Mapping mitonuclear epistasis using a novel recombinant yeast population.

Genetic variation in mitochondrial and nuclear genomes can perturb mitonuclear interactions and lead to phenotypic differences between individuals and populations. Despite their importance to most complex traits, it has been difficult to identify the interacting mitonuclear loci. Here, we present a novel advanced intercrossed population of Saccharomyces cerevisiae yeasts, called the Mitonuclear Recombinant Collection (MNRC), designed explicitly for detecting mitonuclear loci contributing to complex traits. For validation, we focused on mapping genes that contribute to the spontaneous loss of mitochondrial DNA (mtDNA) that leads to the petite phenotype in yeast. We found that rates of petite formation in natural populations are variable and influenced by genetic variation in nuclear DNA, mtDNA and mitonuclear interactions. We mapped nuclear and mitonuclear alleles contributing to mtDNA stability using the MNRC by integrating a term for mitonuclear epistasis into a genome-wide association model. We found that the associated mitonuclear loci play roles in mitotic growth most likely responding to retrograde signals from mitochondria, while the associated nuclear loci with main effects are involved in genome replication. We observed a positive correlation between growth rates and petite frequencies, suggesting a fitness tradeoff between mitotic growth and mtDNA stability. We also found that mtDNA stability was correlated with a mobile mitochondrial GC-cluster that is present in certain populations of yeast and that selection for nuclear alleles that stabilize mtDNA may be rapidly occurring. The MNRC provides a powerful tool for identifying mitonuclear interacting loci that will help us to better understand genotype-phenotype relationships and coevolutionary trajectories.

Evolutionary Trajectories are Contingent on Mitonuclear Interactions.

Critical mitochondrial functions, including cellular respiration, rely on frequently interacting components expressed from both the mitochondrial and nuclear genomes. The fitness of eukaryotic organisms depends on a tight collaboration between both genomes. In the face of an elevated rate of evolution in mtDNA, current models predict that the maintenance of mitonuclear compatibility relies on compensatory evolution of the nuclear genome. Mitonuclear interactions would therefore exert an influence on evolutionary trajectories. One prediction from this model is that the same nuclear genome evolving with different mitochondrial haplotypes would follow distinct molecular paths toward higher fitness. To test this prediction, we submitted 1,344 populations derived from 7 mitonuclear genotypes of Saccharomyces cerevisiae to >300 generations of experimental evolution in conditions that either select for a mitochondrial function or do not strictly require respiration for survival. Performing high-throughput phenotyping and whole-genome sequencing on independently evolved individuals, we identified numerous examples of gene-level evolutionary convergence among populations with the same mitonuclear background. Phenotypic and genotypic data on strains derived from this evolution experiment identify the nuclear genome and the environment as the main determinants of evolutionary divergence, but also show a modulating role for the mitochondrial genome exerted both directly and via interactions with the two other components. We finally recapitulated a subset of prominent loss-of-function alleles in the ancestral backgrounds and confirmed a generalized pattern of mitonuclear-specific and highly epistatic fitness effects. Together, these results demonstrate how mitonuclear interactions can dictate evolutionary divergence of populations with identical starting nuclear genotypes.

Stem cell plasticity and regenerative potential regulation through Ca2+-mediated mitochondrial nuclear crosstalk.

The multi-lineage differentiation potential is one of the prominent mechanisms through which stem cells can repair damaged tissues. The regenerative potential of stem cells is the manifestation of several changes at the structural and molecular levels in stem cells that are regulated through intricate mitochondrial-nuclear interactions maintained by Ca2+ ion signaling. Despite the exhilarating evidences strengthening the versatile and indispensible role of Ca2+ in regulating mitochondrial-nuclear interactions, the extensive details of signaling mechanisms remains largely unexplored. In this review we have discussed the effect of Ca2+ ion mediated mitochondrial-nuclear interactions participating in stem plasticity and its regenerative potential.

Mitochondrial-nuclear coadaptation revealed through mtDNA replacements in Saccharomyces cerevisiae.

