A genome wide study in fission yeast reveals nine PPR proteins that regulate mitochondrial gene expression.

Pentatricopeptide repeat (PPR) proteins are particularly numerous in plant mitochondria and chloroplasts, where they are involved in different steps of RNA metabolism, probably due to the repeated 35 amino acid PPR motifs that are thought to mediate interactions with RNA. In non-photosynthetic eukaryotes only a handful of PPR proteins exist, for example the human LRPPRC, which is involved in a mitochondrial disease. We have conducted a systematic study of the PPR proteins in the fission yeast Schizosaccharomyces pombe and identified, in addition to the mitochondrial RNA polymerase, eight proteins all of which localized to the mitochondria, and showed some association with the membrane. The absence of all but one of these PPR proteins leads to a respiratory deficiency and modified patterns of steady state mt-mRNAs or newly synthesized mitochondrial proteins. Some cause a general defect, whereas others affect specific mitochondrial RNAs, either coding or non-coding: cox1, cox2, cox3, 15S rRNA, atp9 or atp6, sometimes leading to secondary defects. Interestingly, the two possible homologs of LRPPRC, ppr4 and ppr5, play opposite roles in the expression of the cox1 mt-mRNA, ppr4 being the first mRNA-specific translational activator identified in S. pombe, whereas ppr5 appears to be a general negative regulator of mitochondrial translation.

The human mitochondrial ribosome recycling factor is essential for cell viability.

The molecular mechanism of human mitochondrial translation has yet to be fully described. We are particularly interested in understanding the process of translational termination and ribosome recycling in the mitochondrion. Several candidates have been implicated, for which subcellular localization and characterization have not been reported. Here, we show that the putative mitochondrial recycling factor, mtRRF, is indeed a mitochondrial protein. Expression of human mtRRF in fission yeast devoid of endogenous mitochondrial recycling factor suppresses the respiratory phenotype. Further, human mtRRF is able to associate with Escherichia coli ribosomes in vitro and can associate with mitoribosomes in vivo. Depletion of mtRRF in human cell lines is lethal, initially causing profound mitochondrial dysmorphism, aggregation of mitoribosomes, elevated mitochondrial superoxide production and eventual loss of OXPHOS complexes. Finally, mtRRF was shown to co-immunoprecipitate a large number of mitoribosomal proteins attached to other mitochondrial proteins, including putative members of the mitochondrial nucleoid.

mtRF1a is a human mitochondrial translation release factor decoding the major termination codons UAA and UAG.

Human mitochondria contain their own genome, encoding 13 polypeptides that are synthesized within the organelle. The molecular processes that govern and facilitate this mitochondrial translation remain unclear. Many key factors have yet to be characterized-for example, those required for translation termination. All other systems have two classes of release factors that either promote codon-specific hydrolysis of peptidyl-tRNA (class I) or lack specificity but stimulate the dissociation of class I factors from the ribosome (class II). One human mitochondrial protein has been previously identified in silico as a putative member of the class I release factors. Although we could not confirm the function of this factor, we report the identification of a different mitochondrial protein, mtRF1a, that is capable in vitro and in vivo of terminating translation at UAA/UAG codons. Further, mtRF1a depletion in HeLa cells led to compromised growth in galactose and increased production of reactive oxygen species.

Studying mitochondria in an attractive model: Schizosaccharomyces pombe.

The fission yeast Schizosaccharomyces pombe, widely used for studies of cell cycle control and differentiation, provides an alternative and complementary model to the budding yeast Saccharomyces cerevisiae for studies of nucleo-mitochondrial interactions. There are striking similarities between S. pombe and mammalian cells, in both their respiratory physiology and their mitochondrial genome structure. This technical review briefly lists the general and specific properties that are helpful to know when starting to use fission yeast as a model system for mitochondrial studies. In addition, advice is given for cell growth and genetic techniques, tips for disruption of genes involved in respiration are presented. and a basic differential centrifugation protocol is provided for the isolation of purified mitochondria that are suitable for diverse applications such as subfractionation and in vitro import.

The COX18 gene, involved in mitochondrial biogenesis, is functionally conserved and tightly regulated in humans and fission yeast.

The biogenesis of cytochrome c oxidase requires coordination between the nucleus and mitochondria because both these compartments provide the structural subunits of this enzyme. In addition, synthesis, membrane insertion and assembly of the mitochondrially encoded subunits are controlled in a concerted way by numerous nuclear-encoded factors, including Oxa1 and Cox18, which play successive roles in Cox2 assembly in Saccharomyces cerevisiae. These two factors share a weak structural similarity and define two sub-branches of the Oxa1/YidC/Alb3 gene family, whose members facilitate the membrane insertion of various hydrophobic proteins into diverse biological membranes. In this study, we have analyzed a second human and a third fission yeast member of the family. We show, by deletion in the fission yeast genome, as well as expression and functional complementation experiments in both yeasts, that these new genes belong to the COX18 rather than to the OXA1 sub-branch. So far, the fission yeast gene cox18Sp+ is the smallest functional member of this gene family. COX18Hs gives rise to various mRNAs with different coding capacities, and we show that cox18Sp+ and COX18Hs are expressed at a low level and appear to be stringently regulated. This transcriptional control contrasts with the constitutive abundance of the OXA1 mRNAs and might reflect major functional differences between these nevertheless structurally related genes.

High-efficiency biolistic transformation of Chlamydomonas mitochondria can be used to insert mutations in complex I genes.

Mitochondrial transformation of Chlamydomonas reinhardtii has been optimized by using a particle-gun device and cloned mitochondrial DNA or PCR fragments. A respiratory-deficient strain lacking a 1.2-kb mitochondrial DNA region including the left telomere and part of the cob gene could be rescued as well as a double-frameshift mutant in the mitochondrial cox1 and nd1 genes. High transformation efficiency has been achieved (100-250 transformants per microgram of DNA), the best results being obtained with linearized plasmid DNA. Molecular analysis of the transformants suggests that the right telomere sequence can be copied to reconstruct the left telomere by recombination. In addition, both nondeleterious and deleterious mutations could be introduced. Myxothiazol-resistant transformants have been created by introducing a nucleotide substitution into the cob gene. Similarly, an in-frame deletion of 23 codons has been created in the nd4 mitochondrial gene of both the deleted and frameshift recipient strains. These 23 codons are believed to encode the first transmembrane segment of the ND4 protein. This Deltand4 mutation causes a misassembly of complex I, with the accumulation of a subcomplex that is 250-kDa smaller than the wild-type complex I. The availability of efficient mitochondrial transformation in Chlamydomonas provides an invaluable tool for the study of mitochondrial biogenesis and, more specifically, for site-directed mutagenesis of mitochondrially encoded subunits of complex I, of special interest because the yeast Saccharomyces cerevisiae, whose mitochondrial genome can be manipulated virtually at will, is lacking complex I.