Genes required for respiratory growth
Two independent screens of the yeast deletion library have previously revealed two partially overlapping sets of pet
genes. By plating the homozygous diploid yeast deletion library on media containing glycerol as a carbon source, Dimmer et al
] identified 341 deletion mutants that were unable to grow. In a very similar approach, Luban et al
] identified a set of 355 respiratory-deficient clones by screening the MAT
a yeast deletion library. While about two-thirds of the mutants in each screen were found to be respiratory-deficient also in the other screen, a surprisingly large number of mutants were isolated only once [17
]. It seems unlikely that this is due to differences in the genetic background, because both screens have been conducted in largely isogenic strains, BY4743 and BY4741 [18
]. Here, we screened the MAT
α deletion library (BY4742 background) to obtain a third set of respiratory-deficient mutants. This was then compared with the data obtained by Dimmer et al
] and Luban et al
]. The MAT
α deletion library contained 319 mutants that were unable to grow on glycerol-containing medium (Additional data file 1). Of these, 176 are common to all three sets of pet
genes (Figure ). In the following we will refer to these genes as highly penetrant pet
genes. 125 genes have been identified in two of three screens, and 237 genes have been identified only once (pet
genes unique to this study are listed in Additional data file 2). Nineteen additional pet
genes (not included in the set of 176 highly penetrant pet
genes) were only covered by one or two libraries. Based on data from the Saccharomyces Genome Database [19
] and manual annotation, we grouped all genes according to their frequency of occurrence in pet
screens and the intracellular location and function of their gene products (Additional data file 3).
Figure 1 Nuclear pet genes of S. cerevisiae. (a) The numbers of pet mutants identified in three screens of the yeast deletion library are indicated. References: Dimmer et al. , Luban et al. . (b) The intracellular location of proteins encoded by pet genes (more ...)
Strikingly, 129 out of the 176 pet genes found in all three screens encode proteins known to be located in mitochondria, corresponding to 73.3% (Figure ; Additional data file 3). The fraction of genes encoding mitochondrial proteins was reduced to 52.1% for pet genes found in two of three screens, and as low as 14.7% for pet genes that were found only once (Figure ; Additional data file 3). This demonstrates a clear correlation of the penetrance of pet phenotypes with mitochondrial functions of the affected gene products. The majority of the 176 pet genes found in all libraries encode proteins devoted to maintenance and expression of the mitochondrial genome and assembly of the respiratory chain (Figure ; Additional data file 3). Thirteen open reading frames (ORFs) are unlikely to encode proteins, because they overlap with other known genes (Additional data file 3), reducing the number of protein-coding genes to 163.
Differences of growth behavior of strains taken from different versions of the deletion libraries could either reflect inherent properties of the mutant strains, they could be due to technical differences between the various screens, or they could mean that a given deletion in one collection is wrong (as it has occasionally been observed by us and others; for example, strains not correct in the MATα library include Δrpo41 lacking the mitochondrial RNA polymerase). We reasoned that incorrect mutants will be enriched among strains that showed respiratory competence in one screen but were respiratory-deficient in the two other screens because it is more likely that a specific phenotype is obscured rather than generated by chance. To test this, we checked the genotypes of 29 mutants taken from the MATα library by PCR. Nineteen randomly chosen mutants were tested that were respiratory-competent in the MATα library, but respiratory-deficient in the MATa and homozygous diploid library. Of these, six mutants (Δyal012w, Δybl038w, Δydl202w, Δydr268w, Δyor205c, and Δypl029w) contained exclusively the wild-type allele, seven mutants (Δydr231c, Δydr332w, Δyil036w, Δyjr090c, Δymr066w, Δypr047w, and Δypr124w) contained a mixture of deletion and wild-type alleles, and six mutants were found to have the correct genotype (Δyal047c, Δybr163w, Δydr323c, Δykl148c, Δyml081c-a, and Δyml129c). In addition, we tested ten mutants that showed a pet phenotype only in the MATα library, but not in the MATa and homozygous diploid library, and ten mutants, that showed a pet phenotype in all three screens. All mutants of the two latter groups were found to have the correct genotype. This means whenever a wrong deletion was detected, a pet phenotype was obscured by the presence of the wild-type allele, whereas all respiratory-deficient mutants tested were found to have the correct genotype. We conclude that several discrepancies of growth phenotypes can be ascribed to wrong genotypes that are present in the deletion library. However, the fact that a relatively large number of mutants with confirmed correct genotypes show differences in their growth behavior points to a pronounced phenotypic plasticity of pet mutants. Furthermore, the correlation of the penetrance of pet phenotypes with mitochondrial localization of gene products (Figure ) is a clear indication that the phenotypic variability is not only due to wrong deletions present in the mutant libraries, but also reflects biological processes.
