of mit- mutants (G. D. Clark-Walker and X. J. Chen, unpublished data), it is unlikely that a second mutation in a nuclear gene is a prerequisite for the formation of mit- genomes. The reason for the rare occurrence of mit- mutations in petite-negative yeasts remains unknown.

C. A Vital Role for mtDNA in Petite-Negative Yeasts

In the face of evidence presented earlier that it is possible to disrupt chromosomal genes encoding components of the electron transport chain


and ATP synthase, a question is raised as to whether the mitochondrial genome is essential for petite-negative yeasts? Previous studies have shown that severe treatment of petite-negative yeasts with acriflavin or EB results in the production of nonviable microcolonies (Bulder, 1964b; Heritage and Whittaker, 1977). The implication has been that nonviable microcolonies have lost mtDNA, but no direct test of this idea has been made. However, the question has been approached by a genetic experiment whereby the K. lactis homolog of the S. cerevisiae mitochondrial genome maintenance gene, MGMlOl (Chen et al., 1993), has been disrupted (Clark-Walker and Chen, 1996). Meiotic segregants containing the disrupted gene form microcolonies of 6-8000 nonviable cells. Likewise, in S. pombe, disruption of another mitochondrial genome maintenance gene, M G M l ( Jones and Fangman, 1992; Guan et ul., 1993), yields nonviable cells depleted in mt- DNA (Pelloquin et al., 1998). In other words, it has been demonstrated that the loss of mtDNA is lethal in these petite-negative yeasts.

Likewise the importance of mitochondrial protein synthesis to survival of K. lactis and S. pombe has also been examined. Early studies have pointed out the importance of mitochondrial protein synthesis for growth of K. Zuctis based on the inhibitory effects of erythromycin, chlorampheni- col, and tetracycline (Morgan and Whittaker, 1978). In some well-defined Rag+ (fermentatively competent) strains, indeed, erythromycin can totally inhibit growth on glucose medium (X. J. Chen, unpublished data). In a more recent work, Pel and collaborators (1996) disrupted the K. lactis MRFl gene encoding a mitochondrial peptide chain release factor and found that the resulting cells die after 10-13 generations. In fact, in S. pombe, screening for p- mutants from the mitochondrial mutator strains identified deletion mutants exclusively in coxl, ~ 0 x 2 , cox.?, and cob genes, but never in genes required for mitochondrial protein synthesis (tRNA and rRNA genes; Ahne et al., 1984). Taken together, these findings strongly indicate that a mitochondrial translation product(s) plays a vital role in K. lactis and S. pombe.

D. The “Two-Component” Model

If mtDNA and mitochondrial protein synthesis are vital for K. Zuctis and S. pombe and if processes of electron transport and oxidative phosphorylation are not essential, then a simple explanation for the difference between petite-positive and petite-negative yeasts could be that mtDNA in the latter category codes for a vital gene that is not present in the former class. However, examination of the complete sequence of S. pombe mtDNA (Paquin et al., 1997) and nucleotide sequences of K. lactis mtDNA analyzed so far does not reveal a novel gene(s) in comparison with the petite-positive


yeast S. cerevisiue (Foury et af., 1998). As in S. cerevisiue, the mitochondrial genomes of S. pombe and K. factis encode (1) cytochrome b and cytochrome c oxidase subunits 1,2, and 3, which are integral components of the electron transport chain, (2) the subunits 6,8, and 9 of the Fo sector of ATP synthase, and (3) the ribosomal protein Varl.

To address the question raised by the issue described earlier, a “two- component” model has been proposed to explain pO-lethality in petite- negative yeasts. According to the model, although inactivation of the electron transport chain and the ATP synthase is not lethal, the simultaneous loss of mtDNA genes for both components of the electron transport and oxidative phosphorylation pathways cannot be tolerated. Strong support for this model comes from observations in K. factis that a combined disruption of the cytochrome c gene (CYCZ) and the A TP3 or A TP5 genes, encoding integral components of the F1 and Fo complexes, respectively, produces nonviable cells (Clark-Walker and Chen, manuscript in preparation).

V. Nuclear Mutations Predisposing Petite-Negative Yeasts to the Formation of Cytoplasmic Petites

Recent work has demonstrated that petite-negative species such as K. factis and S. pombe can be converted into petite-positive yeasts provided that specific “gain-of-function’’ mutations are introduced in the nuclear genome. The discovery of these mutations, which allow cells to bypass the requirement for mtDNA, has opened a new chapter in research on petite mutation. It is anticipated that this research will provide insight into how the structural and functional integrity of the mitochondrial inner membrane is maintained, particularly in cells lacking a functional electron transport chain and ATP synthase.

A. atp [mgi] Mutations of Kluyveromyces lactis

Isolation of utp (formerly mgi) mutants that can suppress po-lethality in K. Iuctis was achieved by exposure of cells to a high concentration of EB (Chen and Clark-Walker, 1993). Three out of the four mutants isolated in the initial experiment were found to be po, whereas the fourth mutant was found to contain a rearranged mitochondrial genome (Clark-Walker et uf., 1997). Genetic analysis of the three pa mutants revealed that the ability to form p-lpo colonies on EB treatment segregated at a ratio 2’ : 2- in meiotic progeny. It was thus established in K. factis that the ability to produce respiratory deficient mutants is influenced by nuclear genes. This class of


nuclear mutations was initially designated mgi (for mitochondrial genome integrity) but has been renamed atp because they occur in the mitochondrial F1-ATPase (see later).

