Translational activators and mitoribosomal isoforms cooperate to mediate mRNA-specific translation in Schizosaccharomyces pombe mitochondria.

Mitochondrial mRNAs encode key subunits of the oxidative phosphorylation complexes that produce energy for the cell. In Saccharomyces cerevisiae, mitochondrial translation is under the control of translational activators, specific to each mRNA. In Schizosaccharomyces pombe, which more closely resembles the human system by its mitochondrial DNA structure and physiology, most translational activators appear to be either lacking, or recruited for post-translational functions. By combining bioinformatics, genetic and biochemical approaches we identified two interacting factors, Cbp7 and Cbp8, controlling Cytb production in S. pombe. We show that their absence affects cytb mRNA stability and impairs the detection of the Cytb protein. We further identified two classes of Cbp7/Cbp8 partners and showed that they modulated Cytb or Cox1 synthesis. First, two isoforms of bS1m, a protein of the small mitoribosomal subunit, that appear mutually exclusive and confer translational specificity. Second, a complex of four proteins dedicated to Cox1 synthesis, which includes an RNA helicase that interacts with the mitochondrial ribosome. Our results suggest that S. pombe contains, in addition to complexes of translational activators, a heterogeneous population of mitochondrial ribosomes that could specifically modulate translation depending on the mRNA translated, in order to optimally balance the production of different respiratory complex subunits.

Artemisinin and its derivatives target mitochondrial c-type cytochromes in yeast and human cells.

Artemisinin and its derivatives kill malaria parasites and inhibit the proliferation of cancer cells. In both processes, heme was shown to play a key role in artemisinin bioactivation. We found that artemisinin and clinical artemisinin derivatives are able to compensate for a mutation in the yeast Bcs1 protein, a key chaperon involved in biogenesis of the mitochondrial respiratory complex III. The equivalent Bcs1 variant causes an encephalopathy in human by affecting complex III assembly. We show that artemisinin derivatives decrease the content of mitochondrial cytochromes and disturb the maturation of the complex III cytochrome c1. This last effect is likely responsible for the compensation by decreasing the detrimental over-accumulation of the inactive pre-complex III observed in the bcs1 mutant. We further show that a fluorescent dihydroartemisinin probe rapidly accumulates in the mitochondrial network and targets cytochromes c and c1 in yeast, human cells and isolated mitochondria. In vitro this probe interacts with purified cytochrome c only under reducing conditions and we detect cytochrome c-dihydroartemisinin covalent adducts by mass spectrometry analyses. We propose that reduced mitochondrial c-type cytochromes act as both targets and mediators of artemisinin bioactivation in yeast and human cells.

Complete Sequence of the Intronless Mitochondrial Genome of the Saccharomyces cerevisiae Strain CW252.

The mitochondrial genomes of Saccharomyces cerevisiae strains contain up to 13 introns. An intronless recombinant genome introduced into the nuclear background of S. cerevisiae strain W303 gave the S. cerevisiae CW252 strain, which is used to model mitochondrial respiratory pathologies. The complete sequence of this mitochondrial genome was obtained using a hybrid assembling methodology.

Development of an in silico method for the identification of subcomplexes involved in the biogenesis of multiprotein complexes in Saccharomyces cerevisiae.

BACKGROUND: Large sets of protein-protein interaction data coming either from biological experiments or predictive methods are available and can be combined to construct networks from which information about various cell processes can be extracted. We have developed an in silico approach based on these information to model the biogenesis of multiprotein complexes in the yeast Saccharomyces cerevisiae. RESULTS: Firstly, we have built three protein interaction networks by collecting the protein-protein interactions, which involved the subunits of three complexes, from different databases. The protein-protein interactions come from different kinds of biological experiments or are predicted. We have chosen the elongator and the mediator head complexes that are soluble and exhibit an architecture with subcomplexes that could be functional modules, and the mitochondrial bc 1 complex, which is an integral membrane complex and for which a late assembly subcomplex has been described. Secondly, by applying a clustering strategy to these networks, we were able to identify subcomplexes involved in the biogenesis of the complexes as well as the proteins interacting with each subcomplex. Thirdly, in order to validate our in silico results for the cytochrome bc1 complex we have analysed the physical interactions existing between three subunits by performing immunoprecipitation experiments in several genetic context. CONCLUSIONS: For the two soluble complexes (the elongator and mediator head), our model shows a strong clustering of subunits that belong to a known subcomplex or module. For the membrane bc 1 complex, our approach has suggested new interactions between subunits in the early steps of the assembly pathway that were experimentally confirmed. Scripts can be downloaded from the site: http://bim.igmors.u-psud.fr/isips .