Eight highly penetrant pet
, and YPR116w
) encode previously uncharacterized proteins, and earlier studies have revealed a respiratory-deficient phenotype for two additional ORFs of unknown function that were not covered by all three yeast deletion libraries, YNL213c
] and YJL062w-a
]. We confirmed the identity of these mutant strains by PCR and named the genes RRG1
(for 'Required for respiratory growth') as the functional analysis described below proves that their products are novel factors required for respiratory growth.
Comparative growth analysis on different non-fermentable carbon sources
We asked whether the respiratory-deficient phenotype observed for the 319 pet mutants isolated from the MATα deletion library is specific to glycerol metabolism or reflects a general lack of respiration competence. To test this, we plated the mutants also on complete media containing lactate or ethanol as sole carbon sources. The vast majority (305 strains, corresponding to 95.6% of the pet mutants) failed to grow on all non-fermentable carbon sources that were tested. Of the remainder, seven mutants showed a growth defect only on glycerol-containing medium, seven on glycerol or ethanol-containing media, and one mutant on glycerol or lactate containing media (Additional data file 4). As pet phenotypes are highly reproducible even on different carbon sources we conclude that our screen gives a largely accurate estimate of respiratory deficiencies in the MATα deletion library.
Restoration of respiratory activity by mating with Δmip1 and by cytoduction of [rho+] mitochondria
In order to define the genetic basis of respiratory deficiency, we subjected the complete set of 319 pet
mutants isolated from the MAT
α deletion library to various functional tests (Figure ). As a petite
phenotype is often associated with the complete or partial loss of the mitochondrial genome [13
], we first asked whether the pet
mutants contain functional mtDNA. To test this, pet
mutants were mated with a strain lacking the mtDNA polymerase Mip1. As the Δmip1
strain is [rho0
], resulting heterozygous diploid strains are able to grow on glycerol-containing medium only if functional mtDNA is provided by the pet
mutant mating partner. We observed restoration of respiratory activity in 157 heterozygous diploid strains demonstrating that the parental pet
strains possessed an intact mitochondrial genome. In contrast, 162 strains failed to grow on glycerol-containing medium after mating, suggesting that the parental pet
mutants were [rho-
] or [rho0
Figure 2 Summary of the systematic functional analysis of 319 pet mutants isolated from the MATα yeast deletion library. Grey boxes indicate groups of mutants that were further analyzed, black boxes indicate the final level of resolution of functional (more ...)
The complementation test with Δmip1 does not discern whether the protein encoded by the pet gene is obligatorily required for maintenance of mtDNA, or whether a functional mitochondrial genome had been spontaneously lost in the pet mutant during many generations of growth. To discriminate between these possibilities, we replenished cells with mitochondria containing a wild-type [rho+] mitochondrial genome by cytoduction. In brief, pet mutants were crossed with a [rho+] donor strain that carries a kar1 mutation to prevent karyogamy in the zygote. After counterselection against nuclear chromosomes of the donor strain, growth of the haploid progeny was assessed on glycerol-containing media. Restoration of respiratory activity after cytoduction was observed in 67 pet mutants, whereas 252 strains failed to grow on non-fermentable carbon sources.