BACKGROUND: Mitochondrial function requires numerous genetic interactions between mitochondrial- and nuclear- encoded genes. While selection for optimal mitonuclear interactions should result in coevolution between both genomes, evidence for mitonuclear coadaptation is challenging to document. Genetic models where mitonuclear interactions can be explored are needed. RESULTS: We systematically exchanged mtDNAs between 15 Saccharomyces cerevisiae isolates from a variety of ecological niches to create 225 unique mitochondrial-nuclear genotypes. Analysis of phenotypic profiles confirmed that environmentally-sensitive interactions between mitochondrial and nuclear genotype contributed to growth differences. Exchanges of mtDNAs between strains of the same or different clades were just as likely to demonstrate mitonuclear epistasis although epistatic effect sizes increased with genetic distances. Strains with their original mtDNAs were more fit than strains with synthetic mitonuclear combinations when grown in media that resembled isolation habitats. CONCLUSIONS: This study shows that natural variation in mitonuclear interactions contributes to fitness landscapes. Multiple examples of coadapted mitochondrial-nuclear genotypes suggest that selection for mitonuclear interactions may play a role in helping yeasts adapt to novel environments and promote coevolution.

Mitochondrial Recombination Reveals Mito-Mito Epistasis in Yeast.

Genetic variation in mitochondrial DNA (mtDNA) provides adaptive potential although the underlying genetic architecture of fitness components within mtDNAs is not known. To dissect functional variation within mtDNAs, we first identified naturally occurring mtDNAs that conferred high or low fitness in Saccharomyces cerevisiae by comparing growth in strains containing identical nuclear genotypes but different mtDNAs. During respiratory growth under temperature and oxidative stress conditions, mitotype effects were largely independent of nuclear genotypes even in the presence of mito-nuclear interactions. Recombinant mtDNAs were generated to determine fitness components within high- and low-fitness mtDNAs. Based on phenotypic distributions of isogenic strains containing recombinant mtDNAs, we found that multiple loci contributed to mitotype fitness differences. These mitochondrial loci interacted in epistatic, nonadditive ways in certain environmental conditions. Mito-mito epistasis (i.e., nonadditive interactions between mitochondrial loci) influenced fitness in progeny from four different crosses, suggesting that mito-mito epistasis is a widespread phenomenon in yeast and other systems with recombining mtDNAs. Furthermore, we found that interruption of coadapted mito-mito interactions produced recombinant mtDNAs with lower fitness. Our results demonstrate that mito-mito epistasis results in functional variation through mitochondrial recombination in fungi, providing modes for adaptive evolution and the generation of mito-mito incompatibilities.

Mitochondrial Recombination and Introgression during Speciation by Hybridization.

Genome recombination is a major source of genotypic diversity and contributes to adaptation and speciation following interspecies hybridization. The contribution of recombination in these processes has been thought to be largely limited to the nuclear genome because organelles are mostly uniparentally inherited in animals and plants, which prevents recombination. Unicellular eukaryotes such as budding yeasts do, however, transmit mitochondria biparentally, suggesting that during hybridization, both parents could provide alleles that contribute to mitochondrial functions such as respiration and metabolism in hybrid populations or hybrid species. We examined the dynamics of mitochondrial genome transmission and evolution during speciation by hybridization in the natural budding yeast Saccharomyces paradoxus. Using population-scale mitochondrial genome sequencing in two endemic North American incipient species SpB and SpC and their hybrid species SpC*, we found that both parental species contributed to the hybrid mitochondrial genome through recombination. We support our findings by showing that mitochondrial recombination between parental types is frequent in experimental crosses that recreate the early step of this speciation event. In these artificial hybrids, we observed that mitochondrial genome recombination enhances phenotypic variation among diploid hybrids, suggesting that it could play a role in the phenotypic differentiation of hybrid species. Like the nuclear genome, the mitochondrial genome can, therefore, also play a role in hybrid speciation.

Population structure of mitochondrial genomes in Saccharomyces cerevisiae.

BACKGROUND: Rigorous study of mitochondrial functions and cell biology in the budding yeast, Saccharomyces cerevisiae has advanced our understanding of mitochondrial genetics. This yeast is now a powerful model for population genetics, owing to large genetic diversity and highly structured populations among wild isolates. Comparative mitochondrial genomic analyses between yeast species have revealed broad evolutionary changes in genome organization and architecture. A fine-scale view of recent evolutionary changes within S. cerevisiae has not been possible due to low numbers of complete mitochondrial sequences. RESULTS: To address challenges of sequencing AT-rich and repetitive mitochondrial DNAs (mtDNAs), we sequenced two divergent S. cerevisiae mtDNAs using a single-molecule sequencing platform (PacBio RS). Using de novo assemblies, we generated highly accurate complete mtDNA sequences. These mtDNA sequences were compared with 98 additional mtDNA sequences gathered from various published collections. Phylogenies based on mitochondrial coding sequences and intron profiles revealed that intraspecific diversity in mitochondrial genomes generally recapitulated the population structure of nuclear genomes. Analysis of intergenic sequence indicated a recent expansion of mobile elements in certain populations. Additionally, our analyses revealed that certain populations lacked introns previously believed conserved throughout the species, as well as the presence of introns never before reported in S. cerevisiae. CONCLUSIONS: Our results revealed that the extensive variation in S. cerevisiae mtDNAs is often population specific, thus offering a window into the recent evolutionary processes shaping these genomes. In addition, we offer an effective strategy for sequencing these challenging AT-rich mitochondrial genomes for small scale projects.