Ribosome recycling defects modify the balance between the synthesis and assembly of specific subunits of the oxidative phosphorylation complexes in yeast mitochondria.

Mitochondria have their own translation machinery that produces key subunits of the OXPHOS complexes. This machinery relies on the coordinated action of nuclear-encoded factors of bacterial origin that are well conserved between humans and yeast. In humans, mutations in these factors can cause diseases; in yeast, mutations abolishing mitochondrial translation destabilize the mitochondrial DNA. We show that when the mitochondrial genome contains no introns, the loss of the yeast factors Mif3 and Rrf1 involved in ribosome recycling neither blocks translation nor destabilizes mitochondrial DNA. Rather, the absence of these factors increases the synthesis of the mitochondrially-encoded subunits Cox1, Cytb and Atp9, while strongly impairing the assembly of OXPHOS complexes IV and V. We further show that in the absence of Rrf1, the COX1 specific translation activator Mss51 accumulates in low molecular weight forms, thought to be the source of the translationally-active form, explaining the increased synthesis of Cox1. We propose that Rrf1 takes part in the coordination between translation and OXPHOS assembly in yeast mitochondria. These interactions between general and specific translation factors might reveal an evolutionary adaptation of the bacterial translation machinery to the set of integral membrane proteins that are translated within mitochondria.

Yeast as a system for modeling mitochondrial disease mechanisms and discovering therapies.

Mitochondrial diseases are severe and largely untreatable. Owing to the many essential processes carried out by mitochondria and the complex cellular systems that support these processes, these diseases are diverse, pleiotropic, and challenging to study. Much of our current understanding of mitochondrial function and dysfunction comes from studies in the baker's yeast Saccharomyces cerevisiae. Because of its good fermenting capacity, S. cerevisiae can survive mutations that inactivate oxidative phosphorylation, has the ability to tolerate the complete loss of mitochondrial DNA (a property referred to as 'petite-positivity'), and is amenable to mitochondrial and nuclear genome manipulation. These attributes make it an excellent model system for studying and resolving the molecular basis of numerous mitochondrial diseases. Here, we review the invaluable insights this model organism has yielded about diseases caused by mitochondrial dysfunction, which ranges from primary defects in oxidative phosphorylation to metabolic disorders, as well as dysfunctions in maintaining the genome or in the dynamics of mitochondria. Owing to the high level of functional conservation between yeast and human mitochondrial genes, several yeast species have been instrumental in revealing the molecular mechanisms of pathogenic human mitochondrial gene mutations. Importantly, such insights have pointed to potential therapeutic targets, as have genetic and chemical screens using yeast.

Does the study of genetic interactions help predict the function of mitochondrial proteins in Saccharomyces cerevisiae?

Mitochondria are complex organelles of eukaryotic cells that contain their own genome, encoding key subunits of the respiratory complexes. The successive steps of mitochondrial gene expression are intimately linked, and are under the control of a large number of nuclear genes encoding factors that are imported into mitochondria. Investigating the relationships between these genes and their interaction networks, and whether they reveal direct or indirect partners, can shed light on their role in mitochondrial biogenesis, as well as identify new actors in this process. These studies, mainly developed in yeasts, are significant because mammalian equivalents of such yeast genes are candidate genes in mitochondrial pathologies. In practice, studies of physical, chemical and genetic interactions can be undertaken. The search for genetic interactions, either aggravating or alleviating the phenotype of the starting mutants, has proved to be particularly powerful in yeast since even subtle changes in respiratory phenotypes can be screened in a very efficient way. In addition, several high throughput genetic approaches have recently been developed. In this review we analyze the genetic network of three genes involved in different steps of mitochondrial gene expression, from the transcription and translation of mitochondrial RNAs to the insertion of newly synthesized proteins into the inner mitochondrial membrane, and we examine their relevance to our understanding of mitochondrial biogenesis. We find that these genetic interactions are seldom redundant with physical interactions, and thus bring a considerable amount of original and significant information as well as open new areas of research.

A novel mechanism involved in the coupling of mitochondrial biogenesis to oxidative phosphorylation.