Combining the results from the Δmip1 mating test and the cytoduction experiment allowed us to define four classes of pet mutants (Figure ; Additional data file 5). Class I mutants were not rescued either by mating with Δmip1 or by cytoduction; class II mutants were rescued by mating with Δmip1 as well as by cytoduction; class III mutants were rescued only by mating with Δmip1, but not by cytoduction; and class IV mutants were rescued only by cytoduction, but not by mating with Δmip1. The basic properties of these classes are summarized in Table . In the following, the various classes of pet mutants are further examined (Figure ).
Figure 3 Classes of pet mutants. The left column indicates genotypes of haploid pet mutant strains taken from the deletion library carrying a deletion in the nuclear genome (Δyfg1, 'your favourite gene 1') and either no mtDNA ([rho0]; alternatively these (more ...)
Genes required for maintenance of mtDNA
The 118 class I mutants were [rho-] or [rho0] and remained respiratory-deficient after introduction of functional mitochondria. This group of mutants is expected to include all components that are essential for maintenance of a [rho+] genome. In addition, we expected it to contain components deletion of which leads to a gradual loss of mtDNA and, at the same time, induces respiratory deficiency due to lack of functions not directly related to mtDNA maintenance. To discern between these possibilities, we subjected all class I mutants to various functional tests. First, we tested for the presence of mtDNA by DAPI (4',6-diamidino-2-phenylindole) staining immediately after cytoduction. Second, we tested growth on YPG medium after adaptation to the medium by pre-culture on YPG containing low amounts of glucose. And third, we tested mitochondrial protein translation activity by SDS-PAGE and autoradiography after labeling cycloheximide-treated cells with 35S methionine.
Genes essential for maintenance of mtDNA were defined by the following criteria: At least 95% of the cells observed by DAPI staining after cytoduction were devoid of mtDNA and the remainder contained less than five mtDNA nucleoids per cell. This phenotype was observed after cytoduction in the Δmip1
mutant lacking the mtDNA polymerase and, therefore, is indicative of instantaneous loss of mtDNA. In addition, cells lacking genes essential for maintenance of mtDNA were expected to be unable to grow on YPG after adaptation to the carbon source, and they were unable to produce even trace amounts of mitochondria-encoded proteins. Sixteen mutants were identified that matched these criteria (Table ). We propose that the gene products lacking in these mutants are particularly important for maintenance of mtDNA. As expected, this group includes several components known to be involved in mtDNA metabolism: the mtDNA polymerase Mip1 [22
]; mtDNA helicases Hmi1 [23
] and Pif1 [24
]; Apn1, a DNA repair protein active in the nucleus and mitochondria [25
]; and aconitase, Aco1, an enzyme of the citric acid cycle that has an additional role in mtDNA maintenance [26
Genes essential for maintenance of mtDNA
It has been observed that a block of mitochondrial protein synthesis leads to a rapid and quantitative loss of mtDNA [27
]. However, the reasons for this phenomenon are still unknown. Here, we observed instantaneous loss of mtDNA in cells lacking Mrpl37, Mtf1, Mtg2, Rsm24, and Slm5, which are all required for mitochondrial transcription or translation, and in a deletion mutant lacking the dubious ORF YKL091w
, which overlaps with the MRPL38
gene encoding a mitochondrial ribosomal protein. Loss of mtDNA at a relatively high rate was also observed in several other class I mutants lacking components of the mitochondrial protein synthesis machinery. These findings underscore the importance of mitochondrial protein synthesis for maintenance of mtDNA. Moreover, rapid loss of mtDNA was observed in the Δatp4
mutant lacking ATPase subunit b. This is consistent with earlier observations [28
]; however, the molecular reasons are not understood [29
]. Also, Δpet100
mutants lacking a factor required for cytochrome c
oxidase assembly showed rapid loss of mtDNA. As loss of mtDNA in Δatp4
, and Δslm5
occurs instantaneously (as rapid as in Δmip1
) we consider it likely that replication and/or inheritance of mtDNA is actively suppressed in these strains. These results point to an active role of Atp4, Mrpl37, Mtf1, Mtg2, Pet100, Rsm24, and Slm5 in regulating mtDNA abundance in yeast mitochondria.