Mitochondrial-nuclear epistasis contributes to phenotypic variation and coadaptation in natural isolates of Saccharomyces cerevisiae.

Mitochondria are essential multifunctional organelles whose metabolic functions, biogenesis, and maintenance are controlled through genetic interactions between mitochondrial and nuclear genomes. In natural populations, mitochondrial efficiencies may be impacted by epistatic interactions between naturally segregating genome variants. The extent that mitochondrial-nuclear epistasis contributes to the phenotypic variation present in nature is unknown. We have systematically replaced mitochondrial DNAs in a collection of divergent Saccharomyces cerevisiae yeast isolates and quantified the effects on growth rates in a variety of environments. We found that mitochondrial-nuclear interactions significantly affected growth rates and explained a substantial proportion of the phenotypic variances under some environmental conditions. Naturally occurring mitochondrial-nuclear genome combinations were more likely to provide growth advantages, but genetic distance could not predict the effects of epistasis. Interruption of naturally occurring mitochondrial-nuclear genome combinations increased endogenous reactive oxygen species in several strains to levels that were not always proportional to growth rate differences. Our results demonstrate that interactions between mitochondrial and nuclear genomes generate phenotypic diversity in natural populations of yeasts and that coadaptation of intergenomic interactions likely occurs quickly within the specific niches that yeast occupy. This study reveals the importance of considering allelic interactions between mitochondrial and nuclear genomes when investigating evolutionary relationships and mapping the genetic basis underlying complex traits.

A conserved Na(+) binding site of the sodium-coupled neutral amino acid transporter 2 (SNAT2).

The SLC38 family of solute transporters mediates the coupled transport of amino acids and Na(+) into or out of cells. The structural basis for this coupled transport process is not known. Here, a profile-based sequence analysis approach was used, predicting a distant relationship with the SLC5/6 transporter families. Homology models using the LeuT(Aa) and Mhp1 transporters of known structure as templates were established, predicting the location of a conserved Na(+) binding site in the center of membrane helices 1 and 8. This homology model was tested experimentally in the SLC38 member SNAT2 by analyzing the effect of a mutation to Thr-384, which is predicted to be part of this Na(+) binding site. The results show that the T384A mutation not only inhibits the anion leak current, which requires Na(+) binding to SNAT2, but also dramatically lowers the Na(+) affinity of the transporter. This result is consistent with a previous analysis of the N82A mutant transporter, which has a similar effect on anion leak current and Na(+) binding and which is also expected to form part of the Na(+) binding site. In contrast, random mutations to other sites in the transporter had little or no effect on Na(+) affinity. Our results are consistent with a cation binding site formed by transmembrane helices 1 and 8 that is conserved among the SLC38 transporters as well as among many other bacterial and plant transporter families of unknown structure, which are homologous to SLC38.

Translocation and assembly of mitochondrially coded Saccharomyces cerevisiae cytochrome c oxidase subunit Cox2 by Oxa1 and Yme1 in the absence of Cox18.

Members of the Oxa1/YidC/Alb3 family of protein translocases are essential for assembly of energy-transducing membrane complexes. In Saccharomyces cerevisiae, Oxa1 and its paralog, Cox18, are required for assembly of Cox2, a mitochondrially encoded subunit of cytochrome c oxidase. Oxa1 is known to be required for cotranslational export of the Cox2 N-terminal domain across the inner mitochondrial membrane, while Cox18 is known to be required for post-translational export of the Cox2 C-tail domain. We find that overexpression of Oxa1 does not compensate for the absence of Cox18 at the level of respiratory growth. However, it does promote some translocation of the Cox2 C-tail domain across the inner membrane and causes increased accumulation of Cox2, which remains unassembled. This result suggests that Cox18 not only translocates the C-tail, but also must deliver it in a distinct state competent for cytochrome oxidase assembly. We identified respiring mutants from a cox18Delta strain overexpressing OXA1, whose respiratory growth requires overexpression of OXA1. The recessive nuclear mutations allow some assembly of Cox2 into cytochrome c oxidase. After failing to identify these mutations by methods based on transformation, we successfully located them to MGR1 and MGR3 by comparative hybridization to whole-genome tiling arrays and microarray-assisted bulk segregant analysis followed by linkage mapping. While Mgr1 and Mgr3 are known to associate with the Yme1 mitochondrial inner membrane i-AAA protease and to participate in membrane protein degradation, their absence does not appear to stabilize Cox2 under these conditions. Instead, Yme1 probably chaperones the folding and/or assembly of Oxa1-exported Cox2 in the absence of Mrg1 or Mgr3, since respiratory growth and cytochrome c oxidase assembly in a cox18 mgr3 double-mutant strain overexpressing OXA1 is YME1 dependent.