K. lactis atp mutants can be either respiratory competent or deficient, indicating that the Mgi phenotype is independent of respiratory ability (Chen and Clark-Walker, 1993, 1995, 1996; Clark-Walker et al., submitted for publication). Respiratory competent atp mutants behave as petite- positive yeasts in the following ways. First, they can spontaneously form cytoplasmic respiratory deficient colonies at a frequency of 0 5 5 % (Chen and Clark-Walker, 1993, 1996), which is similar to frequencies observed with S. cerevisiae (Clark-Walker et al., 1981). Second, spontaneous petites have mtDNA with simple deletions, genome rearrangements, and amplifi- cations of some segments as in S. cerevisiae petites (Chen and Clark-Walker, 1993). Finally, the afp mutants can form p- or po colonies at high frequency on treatment with EB.

As a tight correlation was found between the occurrence of the Mgi- phenotype and resistance to a high concentration of EB (Chen and Clark- Walker, 1995), a large screening program for the isolation of atp mutants was undertaken. By exposing K. lactis to EB at a concentration of 16 pg/ ml, all resulting resistant colonies contained an atp mutation in the nuclear genome (Chen and Clark-Walker, 1995; Clark-Walker et al., submitted for publication). In a total of 42 atp mutants, only three loci, designated atpl (mgi2), atp2 (mgil) , and alp3 (mgis), have been found.

ATPl (MGZ2) and ATP3 (MGZ5) genes were isolated by complementation of a respiratory deficient phenotype collateral to the atpl-2 and atp3- 1 alleles (Chen and Clark-Walker, 1995), whereas the ATP2 (MGZl) gene was identified by complementing the cold sensitive phenotype associated with the atp2-1 allele (Chen and Clark-Walker, 1996). ATPl, ATP2, and ATP3 genes encode proteins sharing 86.3, 88.9, and 70.6% identity with the a, p, and y subunits of the mitochondrial F1-ATPase from S. cerevisiae and can complement the respiratory deficient phenotype of S. cerevisiae afpl, atp2, and afp3 mutants (X. J. Chen and G. D. Clark-Walker, unpublished data).

6. A Novel Function Associated with F,-ATPase in K. lactis atp Mutants

Several lines of evidence support the notion that the atp alleles of K. lactis are gain-of-function mutations. First, all 42 a@ mutants examined carry a point mutation in one of the three largest subunits of F1-ATPase (Clark- Walker ef al., submitted for publication). Second, loss-of-function mutations do not have the same phenotype as afp alleles. When genes encoding the

PETITE MUTATION IN YEASTS 21 1

a, p, or y subunits of F1 were disrupted, the resulting null mutants remained petite negative (Chen and Clark-Walker, 1995, 1996). Finally, atp alleles exhibit a dominant phenotype, in accord with the gain-of-function nature of the mutations. When treated by EB, atpl+ heterozygous diploids or a haploid strain carrying an atp allele in addition to a wild-type copy of the gene can readily form small colonies that are mostly po (Chen and Clark- Walker, 1995, 1996; also see Fig. 1).

The presence of atp alleles is clearly required for the viability of cells lacking mtDNA as loss of a plasmid-born atp allele from K. lactis po cells is lethal (Clark-Walker and Chen, 1996). An F1 complex, carrying an atp mutation, can therefore gain a novel function that is responsible for the suppression of pO-lethality. This novel function is distinct from the role of F1 in ATP synthesis as a subclass of atp mutants are respiratory deficient (Chen and Clark-Walker, 1995; Clark-Walker et al., submitted for publication). Further evidence for a novel function for F1 comes from the observa-

FIG. 1 The dominant pO-lethality suppressor phenotype of the K. luctis atpl-2 allele. The K. luctis haploid CK190/2, constructed by integrating a wild-type copy of KIATPl into the chromosome of an atpl-2 strain, was exposed to ethidium bromide by the margin of growth technique (Chen and Clark-Walker, 1996). The small colonies formed after drug treatment are mostly po.


tion that suppression of pO-lethality can occur in the absence of the FO complex, as discussed in the following section.

C. Factors Affecting the pO-Lethality Suppressor Activity of FA-ATPase

1. Suppression of pO-Lethality Requires an Assembled FI, but

The pO-lethality suppressor activity of atp alleles is mediated through the action of an assembled F1. The suppressor activity of strains carrying atpl- 2, atp2-1, atp2-3 alleles, with mutations in the a and 0 subunits, respectively, is totally abolished by disruption of the gene encoding the y subunit of F1 (Chen and Clark-Walker, 1995, 1996). Likewise, disruption of the gene encoding the 0 subunit converts an atp3-1 mutant to a petite-negative form. In these strains, an assembled core complex of F1, composed of at least a, 0, and y subunits, is required for the novel function of F1. However, recent work has revealed a second class of atpl and atp2 alleles that function in the absence of the y subunit (X. J. Chen and G. D. Clark-Walker, unpublished data). When the y subunit is introduced into these mutant strains, the pO-lethality suppressor phenotype is inhibited. The significance of these observations in identifying the mechanism of suppression is under investigation.

The presence of the 6 subunit of F1 can affect the manifestation of the Mgi phenotype in an allele-specific manner (Hansbro et al., 1998). Based on the growth of cells after elimination of mtDNA by EB, it was found that disruption of the gene encoding 6 completely abolishes the Mgi phenotype of the atp2-l and atpl-6 alleles, but only partially attenuates the growth of po cells in atp2-9 and atp3-2 strains. However, inactivation of 6 does not affect the Mgi- phenotype of the atpl-1 allele. The role of the 6 protein in the assembly of FI has been reported in S. cerevisiae (Giraud and Velours, 1997). Whether the presence of the 6 subunit modulates the Mgi- phenotype by affecting the assembly of a mutant F1 in K. lacris has yet to be examined.