Other factors required for mtDNA inheritance are Mgm1, Doc1 and the newly identified protein Rrg5. Mgm1 is a dynamin-related protein required for mitochondrial genome maintenance by mediating mitochondrial fusion [30
]. Doc1 is involved in cyclin proteolysis as a processivity factor required for the ubiquitination activity of the anaphase promoting complex (APC) [33
]. Intriguingly, Doc1 has been found in the mitochondrial proteome [6
]. Thus, it is tempting to speculate that it links mtDNA replication and/or inheritance to the cell cycle. The RRG5
) encodes a protein of unknown function. Its sequence does not show similarities to any characterized protein. As the Rrg5 protein has been localized to mitochondria [6
], we propose that it is a novel factor essential for maintenance of mtDNA.
In addition to class I mutants, 44 pet
mutants were identified that were not complemented by mating with Δmip1
but could be rescued by cytoduction. These strains are able to maintain newly re-introduced mtDNA when they are grown on non-fermentable carbon sources (class IV; Additional data file 5). It is conceivable that these mutants have a tendency to spontaneously lose their mitochondrial genome when they are grown on fermentable carbon sources for longer times. This has been observed previously for Δmdm31
mutants that showed a pet
phenotype in the screen performed by Dimmer et al
], but not in the screens performed by Luban et al
] and in the screen reported here. Freshly made Δmdm31
deletion mutants have been found to be able to maintain [rho+
] mtDNA [36
]. However, mtDNA is not stably inherited and is gradually lost after several generations of growth in glucose-containing medium [36
]. To test this systematically for all class IV mutants, we replenished mtDNA by cytoduction and then passaged the strains in liquid YPD medium for 10 days to allow for loss of mtDNA. Presence or absence of mtDNA was assayed by DAPI staining immediately after cytoduction and after 10 days of replicative growth. In all strains, at least 90% of the cells contained mtDNA directly after cytoduction. Continued growth in glucose-containing medium led to increased loss of mtDNA in many mutants (Additional data file 6), suggesting that gradual loss of mtDNA accounts for the pet
phenotype in many class IV mutants. Only few mutants maintained mtDNA as stably as the wild type (Additional data file 6). We consider it possible that these mutants require more generation times or special growth conditions to induce loss of mtDNA, or that these mutants rapidly accumulate mtDNA point mutations or deletions rendering the mitochondrial genome non-functional over time. Interestingly, 77% of the class IV mutants have not been found in the screens by Dimmer et al
] and Luban et al
], suggesting that many of the affected genes are only indirectly related to maintenance of respiratory activity.
Genes required for protein translation in mitochondria
Next, we asked which genes are required for mitochondrial protein synthesis. Mutants defective in this process are expected to be found in either class I or class III. Class I contains mutants that have lost their mtDNA as a consequence of blocked mitochondrial translation activity, whereas class III contains mutants that are defective in translation but maintain an intact mitochondrial genome. In order to be able to test for mitochondrial protein synthesis activity, we replenished wild-type mtDNA in class I mutants by cytoduction. After this treatment, mtDNA could be visualized by DAPI staining in 102 mutants, whereas 16 mutants lacking genes essential for maintenance of mtDNA immediately became [rho0
] (see above; Table ). For class III mutants, we reasoned that some strains might be unable to grow on medium containing glycerol as the sole carbon source because of synergistic effects of compromised mitochondrial function in combination with catabolite repression, which reduces the expression of genes required for respiration [8
]. Therefore, we first relieved catabolite repression in all class III mutants by growth on glycerol-containing medium supplemented with limiting amounts of fermentable carbon source (3% glycerol/0.1% glucose) before replicating the strains on glycerol-containing medium. After this treatment, 77 strains were able to grow on plates containing glycerol as the sole carbon source (Additional data file 7). We conclude that the gene products lacking in these mutants are dispensable for respiration.