Roles of Oxa1-related inner-membrane translocases in assembly of respiratory chain complexes.

Members of the family of the polytopic inner membrane proteins are related to Saccharomyces cerevisiae Oxa1 function in the assembly of energy transducing complexes of mitochondria and chloroplasts. Here we focus on the two mitochondrial members of this family, Oxa1 and Cox18, reviewing studies on their biogenesis as well as their functions, reflected in the phenotypic consequences of their absence in various organisms. In yeast, cytochrome c oxidase subunit II (Cox2) is a key substrate of these proteins. Oxa1 is required for co-translational translocation and insertion of Cox2, while Cox18 is necessary for the export of its C-terminal domain. Genetic and biochemical strategies have been used to investigate the functions of distinct domains of Oxa1 and to identify its partners in protein insertion/translocation. Recent work on the related bacterial protein YidC strongly indicates that it is capable of functioning alone as a translocase for hydrophilic domains and an insertase for TM domains. Thus, the Oxa1 and Cox18 probably catalyze these reactions directly in a co- and/or posttranslational way. In various species, Oxa1 appears to assist in the assembly of different substrate proteins, although it is still unclear how Oxa1 recognizes its substrates, and whether additional factors participate in this beyond its direct interaction with mitochondrial ribosomes, demonstrated in S. cerevisiae. Oxa1 is capable of assisting posttranslational insertion and translocation in isolated mitochondria, and Cox18 may posttranslationally translocate its only known substrate, the Cox2 C-terminal domain, in vivo. Detailed understanding of the mechanisms of action of these two proteins must await the resolution of their structure in the membrane and the development of a true in vitro mitochondrial translation system.

Translocation of mitochondrially synthesized Cox2 domains from the matrix to the intermembrane space.

The N-terminal and C-terminal domains of mitochondrially synthesized cytochrome c oxidase subunit II, Cox2, are translocated through the inner membrane to the intermembrane space (IMS). We investigated the distinct mechanisms of N-tail and C-tail export by analysis of epitope-tagged Cox2 variants encoded in Saccharomyces cerevisiae mitochondrial DNA. Both the N and C termini of a truncated protein lacking the Cox2 C-terminal domain were translocated to the IMS via a pathway dependent upon the conserved translocase Oxa1. The topology of this Cox2 variant, accumulated at steady state, was largely but not completely unaffected in mutants lacking proteins required for export of the C-tail domain, Cox18 and Mss2. C-tail export was blocked by truncation of the last 40 residues from the C-tail domain, indicating that sequence and/or structural features of this domain are required for its translocation. Mss2, a peripheral protein bound to the inner surface of the inner membrane, coimmunoprecipitated with full-length newly synthesized Cox2, whose leader peptide had already been cleaved in the IMS. Our data suggest that the C-tail domain is recognized posttranslationally by a specialized translocation apparatus after the N-tail has been translocated by Oxa1.

Mitochondrial-type assembly of FeS centers in the hydrogenosomes of the amitochondriate eukaryote Trichomonas vaginalis.

Mitochondria are the site of assembly of FeS centers of mitochondrial and cytosolic FeS proteins. Various microaerophilic or anaerobic unicellular eukaryotes lack typical mitochondria ("amitochondriate" protists). In some of these organisms, a metabolically different organelle, the hydrogenosome, is found, which is thought to derive from the same proteobacterial ancestor as mitochondria. Here, we show that hydrogenosomes of Trichomonas vaginalis, a human genitourinary parasite, contain a key enzyme of FeS center biosynthesis, cysteine desulfurase (TviscS-2), which is phylogenetically related to its mitochondrial homologs. Hydrogenosomes catalyze the enzymatic assembly and insertion of FeS centers into apoproteins, as shown by the reconstruction of the apoform of [2Fe-2S]ferredoxin and the incorporation of 35S from labeled cysteine. Our results indicate that the biosynthesis of FeS proteins is performed by a homologous system in mitochondriate and amitochondriate eukaryotes and that this process is inherited from the proteobacterial ancestor of mitochondria.