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 mutation associated with CMT2A neuropathy causes defects in Fzo1 GTP hydrolysis, ubiquitylation, and protein turnover.

Charcot-Marie-Tooth disease type 2A (CMT2A) is caused by mutations in the gene MFN2 and is one of the most common inherited peripheral neuropathies. Mfn2 is one of two mammalian mitofusin GTPases that promote mitochondrial fusion and maintain organelle integrity. It is not known how mitofusin mutations cause axonal degeneration and CMT2A disease. We used the conserved yeast mitofusin FZO1 to study the molecular consequences of CMT2A mutations on Fzo1 function in vivo and in vitro. One mutation (analogous to the CMT2A I213T substitution in the GTPase domain of Mfn2) not only abolishes GTP hydrolysis and mitochondrial membrane fusion but also reduces Mdm30-mediated ubiquitylation and degradation of the mutant protein. Importantly, complexes of wild type and the mutant Fzo1 protein are GTPase active and restore ubiquitylation and degradation of the latter. These studies identify diverse and unexpected effects of CMT2A mutations, including a possible role for mitofusin ubiquitylation and degradation in CMT2A pathogenesis, and provide evidence for a novel link between Fzo1 GTP hydrolysis, ubiquitylation, and mitochondrial fusion.

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.

Yeast mitochondrial biogenesis: a role for the PUF RNA-binding protein Puf3p in mRNA localization.

The asymmetric localization of mRNA plays an important role in coordinating posttranscriptional events in eukaryotic cells. We investigated the peripheral mitochondrial localization of nuclear-encoded mRNAs (MLR) in various conditions in which the mRNA binding protein context and the translation efficiency were altered. We identified Puf3p, a Pumilio family RNA-binding protein, as the first trans-acting factor controlling the MLR phenomenon. This allowed the characterization of two classes of genes whose mRNAs are translated to the vicinity of mitochondria. Class I mRNAs (256 genes) have a Puf3p binding motif in their 3'UTR region and many of them have their MLR properties deeply affected by PUF3 deletion. Conversely, mutations in the Puf3p binding motif alter the mitochondrial localization of BCS1 mRNA. Class II mRNAs (224 genes) have no Puf3p binding site and their asymmetric localization is not affected by the absence of PUF3. In agreement with a co-translational import process, we observed that the presence of puromycin loosens the interactions between most of the MLR-mRNAs and mitochondria. Unexpectedly, cycloheximide, supposed to solidify translational complexes, turned out to destabilize a class of mRNA-mitochondria interactions. Classes I and II mRNAs, which are therefore transported to the mitochondria through different pathways, correlated with different functional modules. Indeed, Class I genes code principally for the assembly factors of respiratory chain complexes and the mitochondrial translation machinery (ribosomes and translation regulators). Class II genes encode proteins of the respiratory chain or proteins involved in metabolic pathways. Thus, MLR, which is intimately linked to translation control, and the activity of mRNA-binding proteins like Puf3p, may provide the conditions for a fine spatiotemporal control of mitochondrial protein import and mitochondrial protein complex assembly. This work therefore provides new openings for the global study of mitochondria biogenesis.

Redundancy in the function of mitochondrial phosphate transport in Saccharomyces cerevisiae and Arabidopsis thaliana.

Most cellular ATP is produced within the mitochondria from ADP and Pi which are delivered across the inner-membrane by specific nuclearly encoded polytopic carriers. In Saccharomyces cerevisiae, some of these carriers and in particular the ADP/ATP carrier, are represented by several related isoforms that are distinct in their pattern of expression. Until now, only one mitochondrial Pi carrier (mPic) form, encoded by the MIR1 gene in S. cerevisiae, has been described. Here we show that the gene product encoded by the YER053C ORF also participates in the delivery of phosphate to the mitochondria. We have called this gene PIC2 for Pi carrier isoform 2. Overexpression of PIC2 compensates for the mitochondrial defect of the double mutant Deltamir1 Deltapic2 and restores phosphate transport activity in mitochondria swelling experiments. The existence of two isoforms of mPic does not seem to be restricted to S. cerevisiae as two Arabidopsis thaliana cDNAs encoding two different mPic-like proteins are also able to complement the double mutant Deltamir1 Deltapic2. Finally, we demonstrate that Pic2p is a mitochondrial protein and that its steady state level increases at high temperature. We propose that Pic2p is a minor form of mPic which plays a role under specific stress conditions.

The Rieske FeS protein encoded and synthesized within mitochondria complements a deficiency in the nuclear gene.

The Rieske FeS protein, an essential catalytic subunit of the mitochondrial cytochrome bc1 complex, is encoded in yeast by the nuclear gene RIP1, whose deletion leads to a respiratory-deficient phenotype. By using biolistic transformation, we have relocated the nuclear RIP1 gene into mitochondria. To allow its expression within the organelle and to direct its integration downstream of the cox1 gene, we have fused the 3' end of the Saccharomyces douglasii cox1 gene upstream of the mitochondrial copy of RIP1 (RIP1m) flanked by the Saccharomyces cerevisiae cox1 promoter and terminator regions. We show that RIP1m integrated between the cox1 and atp8 genes is mitotically stable and expressed, and it complements a deletion of the nuclear gene. Immunodetection experiments demonstrate that the mitochondrial genome containing RIP1m is able to produce the Rip1 protein in lower steady-state amounts than the wild type but still sufficient to maintain a functional cytochrome bc1 complex and respiratory competence to a RIP1-deleted strain. Thus, this recombined mitochondrial genome is a fully functional mitochondrial chromosome with an extended gene content. This successful mitochondrial expression of a nuclear gene essential for respiration can be viewed at the evolutionary level as an artificial reversal of evolutionary events.

A pathogenic cytochrome b mutation reveals new interactions between subunits of the mitochondrial bc1 complex.

Energy transduction in mitochondria involves five oligomeric complexes embedded within the inner membrane. They are composed of catalytic and noncatalytic subunits, the role of these latter proteins often being difficult to assign. One of these complexes, the bc1 complex, is composed of three catalytic subunits including cytochrome b and seven or eight noncatalytic subunits. Recently, several mutations in the human cytochrome b gene have been linked to various diseases. We have studied in detail the effects of a cardiomyopathy generating mutation G252D in yeast. This mutation disturbs the biogenesis of the bc1 complex at 36 degrees C and decreases the steady-state level of the noncatalytic subunit Qcr9p. In addition, the G252D mutation and the deletion of QCR9 show synergetic defects that can be partially bypassed by suppressor mutations at position 252 and by a new cytochrome b mutation, P174T. Altogether, our results suggest that the supernumerary subunit Qcr9p enhances or stabilizes the interactions between the catalytic subunits, this role being essential at high temperature.