The presence of a Fo sector is not required for pO-lethality suppressor activity of F1. Because subunits 6 , 8, and 9 of Fo are encoded by mtDNA, it is unlikely that F1 carrying an atp mutation executes its function through Fo in po cells. This notion has been supported further by the observation that the disruption of K. lactis nuclear ATP4, ATP5, and ATP7 genes, encoding subunits b, OSCP, and d, does not abolish the growth of atp mutants on elimination of mtDNA by EB (Chen er al., 1998). In these strains, the six major Fo proteins, namely subunits 6,8,9,b, OSCP, and d, are all absent. The novel F1 function is thus independent of Fo. Whether

Is Independent of Fo


the mutant F1 complex exists in a free form in the mitochondrial matrix or interacts with a non-Fo membrane component has yet to be determined.

2. ATP Hydrolyzing Activity

F1-associated ATP hydrolyzing activity is likely to be essential for the po- lethality suppressor function of altered FI as a mutation in the ATP-binding site of the fi subunit abolishes the Mgi- phenotype of a atpl-2 strain (G. D. Clark-Walker, unpublished data). This is consistent with the fact that all suppressor mutants retain some F1-related ATPase activity. However no direct correlation was found between F1-related ATPase activity and suppression of pO-lethality as mitochondria from strains with different suppressor mutations can have ATPase activities higher or lower than a wild- type strain (Clark-Walker etal., submitted for publication). A strain carrying the T191S mutation in the p subunit, with a mitochondrial ATPase activity greater than the suppressor strains examined so far and twofold higher than a wild-type strain, does not suppress pO-lethality. These observations suggest that the F1-associated ATPase activity is essential for the suppressor function, but ATP hydrolysis might not be the primary cause for the novel function of F1.

3. Mutation Sites

Different pO-lethality suppressor activities can be observed in different atp mutants judging from the growth rate of po cells. In a total of 42 atp alleles the mutable amino acids are confined to only seven positions, with Pro- 328, Ala-333, and Phe-443 in the (Y subunit; Val-306 and Arg-435 in the p subunit; and Thr-275 and Ile-281 in the y protein (Clark-Walker et al., submitted for publication). All mutable residues are highly conserved through evolution, as can be seen in Fig. 2. A curious aspect of mutations at Arg-435 in the p subunit is that this amino acid can be substituted by five residues, ranging from the similarly charged Lys to nonpolar Gly. Thus it seems that the critical change for the suppression of pO-lethality is the removal of a property provided by pR43.5. When projected onto the crystal- lographic structure of the bovine F1 complex (Abrahams et al., 1994), the mutable amino acids are found in two regions. One location lies adjacent to the membrane surface at the “base” of F1 and the other position is near the top or matrix proximal region. These two subdomains are therefore critical for the pO-lethality suppressor function of the F1 complex.

D. ptp Mutants of S. pornbe

Two nuclear mutations, ptpl-1 andptp2-1, have been described in S. pombe that allow the growth of cells in the absence of mtDNA (Haffter and Fox,

a- subunit KlATPl 298 ScATPl 295 SpATPl 286 BtATPl 2 5 8 HsATPl 301 BaATPl 250 EcATPl 250

KlATPl 407 ScATPl 404 SpATPl 395 BtATPl 367 HsATPl 410 BaATPl 359 EcATPl 370

p- subunit KlATP2 267 ScATP2 273 SpATP2 286 BtATP2 240 HsATP2 290 EcATP2 228 BaATP2 236

atpl-1 (F->S) atpl-5 (F->L)

atp2-9 (V->F)

KlATPZ 387 ScATP2 393 SpATP2 406 BtATP2 360 HsATP2 410 EcATP2 341 BaATP2 356

atpa-1 (R->G) atp2-3 (R->I) atp2-6 (R->T) atp2-7 (R->V) atp2-8 (R->K)

y- subunit KlATP3 266 ScATP3 288 BtATP3 2 5 0 HsATP3 275 BaATP3 263 EcATP3 265

atp3-1(T->A) atp3-2 (I->T)

FIG. 2 Evolutionary conservation of amino acid residues in the a, B, and y subunits of F1- ATPase that are subjected to mutations in utp strains of K. lactis. Sequences from K. lacris (KI), S. cerevisiae (Sc), S. pornbe (Sp), cow (Bt), humans (Hs), E. coli (Ec), and the thermophilic Bacillus PS3 (Ba) are aligned. Numbering of amino acids for genes from K . lactis, S. cerevisiue, S. pornbe, and humans starts from the first Met in the precursors. Numbering for the bovine proteins refers to ones in the crystal structure (Abraham et uL, 1994). Arrows indicate the position of residues changed in K. Zactis atp mutants. The amino acid valine, which is the site for the afp2-9 mutation, is not totally conserved as an isoleucine residue is present in BaATP2. The sequence for the y subunit of S. pornbe is currently unavailable.


1992). These mutants have been isolated by a long-term incubation of cells in liquid medium containing glucose and EB. In an independent study, it was shown that po cells can arise from a S. pombe strain with a mitochondrially encoded mutator mutation (Massardo et al., 1994). As the frequency of po cells was not established precisely, it is uncertain if nuclear mutations are present in these isolates that support the isolation of po cells.

The ptpl-1 mutation has been shown to be required for the viability of cells defective in the RNase MRP RNA gene that participates in mitochondrial DNA metabolism (Paluh and Clayton, 1996). However, the nature of the ptp mutations remains unknown. Whether the ptp mutations occur in F1-ATPase subunits has yet to be investigated.