Then, we tested mitochondrial translation in a total number of 159 deletion mutants (102 class I mutants with replenished mtDNA and 57 class III mutants unable to grow on glycerol-containing medium after adaptation to the carbon source). Strains were grown to logarithmic growth phase in medium containing fermentable carbon sources, before cytosolic translation was stopped by the addition of cycloheximide. Newly synthesized mitochondrial proteins were labeled with 35S methionine, and cell extracts were analyzed by SDS-PAGE and autoradiography.
Mitochondrial translation products could not be detected in 88 mutants (Table ). We conclude that these genes are required for mitochondrial protein synthesis. Encoded proteins include 39 subunits of the mitochondrial ribosome and several additional components required for mitochondrial transcription, translation or assembly of the respiratory chain [37
]. In addition, mitochondrial translation activity was absent in several mutants lacking proteins known to be required for mtDNA inheritance, such as Fzo1 [38
], Mhr1 [40
], Msh1 [41
], or Mgm101 [42
]. Supposedly, in these strains - and likely also in other class I mutants - the mitochondrial genome had been largely lost or damaged during growth of the strains in the time between cytoduction and the labeling reaction. It should be noted that strain-dependent effects might also play a role, because, for example, Δpet309
was observed to be completely translation-inactive here, whereas mitochondrial translation products could be observed when this mutant was constructed in the W303 genetic background [43
]. Five genes (RRG1
, and RRG8
) encode uncharacterized proteins, and two dubious ORFs (YDR114c
) overlap with genes encoding mitochondrial ribosomal proteins. A possible role of Rrg1, Rrg2, Rrg6, and Rrg8 as novel components required for mitochondrial protein synthesis is discussed below.
Genes essential for mitochondrial translation
Specific alterations of the pattern of newly translated mitochondrial proteins were observed in ten mutants (Figure and Table ). A role in the expression of specific mitochondria-synthesized proteins has already been described for Aep2 [44
], Cbs2 [45
], Mrs1 [46
], Mss51 [47
], Pet54 [48
], and Pet494 [50
]. We observed that the pattern of mitochondrial translation products was also altered in Δcoq3
, and Δvma8
mutants. Coq3 is required for the biosynthesis of ubiquinone (coenzyme Q) in mitochondria [51
]. We observed that mutant cells show a strong reduction of Cox1 (Figure , lane 11). Cyc3 is the mitochondrial cytochrome c
heme lyase that attaches the heme cofactor to apo-cytochrome c
in the intermembrane space [52
]. Strikingly, mutant mitochondria show a strong reduction of Cox1 and cytochrome b
and generate an additional protein band above Cox3 (Figure , lane 2), pointing to a role of Cyc3 also in the biogenesis of other mitochondrial proteins. Rrg10 is an uncharacterized mitochondrial protein that might play a specific role in the expression of the mitochondrial COX1
gene (Figure , lane 7), as discussed below. Cox1 and Atp6 are also reduced in the Δvma8
mutant lacking a subunit of the vacuolar H+
], suggesting that expression of these proteins is particularly sensitive to changes in cell metabolism (Figure , lane 4).
Figure 4 Mitochondrial protein synthesis in pet mutants showing an altered translation pattern. Yeast strains were grown in raffinose-containing minimal medium to logarithmic growth phase, cytosolic translation was stopped by the addition of cycloheximide, and (more ...)