Mitochondria are essential organelles that are central to a multitude of cellular processes, including oxidative phosphorylation (OXPHOS), which produces most of the ATP in animal cells. Thus it is important to understand not only the mechanisms and biogenesis of this energy production machinery but also how it is regulated in both physiological and pathological contexts. A recent study by Ostojic et al. [Cell Metabolism (2013) 18, 567-577] has uncovered a regulatory loop by which the biogenesis of a major enzyme of the OXPHOS pathway, the respiratory complex III, is coupled to the energy producing activity of the mitochondria.

The energetic state of mitochondria modulates complex III biogenesis through the ATP-dependent activity of Bcs1.

Our understanding of the mechanisms involved in mitochondrial biogenesis has continuously expanded during the last decades, yet little is known about how they are modulated to optimize the functioning of mitochondria. Here, we show that mutations in the ATP binding domain of Bcs1, a chaperone involved in the assembly of complex III, can be rescued by mutations that decrease the ATP hydrolytic activity of the ATP synthase. Our results reveal a Bcs1-mediated control loop in which the biogenesis of complex III is modulated by the energy-transducing activity of mitochondria. Although ATP is well known as a regulator of a number of cellular activities, we show here that ATP can be also used to modulate the biogenesis of an enzyme by controlling a specific chaperone involved in its assembly. Our study further highlights the intramitochondrial adenine nucleotide pool as a potential target for the treatment of Bcs1-based disorders.

An in silico approach combined with in vivo experiments enables the identification of a new protein whose overexpression can compensate for specific respiratory defects in Saccharomyces cerevisiae.

BACKGROUND: The mitochondrial inner membrane contains five large complexes that are essential for oxidative phosphorylation. Although the structure and the catalytic mechanisms of the respiratory complexes have been progressively established, their biogenesis is far from being fully understood. Very few complex III assembly factors have been identified so far. It is probable that more factors are needed for the assembly of a functional complex, but that the genetic approaches used to date have not been able to identify them. We have developed a systems biology approach to identify new factors controlling complex III biogenesis. RESULTS: We collected all the physical protein-protein interactions (PPI) involving the core subunits, the supernumerary subunits and the assembly factors of complex III and used Cytoscape 2.6.3 and its plugins to construct a network. It was then divided into overlapping and highly interconnected sub-graphs with clusterONE. One sub-graph contained the core and the supernumerary subunits of complex III, it also contained some subunits of complex IV and proteins participating in the assembly of complex IV. This sub-graph was then split with another algorithm into two sub-graphs. The subtraction of these two sub-graphs from the previous sub-graph allowed us to identify a protein of unknown function Usb1p/Ylr132p that interacts with the complex III subunits Qcr2p and Cor1p. We then used genetic and cell biology approaches to investigate the function of Usb1p. Preliminary results indicated that Usb1p is an essential protein with a dual localization in the nucleus and in the mitochondria, and that the over-expression of this protein can compensate for defects in the biogenesis of the respiratory complexes. CONCLUSIONS: Our systems biology approach has highlighted the multiple associations between subunits and assembly factors of complexes III and IV during their biogenesis. In addition, this approach has allowed the identification of a new factor, Usb1p, involved in the biogenesis of respiratory complexes, which could not have been found using classical genetic screens looking for respiratory deficient mutants. Thus, this systems biology approach appears to be a fruitful new way to study the biogenesis of mitochondrial multi-subunit complexes.

Deleterious effect of the Qo inhibitor compound resistance-conferring mutation G143A in the intron-containing cytochrome b gene and mechanisms for bypassing it.

The mutation G143A in the inhibitor binding site of cytochrome b confers a high level of resistance to fungicides targeting the bc(1) complex. The mutation, reported in many plant-pathogenic fungi, has not evolved in fungi that harbor an intron immediately after the codon for G143 in the cytochrome b gene, intron bi2. Using Saccharomyces cerevisiae as a model organism, we show here that a codon change from GGT to GCT, which replaces glycine 143 with alanine, hinders the splicing of bi2 by altering the exon/intron structure needed for efficient intron excision. This lowers the levels of cytochrome b and respiratory growth. We then investigated possible bypass mechanisms that would restore the respiratory fitness of a resistant mutant. Secondary mutations in the mitochondrial genome were found, including a point mutation in bi2 restoring the correct exon/intron structure and the deletion of intron bi2. We also found that overexpression of nuclear genes MRS2 and MRS3, encoding mitochondrial metal ion carriers, partially restores the respiratory growth of the G143A mutant. Interestingly, the MRS3 gene from the plant-pathogenic fungus Botrytis cinerea, overexpressed in an S. cerevisiae G143A mutant, had a similar compensatory effect. These bypass mechanisms identified in yeast could potentially arise in pathogenic fungi.