VI. Genes Required for Viability of Petites in S. cerevisiae

Although the conversion of petite-negative yeasts to petite-positive forms has been described only recently, it has been known for some time with S. cerevisiae that petite mutants cannot survive in the presence of opl (Kova- cova et al., 1968) or Z l X l (peZ1) mutations (Subik, 1974). Similarly, mutations affecting the fermentative pathway can also cause S. cerevisiae to become petite negative. This second category of genes, such as those encoding alcohol dehydrogenase I and pyruvate decarboxylase (Ciriacy, 1976; Lancashire et al., 1981), will not be discussed in this review. However, in addition to opl and pell, it is now known that mutations in three other loci, not concerned with fermentative growth, can affect the survival of petites in baker's yeast. As described later, disruption of AAC2, PGSl/ PELl, YMEl, ATPl, and ATP2 can turn S. cerevisiae into a petite- negative form.

A. AAC2

ADPlATP translocation across the mitochondrial inner membrane is a key element of oxidative phosphorylation. It is required for the export of ATP synthesized in mitochondria and in the import of ATP into the organelle under conditions where respiration is repressed. S. cerevisiae has three homologous genes encoding ADP/ATP translocases. AA CI (Adrian et al., 1986) andAAC2 genes (Lawson and Douglas, 1988) are expressed preferentially under aerobic conditions, whereas the derepression of AAC3 occurs exclusively under anaerobic conditions (Kolarov et al., 1990; Drgon et al., 1991). The AAC2 gene encodes the bulk of the mitochondrial translocator

21 6 XIN JIE CHEN AND G. DESMOND CLARK-WALKER

in respiring cells. In contrast to AACl and AAC3, whose inactivation does not significantly affect growth on a non fermentable carbon source or on glucose under anaerobic conditions, disruption of AAC2 yields a respiratory deficient phenotype (Lawson et al., 1990; Drgon et al., 1991). However, overexpression of AACl and AAC3 can compensate for the respiratory deficiency of aac2 mutants (Lawson and Douglas, 1988; Kolarov et al., 1990). Under anaerobic conditions, am2 mutants can grow provided that a wild-type AAC3 is present (Kolarov et al., 1990).

The AAC2 gene is well known because of the opl mutation that was first isolated as a strain with a possible defect in oxidative phosphorylation (Kovac et al., 1967) but subsequently turned out to be affected in nucleotide translocation across the mitochondrial membrane (Kovac et al., 1972). The opl mutant has a lesion in the AAC2 gene (Kolarov et al., 1990; Lawson et al., 1990) and does not tolerate p- mutations as acriflavin treatment resulted in nonviable cells (Kovacova et al., 1968).

The petite-negative nature of opl/aac2 mutants has been explained by the role of AAC2 in the import of ATP into mitochondria. Because intramitochondrial ATP is essential for mitochondrial biogenesis (e.g., for protein import; Nelson and Schatz, 1979; Hwang and Schatz, 1989; Cyr et al., 1993), import of ATP from the cytosol is essential for viability of p-/po cells. Therefore, incubation of p- mutants from wild-type S. cerevisiae with bongkrekic acid, an inhibitor of the ADP/ATP translocator(s), resulted in a complete inhibition of cell growth (Subik et al., 1972; Kolarov and Klingen- berg, 1974). Likewise, a combined application of inhibitors of electron transport such as antimycin A with bongkrekic acid also gives rise to nonviable cells because of the depletion of intramitochondrial ATP (Gbelska et al., 1983). One puzzling issue concerning the role of the ADP/ATP exchange process for the viability of petites has been that mit- mutants, defective in the electron transport chain (Kotylak and Slonimski, 1977; reviewed by Dujon, 1981), can be isolated from opl mutants. These observations imply that the loss of intramitochondrial ATP synthesis can be tolerated in opl strains defective in ADP/ATP exchange. Therefore, the role AAC2 in ATP import cannot fully explain the petite-negative phenotype of opl mutants, although it is arguable that the opl allele or the cytoplasmic mit- mutations might be leaky. In this respect, it would be interesting to determine whether mit- or nuclear pet mutations can be tolerated in aac2 null mutants. Here, one has also to bear in mind that the application of bongkrekic acid, which may inhibit the activity of all three translocases, cannot totally reflect physiological conditions in opl mutants. In opl cells, the low-level expression of AACl or AAC3 is unable to support respiratory growth, but sufficient ATP may still be imported to maintain other functions of mitochondria (see later). This may well reconcile the observations that the growth of p- mutants can be totally inhibited by bongkrekic acid but that mit- mutations can be tolerated by opl mutants. If this were the case, it can be suggested


that the role of AAC2 in po cells must be other than just maintaining intramitochondrial ATP levels.

In fact, a role of AAC2-mediated ADP/ATP translocation in the maintenance of the mitochondrial inner membrane potential, A$, has been proposed. It is now accepted that A$ is essential for mitochondrial biogenesis as protein import and sorting are A$ dependent (for a review, see Neupert, 1997). In p+ yeast cells, A$ is generated by proton pumping coupled to electron transport (Mitchell, 1979) or, under anaerobic conditions, by the reversible proton translocation through the FIFO-ATP synthase at the ex- pense of ATP hydrolysis (Kovac et al., 1972). In p-/po cells, both A$- generating pathways are absent and a third mechanism has to be in place to energize the membrane. Although transport systems for phosphate (Pi/ Ht symport or Pi/OH- antiport, reviewed by Wohlrab, 1986; Wehrle and Pedersen, 1989) and for inorganic pyrophosphate (Pereira-da-Silva et aZ., 1993) have been reported to be electrogenic, these systems do not appear to have a significant role in the generation of A$in po mitochondria (Giraud and Velours, 1997). However, the import of ATP through the ADP/ATP translocase and the hydrolysis of ATP by FI-ATPase are believed to play an important role in the formation of the transmembrane potential in petites (Chen and Douglas, 1989; Giraud and Velours, 1997).