Genes required for expression of specific mitochondrial translation products
Other genes important for respiration
In sum, 61 respiratory-deficient mutants showed a wild-type-like mitochondrial translation pattern (Additional data file 8). We conclude that these genes are not essential for mitochondrial genome maintenance or mitochondrial protein synthesis. This group contains 32 genes encoding known mitochondrial proteins, many of which are required for assembly of the respiratory chain. Eighteen genes encode known extra-mitochondrial proteins, and 11 ORFs are uncharacterized. Five of the uncharacterized ORFs are unlikely to encode proteins because they overlap with known protein-coding genes, whereas six ORFs (YDL129w, YDL133w, YDL033w/RRG4, YNL213c/RRG9, YOL071w, and YOL083w) might encode novel proteins involved in maintenance of respiratory activity. Possible roles of Rrg4 and Rrg9 in this process are discussed below.
Half of the pet
genes encoding extra-mitochondrial proteins are associated with vacuolar functions (Additional data file 8). Moreover, a surprisingly large number of genes encoding V-ATPase subunits are highly penetrant pet
genes (Figure ; Additional data file 3). What might be the function of the vacuole in maintenance of respiratory activity in yeast? We suggest three possibilities. First, vacuolar functions in metabolite storage or in cytosolic ion and pH homeostasis [54
] might interfere with mitochondrial metabolism. Second, loss of V-ATPase activity has been reported to render cells hypersensitive to oxidative stress [56
], which might have an impact on mitochondrial functions as well. And third, the vacuole is the terminal compartment receiving cellular components destined for degradation by autophagic pathways. As also mitochondria are degraded by autophagy in yeast [59
], it is possible that the vacuole plays an important role in mitochondrial quality control and turn-over. The high number of pet
mutants lacking V-ATPase subunits clearly demonstrates that there is an important - as yet not fully understood - functional relationship between the vacuole and mitochondria.
Contribution of acquired defects to maintenance of respiratory activity
The respiratory-deficient phenotype of 23 pet mutants was rescued by mating with Δmip1 as well as by cytoduction (class II; Additional data file 9). These mutants contained a [rho+] mitochondrial genome, as indicated by the mating experiment. In addition, three independently performed cytoduction experiments suggest that replenishment of cytoplasmic material reproducibly restores and maintains respiratory growth, at least for a few generations. These observations point to the possibility that respiratory competence may involve acquired properties that are not strictly linked to the nuclear or mitochondrial genotype. In order to corroborate this assumption, we tested whether cytoduction with a [rho0] donor strain would also restore respiratory growth. Rescue was observed in 11 strains (Additional data file 9), suggesting that, at least in some cases, cytoplasmic components other than mtDNA are able to improve respiratory functions. We hypothesize that respiratory deficiency may be an acquired phenotype that does not exclusively depend on the genotype.
Among ten class II mutants lacking known mitochondrial proteins (Δcoq5
, and Δsom1
) are four mutants that are specifically defective in the assembly of the cytochrome c
oxidase (COX complex). Cox10 is required for the synthesis of the heme A cofactor [60
], Cox19 is a metallochaperone that delivers copper to the COX complex [62
], Mss2 is required for the membrane translocation of the carboxyl terminus of the mitochondria-encoded Cox2 protein [63
], and Cox16 contributes to assembly of the COX complex by an as yet unknown mechanism [64
]. Intriguingly, all four of these proteins are required for assembly of COX subunits at a post-translational stage. While respiratory-deficiency in Δcox10
, and Δmss2
mutants has been documented before [60
], we asked whether acquired properties might contribute to the loss of respiratory activity in these mutants. To exclude effects due to differences in mtDNA copy number, we first quantified the abundance of the mitochondrial COX3
gene by RT-PCR. We found that mtDNA is stably maintained in Δcox10
, and Δmss2
mutants at a level very similar to wild-type cells (Figure ).
Figure 5 Acquired phenotypes of COX assembly mutants. (a) Quantification of mtDNA. Yeast strains were grown overnight in liquid glucose-containing medium. Total DNA was isolated and the copy number of the mitochondrial COX3 gene was related to that of the nuclear (more ...)