A transcriptome screen in yeast identifies a novel assembly factor for the mitochondrial complex III.

Starting from a transcriptome based study of the spatio-temporal expression of yeast genes encoding mitochondrial proteins of unknown function, we have identified the gene BCA1 (YLR077W). A FISH analysis showed that the BCA1 mRNA co-localized with the mitochondrial network. Cellular fractionation revealed that Bca1 is bound to the mitochondrial inner-membrane and protrudes into the inter-membrane space. We show that Bca1 controls an early step in complex III assembly and that the supra-molecular organization of Bca1 is dependent upon the assembly level of complex III. Thus, Bca1 is a novel assembly factor for the respiratory complex III.

A mutational analysis reveals new functional interactions between domains of the Oxa1 protein in Saccharomyces cerevisiae.

The Oxa1/YidC/Alb3 family plays a key role in the biogenesis of the respiratory and photosynthetic complexes in bacteria and organelles. In Saccharomyces cerevisiae, Oxa1 mediates the co-translational insertion of mitochondrially encoded subunits of the three respiratory complexes III, IV and V within the inner membrane and also controls a late step in complex V assembly. No crystal structure of YidC or Oxa1 is available and little is known about the respective role of each transmembrane segment (TM) and hydrophilic loop of this polytopic protein on the biogenesis of the three complexes. Here, we have generated a collection of random point mutations located in the hydrophobic and hydrophilic domains of the protein and characterized their effects on the assembly of the three respiratory complexes. Our results show mutant-dependent differential effects, particularly on complex V. In order to identify tertiary interactions within Oxa1, we have also isolated revertants carrying second-site compensatory mutations able to restore respiration. This analysis reveals the existence of functional interactions between TM2 and TM5, TM4 and TM5 as well as between TM4 and loop 2, highlighting the key position of TM4 and TM5 in the Oxa1 protein.

Spatio-temporal dynamics of yeast mitochondrial biogenesis: transcriptional and post-transcriptional mRNA oscillatory modules.

Examples of metabolic rhythms have recently emerged from studies of budding yeast. High density microarray analyses have produced a remarkably detailed picture of cycling gene expression that could be clustered according to metabolic functions. We developed a model-based approach for the decomposition of expression to analyze these data and to identify functional modules which, expressed sequentially and periodically, contribute to the complex and intricate mitochondrial architecture. This approach revealed that mitochondrial spatio-temporal modules are expressed during periodic spikes and specific cellular localizations, which cover the entire oscillatory period. For instance, assembly factors (32 genes) and translation regulators (47 genes) are expressed earlier than the components of the amino-acid synthesis pathways (31 genes). In addition, we could correlate the expression modules identified with particular post-transcriptional properties. Thus, mRNAs of modules expressed "early" are mostly translated in the vicinity of mitochondria under the control of the Puf3p mRNA-binding protein. This last spatio-temporal module concerns mostly mRNAs coding for basic elements of mitochondrial construction: assembly and regulatory factors. Prediction that unknown genes from this module code for important elements of mitochondrial biogenesis is supported by experimental evidence. More generally, these observations underscore the importance of post-transcriptional processes in mitochondrial biogenesis, highlighting close connections between nuclear transcription and cytoplasmic site-specific translation.

Functional analysis of yeast bcs1 mutants highlights the role of Bcs1p-specific amino acids in the AAA domain.