The electrogenic nature of ATP/ADP exchange has been demonstrated in a number of early studies (Pfaff and Klingenberg, 1968; Laris, 1977; Klingenberg and Rottenberg, 1977). Basically, an exchange of external ATP4-, produced by the fermentative pathway, against the internal ADP3- results in a net gain of a negative charge on the matrix side of the membrane. In S. cerevisiae, an active ADP/ATP translocation is preserved in anaerobically grown cells and in cytoplasmic respiratory deficient mutants (Kolarov and Klingenberg, 1974; Subik et al., 1974b). In p" mitochondria, a A$potential value of 55 mV can be detected and this potential is generated in an ATP-dependent manner (Dupont et al., 1985).

As discussed earlier, because op l strains do not tolerate large deletions in mtDNA, such strains have been used successfully for the isolation of mit- mutants. However, it does not appear that mit- mutations in the mitochondrially encoded ATP6, 8 and 9 genes can be isolated from op l strains (J. Subik, personal communication). It seems that a functional FIFO- ATP synthase is required in cells lacking AAC2. In summary, despite the extensive studies that have been undertaken since the discovery of the opl mutation, the precise contribution of AA C2 in the petite-positive phenotype of S. cerevisiae is still a topic of active investigation.

6. PGSI [formerly PEL I]

S. cerevisiae pgsl/pell mutants are pfl-lethal (Janitor et al., 1995; Chang et aZ., 1998a). The PGSUPELI locus was first described as a mutant that

21 8 XIN JIE CHEN AND G. DESMOND CLARK-WALKER

does not tolerate EB mutagenesis or elimination of mtDNA (Subik, 1972). As the gene product encoded by PGSUPELI shows some sequence homology to the phosphatidylserine synthase (Pss) of Escherichia coli (Janitor et af., 1995), the gene was initially thought to encode a minor Pss activity in S. cerevisiae. Subsequent studies showed that PGSUPELI encodes a phosphatidylglycerophosphate (PG-P) synthase (Pgs). This was demonstrated clearly by evidence showing that overexpression of PGSUPELI results in a significant increase in in vitro PG-P synthase activity and that expression of an N-terminal truncated derivative of the PGSl/PELl gene can rescue the growth defect of an E. colipgsA mutant (Chang et af., 1998a).

Pgs catalyzes the synthesis of PG-P from CDP-diacylglycerol and glycerol-3-phosphate (Fig. 3), which is a rate-limiting step in the biosynthesis of phosphatidylglycerol (PG) and cardiolipin (CL) (Carman and Zeimetz, 1996; Greenberg and Lopes, 1996; Minskoff and Greenberg, 1997). PG and CL are two anionic phospholipids that are mainly confined to mitochondria1 membranes of yeast cells (Zinser et af., 1991). The biological roles of the two anionic phospholipids in both prokaryotic and eukaryotic organisms have been reviewed by Dowhan (1997). Two possible functions of the phospholipids are noteworthy. First, the anionic phospholipids appear to be required for the unfolding of proteins during translocation across the

Glycerol

Glycerol-3-Phosphate t

t

t t > 1 -Acyl-Glycerol-3-Phosphate

Phosphatidic Acid (PA)

CDP-Diacylglycerol

PGS 7 / P E L 7 + s e F \ + Glycerol-3-Phosphate

PhosPhatidYlserine (PSI Phosphatidylglycerophosphate (PGP)

Phosphatidylethanolarnine t (PE) + Phosphatidylglycerol (PG) 1

1 1

4 CL S 1/C R D 7

Phosphatidyicholine (PC) Cardiolipin (CL)

FIG. 3 Biosynthetic pathways in yeast for cardiolipin, phosphatidylglycerol, phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine. Only the genes encoding the enzymes discussed in the text are listed.


mitochondrial inner membrane. Precursors of mitochondrial proteins can bind specifically to cardiolipin (Ou et al., 1988). Andriamycin, an antibiotic with high affinity to anionic phospholipids, inhibits the import of proteins into mitochondria (Eilers et al., 1989; Endo et al., 1989). In addition, it has also been shown that anionic phospholipids participate in the formation of a helix in the presequences of mitochondrial proteins (Wang and Weiner, 1994; Chupin et al., 1995,1996). Second, CL was found to be bound tightly to a number of mitochondrial proteins and complexes. Activities of these proteins or complexes are sometimes affected by the absence of CL (Hoch, 1992). One example is the interaction of CL with the ADP/ATP translocase (Beyer and Klingenberg, 1985) and a requirement of CL for activity of the nucleotide carrier (Hoffmann et al., 1994).