Next, we tried to rescue the deletion mutants with plasmids encoding wild-type copies of the respective genes under control of their endogenous promoters. Remarkably, after growth on selective medium a substantial number of transformants remained respiratory-defective after complementation with the respective wild-type gene (Figure ). The occurrence of respiratory-deficient clones was not induced by the transformation procedure per se
because transformation of wild-type cells with the same plasmids yielded 100% respiration-competent clones (not shown). In order to test whether Δcox10
, and Δmss2
clones lose properties required for respiratory competence over time, we subjected the deletion mutants to chronological aging [65
], that is, continued incubation of stationary phase cultures. Mutant cells were incubated on glucose-containing medium for several days at room temperature before transformation with the complementing plasmids. Under these conditions, the fraction of clones that could not be rescued increased to 60 to 81% for mutant cells, whereas only 6% of aged wild-type clones were observed to be respiratory-deficient after transformation (Figure ). This suggests that mitochondria in Δcox10
, and Δmss2
cells become irreversibly damaged over time, producing a respiratory-deficient phenotype that cannot be rescued any more. Apparently, this damage is already induced during vegetative growth and is markedly enhanced during aging.
As mitochondrial metabolism and aging are linked to the generation of potentially harmful reactive oxygen species (ROS) [66
] we asked whether ROS accumulate in COX assembly mutants. High levels of ROS generated in yeast cells convert the non-fluorescent compound dihydrorhodamine 123 (DHR) to the oxidized fluorescent chromophore rhodamine 123 [67
]. Upon incubation of young wild-type Δcox10
, and Δmss2
cultures with DHR (8 h in liquid YPD medium) only very few cells showed significant staining (Figure ). After continued incubation (32 h), about 60% of wild-type cells and 90 to 98% of mutant cells showed significant rhodamine staining (Figure ). Very similar results were obtained when aging was allowed for up to 80 h (not shown). Furthermore, we noticed that rhodamine staining in wild-type cells was relatively faint and often restricted to tubular structures (presumably representing the mitochondrial network), whereas the signal was much stronger and dispersed throughout the cytosol in mutant cells (Figure ). We conclude that Δcox10
, and Δmss2
cells produce elevated ROS levels during chronological aging. Presumably, ROS induce irreversible damage to mitochondrial proteins, lipids and/or mtDNA, thereby preventing rescue of the mutant phenotype by transformation with complementing plasmids. On the other hand, replenishment of fresh mitochondria by cytoduction might improve respiratory performance, at least for a limited time. It remains to be shown whether accumulation of ROS-induced damage is a general feature of class II mutants.
Novel components essential for respiratory growth
All previously uncharacterized RRG
genes analyzed herein can be clearly related to mitochondrial functions. Proteins Rrg1, Rrg2, and Rrg5 through Rrg10 have been localized to mitochondria by high-throughput green fluorescent protein (GFP) fusion protein localization [35
] and/or mitochondrial proteome analysis [6
]. The Rrg3 protein carries a putative mitochondrial presequence, whereas the intracellular location of Rrg4 remains unknown. Functional properties of RRG
genes are summarized in Table .
Functional properties of newly described RRG genes
Δrrg1, Δrrg2, Δrrg4, Δrrg5, Δrrg6, Δrrg8, and Δrrg9 are class I pet mutants lacking a functional mitochondrial genome. DAPI staining revealed defects in the organization of mtDNA that emerged early after introduction of wild-type mitochondrial genomes by cytoduction in Δrrg1, Δrrg2, Δrrg6, Δrrg8, and Δrrg9 mutants. Nucleoids appeared larger compared to the wild type, the number of nucleoids per cell was reduced, and several cells were completely devoid of mtDNA (not shown). These observations suggest that Rrg1, Rrg2, Rrg6, Rrg8, and Rrg9 play an important role in maintenance of mtDNA. Immediate and complete loss of mtDNA after cytoduction in the Δrrg5 mutant indicates an essential role of Rrg5 for maintenance of mtDNA (see above).