The mitochondrial protein Bcs1p is conserved from Saccharomyces cerevisiae to humans and its C-terminal region exhibits an AAA (ATPases associated with diverse cellular activities) domain. The absence of the yeast Bcs1p leads to an assembly defect of the iron-sulfur protein (ISP) subunit within the mitochondrial respiratory complex III, whereas human point mutations located all along the protein cause various pathologies. We have performed a structure-function analysis of the yeast Bcs1p by randomly generating a collection of respiratory-deficient point mutants. We showed that most mutations are in the C-terminal region of Bcs1p and have localized them on a theoretical three-dimensional model based on the structure of several AAA proteins. The mutations can be grouped into classes according to their respiratory competence and their location on the three-dimensional model. We have further characterized five mutants, each substituting an amino acid conserved in yeast and mammalian Bcs1 proteins but not in other AAA proteins. The effects on respiratory complex assembly and Bcs1p accumulation were analyzed. Intragenic and extragenic compensatory mutations able to restore complex III assembly to the mutants affecting the AAA domain were isolated. Our results bring new insights into the role of specific residues in critical regions that are also conserved in the human Bcs1p. We show that (1) residues located at the junction between the Bcs1p-specific and the AAA domains are important for the activity and stability of the protein and (2) the residue F342 is important for interactions with other partners or substrate proteins.

Multiple defects in the respiratory chain lead to the repression of genes encoding components of the respiratory chain and TCA cycle enzymes.

Respiratory complexes III, IV and V are formed by components of both nuclear and mitochondrial origin and are embedded in the inner mitochondrial membrane. Their assembly requires the auxiliary factor Oxa1, and the absence of this protein has severe consequences on these three major respiratory chain enzymes. We have studied, in the yeast Saccharomyces cerevisiae, the effect of the loss of Oxa1 function and of other respiratory defects on the expression of nuclear genes encoding components of the respiratory complexes and tricarboxylic acid cycle enzymes. We observed that the concomitant decrease in the level of two respiratory enzymes, complexes III and IV, led to their repression. These genes are known targets of the transcriptional activator complex Hap2/3/4/5 that plays a central role in the reprogramming of yeast metabolism when cells switch from a fermenting, glucose-repressed state to a respiring, derepressed state. We found that the Hap4 protein, the regulatory subunit of the transcriptional complex, was present at a lower level in the oxa1 mutants whereas no change in HAP4 transcript level was observed, suggesting a posttranscriptional modulation. In addition, an altered mitochondrial morphology was observed in mutants with decreased expression of Hap2/3/4/5 target genes. We suggest that the aberrant mitochondrial morphology, presumably caused by the severely decreased level of at least two respiratory enzymes, might be part of the signalling pathway linking the mitochondrial defect and Hap2/3/4/5.

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.

Respiratory mutations lead to different pleiotropic effects on OXPHOS complexes in yeast and in human cells.

Pleiotropic effects in the oxidative phosphorylation pathway (OXPHOS) were investigated in yeast respiratory mutants and in cells from patients with OXPHOS genetic alterations. The main differences between yeast and human cells were (1) the site of the primary defect that was associated with pleiotropic effects, yeast complex V and human complex IV, and (2) the nature of the complex targeted by the secondary effect, yeast complex IV and human complex I. The pleiotropic effects did not correlate with the organization of OXPHOS into supercomplexes and their functional consequences appeared to be a slowing down of the respiratory chain in order to avoid either an increase in the membrane potential or the accumulation of reduced intermediary components of the respiratory chain.

Preparation of respiratory chain complexes from Saccharomyces cerevisiae wild-type and mutant mitochondria : activity measurement and subunit composition analysis.

The mitochondrial oxidative phosphorylation involves five multimeric complexes imbedded in the inner membrane: complex I (Nicotinamide Adenine Dinucleotide (NADH) quinone oxidoreductase), II (succinate dehydrogenase), III (ubiquinol cytochrome c oxido reductase or bc1 complex), IV (cytochrome c oxidase), and V (ATP synthase). These respiratory complexes are conserved from the yeast Saccharomyces cerevisiae to human with the exception of complex I, which is replaced by three NADH dehydrogenases in S. cerevisiae. Here, we provide several protocols allowing an exhaustive characterization of each yeast complex: this chapter describes procedures from mitochondria preparation to measurement of the activity of each complex and analysis of their subunit composition and provides information on the interactions between different complexes.

UCP2 is a mitochondrial transporter with an unusual very short half-life.

This study focused on the stability of UCP2 (uncoupling protein 2), a mitochondrial carrier located in the inner membrane of mitochondrion. UCP2 is very unstable, with a half-life close to 30min, compared to 30h for its homologue UCP1, a difference that may highlight different physiological functions. Heat production by UCP1 in brown adipocytes is generally a long and adaptive phenomenon, whereas control of mitochondrial ROS by UCP2 needs more subtle regulation. We show that a mutation in UCP2 shown to modify its activity, actually decreases its stability.