S. cerevisiae can survive without CL as null mutants of the gene encoding cardiolipin synthase, CLSUCRDI, are viable. In addition, it came as a surprise to find that the mutants can grow on nonfermentable carbon sources such as glycerol and ethanol, although with a slower rate (Jiang et al., 1997; Tuller et al., 1998; Chang et al., 1998b). It has been suggested that in mutant cells, other membrane phospholipids, such as PG, can adequately fulfill the cellular requirement for CL. A fivefold elevation of PG has been detected in strains lacking Cls/Crd activity (Chang et al., 1998b). Indeed, S. cerevisiaepgsl/pell null mutants, lacking both PG and CL, display a much more severe phenotype. The pgsl/pell -disrupted strains are respiratory deficient, temperature sensitive on glucose medium, and have a low level of cytochrome c oxidase (Subik, 1974; Janitor et al., 1995; Chang et al., 1998a). When combined with disruption of the CHOl gene, encoding phosphatidylserine synthase, a lethal phenotype was observed, indicating a functional overlap between anionic phospholipids and phosphadylethanolamine (PE) in maintaining cellular structures (Janitor et al., 1996). Most impor- tantly, pgsl/pell null mutants are pO-lethal (Janitor et al., 1993; Chang et al., 1998a), which is in contrast to clsl/crdl mutants that can tolerate the elimination of mtDNA (Chang et aL, 1998b).

C. YMEl

The S. cerevisiae Y M E l (yeast mitochondrial escape) gene was initially isolated by complementation of a mutant that has increased escape of mitochondrial DNA to the nucleus (Thorsness and Fox, 1993; Thorsness el aL, 1993; reviewed by Thorsness and Weber, 1996). ymel mutants show some defects in the maintenance of their mitochondrial compartment integrity (Campbell et al., 1994). The mutants have an increased turnover of abnormal mitochondria by the vacuole and, subsequently, mtDNA is re- leased from the organelle at a higher frequency and migrates to the nucleus


at an increased rate compared to wild-type cells (Campbell and Thors- ness, 1998).

YMEl encodes an ATP- and zinc-dependent protease belonging to the AAA (ATPase associated with a variety of cellular activities) family of proteins. Ymelp is related closely to the E. coli FtsH protein (Tomoyasu et al., 1993) and contains ATP binding as well as the proteolytic HExxH motif (Nakai et al., 1995; Weber et al., 1996). Ymelp is part of an inner mitochondrial membrane complex of approximately 850 kDa (Leonhard et al., 1996) localized to the mitochondrial inner membrane (Schnall et al., 1994; Weber et al., 1996; Leonhard et al., 1996). The orientation of ATP- binding and proteolytic sites has been reported to be in the intermembrane space (Leonhard et al., 1996), but an orientation in the matrix has also been proposed (Weber et al., 1996).

In addition to DNA escape from mitochondria, mutations in Y M E l have pleiotropic effects. First, the mutants are defective in the degradation of unassembled cytochrome c oxidase subunit 2 (Nakai et al., 1995), indicating that Ymelp has an active role in protein turnover. Second, the mutants have a reduced activity of respiratory chain complexes (Nakai et al., 1995). Third, ymel cells are temperature sensitive on nonfermentable carbon sources and cold sensitive on glucose medium (Thorsness et al., 1993), which may also reflect a vulnerable state of the mitochondrial inner membrane. Finally, ymel mutants are pO-lethal (Thorsness et al., 1993). It remains unclear whether all the phenotypes can be attributed to the defect of ymel mutants in the degradation of membrane proteins. Alternatively, an additional role of Ymelp as a molecular chaperone should be considered (Nakai et al., 1995; Weber et al., 1996; also see review by Rep and Gri- vell, 1996).

D. ATPl and ATP2

The FIFo-ATP synthase is a multisubunit protein complex in the oxidative phosphorylation pathway located on the inner membrane of mitochondria. The yeast enzyme, like its counterparts in bacteria and chloroplasts, consists of two essential domains: the extrinsic and intrinsic membrane complexes, F1 and Fo. ATP is synthesized in the F1 sector by using the energy transmitted from Fo as a result of proton movement from the intermembrane space to the matrix side of the inner membrane (Boyer, 1997). The F1 portion, also called F1-ATPase, is composed of five proteins with the stoichiometry of 3a:3p:ly:16:1~. From the crystal structure of bovine F1-ATPase (Abrahams et al., 1994), it has been found that a and p subunits alternate in a hexameric complex and that a central space formed by the array is occupied by amino- and carboxyl-terminal a helices of the y subunit. ATP is synthesized as a


result of the rotation of the y subunit within the hexameric array (Sabbert et al., 1996; Noji et al., 1997; Yasuda et al., 1998) driven by proton movement through the Fo complex (Elston et al., 1998). The position of subunits 6 and E in F, has not been resolved, although the crystal structure of the E. coli E subunit, which is equivalent to the mitochondria1 6 protein, has been established (Uhlin et al., 1997).

All five subunits of F1-ATPase are encoded by nuclear genes in S. cerevisiae. The A TPI, A TP2, A TP3, A TP6(A TP16), and A TP E ( A TP15) genes encode the a, p, y, 6, and E subunits, respectively (Takeda et al., 1985, 1986; Arselin et al., 1991; GuClin et al., 1993; Giraud and Velours, 1994; Paul et al., 1994), with sizes of 55.0, 51.3, 30.6, 14.6, and 6.6 kDa (Arnold et al., 1998). The Fo complex is composed of at least nine proteins, with subunits 6 (28.0 kDa), 8 (5.8 kDa), and 9 (7.8 kD) encoded by mtDNA (Grivell, 1989) and subunits 4 (or b, 23.2 kDa), 5 (or OSCP, 20.9 kDa), 7 (or d, 19.7 kDa), h (or Atp 14, 10.4 kDa), f (or Atpl7, 10.6 kDa), and j/i (or Atpl8, 6.7 kDa) encoded by nuclear genes (Velours et al., 1988; Uh et al., 1990; Norais et al., 1991; Arselin et al., 1996; Spannagel et al., 1997; Arnold et al., 1998, 1999; Vaillier et al., 1999). Subunits 6, b, and 9 are equivalent to subunits a, b, and c from E. coli whereas OSCP, which confers oligomycin sensitivity to S. cerevisiae ATPase activity, corresponds to the F1 6 subunit of E. coli (Cox. et al., 1992). Three additional subunits, e(or Atp21Kiml1, 10.7 kDa; Arnold et al., 1997), g (or Atp20, 12.9 kDa), and k (or Atpl9, 7.5 kDa), have been found associated with a dimer form of the ATP synthase (Arnold et al., 1998).