Interestingly, Rrg2 contains a pentatricopeptide (PPR) motif. PPR protein-encoding genes can be found in virtually all sequenced eukaryotic genomes, but are particularly abundant in plants. PPR proteins are localized in plastids and mitochondria where they are involved in the control of various stages of gene expression [68
]. Lack of mitochondrial translation activity and early loss of mtDNA observed here are consistent with a role of Rrg2 in control of mitochondrial gene expression.
is a class III pet
mutant able to maintain a [rho+
] genome and wild-type-like mitochondrial protein translation activity. Although a mitochondrial location of Rrg3 has not been shown experimentally, the Mitoprot program [69
] predicts the presence of a mitochondrial presequence with a high probability (0.9484). Mutants lacking Rrg3 (alternative name Aim22) show an increased petite
]. The protein has high homology to lipoate-protein ligase A family members [71
]. Thus, it is conceivable that Rrg3 mediates the attachment of the lipoic acid cofactor to mitochondrial multienzyme complexes, such as pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, glycine decarboxylase or others. Intriguingly, it has recently been reported that lipoate-protein ligase activity is important for maturation of RNase P, an enzyme that processes mitochondrial precursor tRNAs [72
]. It will be interesting to determine whether Rrg3 plays a specific role in this process.
mutant has recently been identified as one of 86 gene deletion mutants that show an increased assembly of Rad52, a central protein of the homologous recombination machinery, in subnuclear foci reflecting DNA repair centers. Therefore, the gene has been named IRC19
(for 'increased recombination centers') [73
]. Interestingly, several other genes related to mitochondrial function were also isolated in this screen, including CBT1
, and YMR31
. It has been suggested that an increase of oxidative damage due to impaired respiratory chain functions might stimulate spontaneous DNA lesions in the nucleus and, therefore, constitutes a functional link between mitochondrial respiration and DNA repair processes in the nucleus [73
]. As a Rrg4-GFP fusion protein can not be visualized in cells [35
], the intracellular location of Rrg4 remains unknown.
gene has recently been found in a screen for components involved in remodelling of the endoplasmic reticulum (ER). It has been named HER2
(Hmg2-induced ER remodelling); however, its molecular role in shaping the ER membrane remained unknown [74
]. As the Rrg6 protein has been localized to mitochondria by both GFP tagging and proteome analysis [6
], we propose that its primary function is related to maintenance of respiratory activity. The protein is highly homologous to bacterial glutamyl-tRNA amidotransferases, and a role in mitochondrial protein synthesis is consistent with our observation that mitochondrial translation is blocked in the Δrrg6
mutant (Table ). Recently, RRG5
(alternative name GEP5
) and RRG6
(alternative names GEP6
) have been shown to genetically interact with genes encoding prohibitin ring complexes in the mitochondrial inner membrane [75
]; however, the functional significance of this interaction is not yet understood.
is a class II pet
mutant presumably acquiring respiratory deficiency independent of its genotype. The RRG7
gene encodes a mitochondrial protein [35
] that has homologs in fungi and other lower eukaryotes. Its function in mitochondrial biogenesis is currently unknown; however, the deletion mutant has been reported to exhibit increased sensitivity to the synthetic tripeptide arsenical 4-(N
glutathionylacetyl)amino) phenylarsenoxide that targets mitochondria by inactivating the adenine nucleotide translocator. This drug inhibits proliferation of actively dividing endothelial cells and is an inhibitor of angiogenesis during tumor formation [76
is a class III pet
mutant able to maintain a [rho+
] genome. The RRG10
gene encodes a small mitochondrial protein [34
] of only 85 amino acid residues. Analysis of the mitochondrial translation pattern revealed a reduction of Cox1, suggesting that Rrg10 plays a specific role in transcription or maturation of mitochondrial mRNAs and/or translation or assembly of mitochondrial gene products.