A number of investigations have shown that mutations in F1-ATPase affect the growth of S. cerevisiae po cells. In an early study, it was found that a strain defective in F1-ATPase has a great tendency to lose its mitochondria1 genome. The resulting double mutants display a slow growth phenotype and do not grow anaerobically (Ebner and Schatz, 1973). Similar observations have also been made in recent studies. S. cerevisiae strains disrupted in the genes encoding the y and 6 subunits of F1-ATPase not only show total conversion to p-/po, but also grow poorly on glucose medium (Weber et al., 1995; Giraud and Velours, 1997; Zhang et al., 1999). These experiments raised the possibility that F1-ATPase has an active role in maintaining the strong growth of p-/po cells. A thorough study in this respect has been conducted showing that disruption of the genes encoding the a and p subunits of F1-ATPase renders S. cerevisiae petite negative (Chen and Clark-Walker, 1999). The latter finding demonstrates clearly that a function associated with F1-ATPase is essential for the viability of petites in S. cerevisiae. This is also consistent with the observation that the growth of S. cerevisiae strains, defective in the assembly of F1-ATPase, is affected severely in a p - background (A. Tzagoloff, personal communication). It can be expected that the disruption of genes such as ATPl l and ATP12,


which specifically coordinate the assembly of F1 (Ackerman and Tzagoloff, 1990; Bowman et al., 1991; Ackerman et al., 1992), would produce a petite- negative phenotype. It is thus not surprising that the inactivation of ATPl l prevents the conversion of cells lacking the y or 6 subunits of F1 into p-/ po (Zhang et al., 1999).

The efficient conversion of S. cerevisiae lacking F1-ATPase y and S subunits into petites has been explained by the passive and uncoupled proton flow through the Fo complex that may cause a collapse of the protonmotive force across the mitochondrial inner membrane (Zhang et al., 1999). As such, cells tend to eliminate the proton pore by eliminating mtDNA that encodes the subunits 6,8, and 9 of Fo. As far as the slow growth phenotype of po Ay and po AScells is concerned, Zhang and co-workers (1999) suggested that it may be due to the inefficiency of the ATP synthase inhibitor protein to control ATP hydrolysis by F1 lacking the y subunit. As a result, the intracellular ATP level is decreased, resulting in slow growth. In contrast, Giraud and Velours (1997) explained the slow growth of cells lacking the 6 subunit by a defect in the assembly of F1. The decreased hydrolysis of ATP in mitochondria by F1 lowers the intramitochondrial ADP level. Because the intramitochondrial ADP level is important for the maintenance of the mitochondrial membrane potential, A#, in po cells through the ADP/ATP exchange by the nucleotide carrier (see later), the growth rate of F1 mutants is affected.

E. Possible Explanation for the pO-Lethal Phenotype in S. cerevisiae Mutants

To understand the mechanism underlying the conversion of S. cerevisiae into a petite-negative form by mutations in AAC2, PGSl/PELl, YMEl, and genes encoding the (Y and p subunits of F1-ATPase, it would be interesting to know whether one can trace a common primary defect in the four types of mutants. One obvious remark from studies of AAC2, PGSl/PELl, and YMEl is that the primary functions of the three genes are all confined to the maintenance of structural and functional integrity of the mitochondrial inner membrane. Mutations in PGSl/PELl, leading to a deficiency in the anionic phospholipids PG and CL, and the accumulation of unassembled proteins in ymel mutants would directly affect the structural integrity of the membrane. Deletion of AAC2, as discussed earlier, may abolish the nucleotide carrier-mediated A# generation pathway across the inner membrane, which is essential for the survival of p-/po cells. In this sense, similar consequences can be expected as a result of the loss of mtDNA. po cells may have a compromised membrane integrity because of the accumulation of nuclear-encoded proteins in the absence of their mtDNA-encoded part-


ners and also have a decreased membrane potential due to the lack of a functional electron transport chain and ATP synthase. In this context, it may not be surprising that a synergistic lethality can be created when a mutation in one of the three genes is combined with elimination of the mitochondria1 genome. The primary cause of cell death is likely to be either the loss of inner membrane function or a collapse of A$.

The role of F1-ATPase in the survival of p-/po cells is not so clear. One possibility that has gained acceptance is that F1-ATPase may be epistatic to AAC2 in the maintenance of A@ (Giraud and Velours, 1997). ATP hydrolysis by F1 is important for producing intramitochondrial ADP that subsequently keeps the electrogenic ADP/ATP exchange process functioning in po cells. Based on this notion, one suggestion is that wild-type petite- negative yeasts such as K. lactis may not have a functional F1 on the elimination of mtDNA. Suppressor mutations in atp alleles could result in the formation of F1 that can hydrolyze ATP in the absence of Fo. This point remains to be clarified.

One puzzling and also intriguing finding is the possible genetic interaction between F1-ATPase and Ymelp. Weber and co-workers (1995) found that the pO-lethal phenotype of S. cerevisiae ymel mutants can be suppressed by the Thr297Ala and Ile303Thr mutations in they subunit of F1. Moreover, these two mutations are identical to the KZatp3-2 and -2 alleles (formerly mgi5-l and -2) that suppress pO-lethality in K. lactis (Chen and Clark- Walker, 1995; Clark-Walker et aZ., submitted for publication; see Fig. 2). The coincidental occurrence of the mutations suggests strongly that S. cerevisiae F1 and the mutant complex in K. Zucris are operating in a similar manner for the suppression of pO-lethality. Ymelp may have direct functional overlap with the mutant F1-ATPase or, alternatively, the function of an unknown protein, which is the substrate of Ymelp, can be replaced by the F1 complex in po cells. It is worthwhile to note that the wild-type F1 complex in po S. cerevisiae strains, which retains substantial ATP hydrolysis activity (Schatz, 1968; Kovac and Weissova, 1968; Criddle and Schatz, 1969; Tzagoloff et al., 1973), is unable to suppress the pO-lethal phenotype of ymel mutants. Consequently, the pO-lethality suppressor activity of the mutant F1 may not be simply the hydrolysis of ATP in mitochondria. It remains possible that a novel property of the complex other than ATP hydrolysis is responsible for the suppression of pO-lethality. It has been reported that the a subunit of F1 shares sequence similarities with molecular chaperones (Luis et aZ., 1990; Alconada et al., 1994). Functionally, the presence of the a subunit is required for the efficient import of proteins into mitochondria (Yuan and Douglas, 1992). It is unclear whether such a molecular chaperone activity of the F1 complex is involved in the suppression of pO-lethality. As can be seen in Fig. 4, in addition to the functional


FIG. 4 Possible functional interactions between nuclear genes encoding F1-ATPase (Y and p subunits, AAC2, YMEI, PGSI/PELI, and the mitochondria1 genome of S. cerevisiae. PG, phosphatidylglycerol; CL, cardiolipin.

interaction between the mutant F1 and Ymelp, it would be interesting to see whether similar interactions occur between F1 and AAC2 or PELl.

VII. Concluding Remarks

Although 50 years have elapsed since the first reports on cytoplasmic inheritance in baker’s yeast, we still have not answered some of the questions raised by these initial observations. Many hundreds of chemicals besides acriflavin can induce the formation of petite mutants but it remains to be determined how these agents act (Ferguson and von Borstel, 1992). The structure of mtDNA in petite mutants has been investigated thoroughly (Locker et al., 1979; Bernardi, 1979; Dujon, 1981; Dujon and Belcour, 1989), and the sites of recombination leading to deletions are known to be short regions of sequence homology (Clark-Walker, 1989; Weiller et al., 1991); however, the steps in recombination and the participating enzymes have been examined in detail only recently (Zweifel and Fangman, 1991; Kleff et al., 1992; Ezekiel and Zassenhans, 1993; Piskur, 1994; Ling et al., 1995; White and Lilley, 1996). Likewise, we know that highly suppressive petites in baker’s yeast retain mtDNA with an intact promoter in a GC-rich seg- ment termed ori/rep because it appears to be an origin of replication (Blanc and Dujon, 1980; Dujon, 1981; de Zamaroczy et al., 1981,1984). However, we do not understand how this element appears preferentially in progeny


(Zweifel and Fangman, 1991; Kleff et al., 1992; Lockshon et al., 1995; Piskur, 1997; Graves et al., 1998; van Dyck and Clayton, 1998).

Another aspect of the petite mutation considered in some detail in this review stems from the early observation that not all yeasts form mitochondrial genome deletion mutants when treated with DNA-targeting drugs. One of the central objectives is to understand how pO-lethality suppressor mutations enable K. lactis to survive the loss of mtDNA and to apply this knowledge to determining how S. cerevisiae has evolved to behave like a naturally occurring suppressor strain. The discovery of seemingly unrelated genes required for the maintenance of a petite-positive phenotype in S. cerevisiae, such as AAC2, PGSYPELl , Y M E l , and ones encoding the a and /3 subunits of F1-ATPase, indicates that understanding the dependence of po mutants on these genes will not be easy. However, it is to be anticipated that more genes of this type will be discovered.

Studies on pO-lethality suppressor mutations in other petite-negative yeasts should uncover whether atp alleles are a peculiarity of K. lactis or can be generalized to other organisms. Thus, it would be interesting to determine the nature o f p t p mutations in S. pombe and also whether genetic changes, similar to suppressor mutations, underlie the isolation of mammalian po cells (Desjardins et al., 1985; King and Attardi, 1989; Martinus et al., 1996).

In broader terms, the knowledge gained from investigations into the petite mutation in yeasts could be relevant to other eukaryotes. It has been documented that mtDNA deletions can be found in human diseases such as myopathies and neuropathies that occur either sporadically or in a specific and heritable way (Holt et al., 1988; Zeviani et al., 1989). Nuclear loci have been identified that predispose cells to deletions in mtDNA and cause autosomal dominant disorders (Suomalainen et al., 1995; Kaukonen et al., 1996). Autosomal recessive mutations have also been reported in some diseases that are responsible for multiple deletions in mtDNA (Mizusawa et al., 1988; Bohlega et al., 1996; Nishino et al., 1999). When contemplating possible genetic alterations in these diseases, mutations in nuclear genes involved directly in replication and transmission of mtDNA are likely candi- dates for change. In addition, the possible involvement of pO-lethality suppressor mutations that predispose mtDNA to deletions needs to be considered.

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[International Review of Cytology] Volume 194 || The Petite Mutation in Yeasts: 50 Years On

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