Copper Import into the Mitochondrial Matrix in Saccharomyces cerevisiae Is Mediated by Pic2, a Mitochondrial Carrier Family Protein

Saccharomyces cerevisiae must import copper into the mitochondrial matrix for eventual assembly of cytochrome c oxidase. This copper is bound to an anionic fluorescent molecule known as the copper ligand (CuL). Here, we identify for the first time a mitochondrial carrier family protein capable of importing copper into the matrix. In vitro transport of the CuL into the mitochondrial matrix was saturable and temperature-dependent. Strains with a deletion of PIC2 grew poorly on copper-deficient non-fermentable medium supplemented with silver and under respiratory conditions when challenged with a matrix-targeted copper competitor. Mitochondria from pic2Δ cells had lower total mitochondrial copper and exhibited a decreased capacity for copper uptake. Heterologous expression of Pic2 in Lactococcus lactis significantly enhanced CuL transport into these cells. Therefore, we propose a novel role for Pic2 in copper import into mitochondria.     

A Novel DNA-Binding Protein Bound to the Mitochondrial Inner Membrane Restores the Null Mutation of Mitochondrial Histone Abf2p in Saccharomyces cerevisiae

The yeast mitochondrial HMG-box protein, Abf2p, is essential for maintenance of the mitochondrial genome. To better understand the role of Abf2p in the maintenance of the mitochondrial chromosome, we have isolated a multicopy suppressor (YHM2) of the temperature-sensitive defect associated with an abf2 null mutation. The function of Yhm2p was characterized at the molecular level. Yhm2p has 314 amino acid residues, and the deduced amino acid sequence is similar to that of a family of mitochondrial carrier proteins. Yhm2p is localized in the mitochondrial inner membrane and is also associated with mitochondrial DNA in vivo. Yhm2p exhibits general DNA-binding activity in vitro. Thus, Yhm2p appears to be novel in that it is a membrane-bound DNA-binding protein. A sequence that is similar to the HMG DNA-binding domain is important for the DNA-binding activity of Yhm2p, and a mutation in this region abolishes the ability of YHM2 to suppress the temperature-sensitive defect of respiration of the abf2 null mutant. Disruption of YHM2 causes a significant growth defect in the presence of nonfermentable carbon sources such as glycerol and ethanol, and the cells have defects in respiration as determined by 2,3,5,-triphenyltetrazolium chloride staining. Yhm2p may function as a member of the protein machinery for the mitochondrial inner membrane attachment site of mitochondrial DNA during replication and segregation of mitochondrial genomes.     

Mitochondrial uncoupling,ROS generation and cardioprotection

Mitochondrial oxidative phosphorylation is incompletely coupled, since protons translocated to the intermembrane space by specific respiratory complexes of the electron transport chain can return to the mitochondrial matrix independently of the ATP synthase —a process known as proton leak— generating heat instead of ATP. Proton leak across the inner mitochondrial membrane increases the respiration rate and decreases the electrochemical proton gradient (Δp), and is an important mechanism for energy dissipation that accounts for up to 25% of the basal metabolic rate. It is well established that mitochondrial superoxide production is steeply dependent on Δp in isolated mitochondria and, correspondingly, mitochondrial uncoupling has been identified as a cytoprotective strategy under conditions of oxidative stress, including diabetes, drug-resistance in tumor cells, ischemia-reperfusion (IR) injury or aging. Mitochondrial uncoupling proteins (UCPs) are able to lower the efficiency of oxidative phosphorylation and are involved in the control of mitochondrial reactive oxygen species (ROS) production. There is strong evidence that UCP2 and UCP3, the UCP1 homologues expressed in the heart, protect against mitochondrial oxidative damage by reducing the production of ROS. This review first analyzes the relationship between mitochondrial proton leak and ROS generation, and then focuses on the cardioprotective role of chemical uncoupling and uncoupling mediated by UCPs. This includes their protective effects against cardiac IR, a condition known to increase ROS production, and their roles in modulating cardiovascular risk factors such as obesity, diabetes and atherosclerosis.     

4-Methylene-2-octyl-5-oxotetrahydrofuran-3-carboxylic Acid (C75), an Inhibitor of Fatty-acid Synthase,Suppresses the Mitochondrial Fatty Acid Synthesis Pathway and Impairs Mitochondrial Function

4-Methylene-2-octyl-5-oxotetrahydrofuran-3-carboxylic acid (C75) is a synthetic fatty-acid synthase (FASN) inhibitor with potential therapeutic effects in several cancer models. Human mitochondrial β-ketoacyl-acyl carrier protein synthase (HsmtKAS) is a key enzyme in the newly discovered mitochondrial fatty acid synthesis pathway that can produce the substrate for lipoic acid (LA) synthesis. HsmtKAS shares conserved catalytic domains with FASN, which are responsible for binding to C75. In our study, we explored the possible effect of C75 on HsmtKAS and mitochondrial function. C75 treatment decreased LA content, impaired mitochondrial function, increased reactive oxygen species content, and reduced cell viability. HsmtKAS but not FASN knockdown had an effect that was similar to C75 treatment. In addition, an LA supplement efficiently inhibited C75-induced mitochondrial dysfunction and oxidative stress. Overexpression of HsmtKAS showed cellular protection against low dose C75 addition, whereas there was no protective effect upon high dose C75 addition. In summary, the mitochondrial fatty acid synthesis pathway has a vital role in mitochondrial function. Besides FASN, C75 might also inhibit HsmtKAS, thereby reducing LA production, impairing mitochondrial function, and potentially having toxic effects. LA supplements sufficiently ameliorated the toxicity of C75, showing that a combination of C75 and LA may be a reliable cancer treatment.     

Mitochondrial Fragmentation Due to Inhibition of Fusion Increases Cyclin B through Mitochondrial Superoxide Radicals

During the cell cycle, mitochondria undergo regulated changes in morphology. Two particularly interesting events are first, mitochondrial hyperfusion during the G₁-S transition and second, fragmentation during entry into mitosis. The mitochondria remain fragmented between late G₂- and mitotic exit. This mitotic mitochondrial fragmentation constitutes a checkpoint in some cell types, of which little is known. We bypass the ‘mitotic mitochondrial fragmentation’ checkpoint by inducing fragmented mitochondrial morphology and then measure the effect on cell cycle progression. Using Drosophila larval hemocytes, Drosophila S2R⁺ cell and cells in the pouch region of wing imaginal disc of Drosophila larvae we show that inhibiting mitochondrial fusion, thereby increasing fragmentation, causes cellular hyperproliferation and an increase in mitotic index. However, mitochondrial fragmentation due to over-expression of the mitochondrial fission machinery does not cause these changes. Our experiments suggest that the inhibition of mitochondrial fusion increases superoxide radical content and leads to the upregulation of cyclin B that culminates in the observed changes in the cell cycle. We provide evidence for the importance of mitochondrial superoxide in this process. Our results provide an insight into the need for mitofusin-degradation during mitosis and also help in understanding the mechanism by which mitofusins may function as tumor suppressors.     

Mitochondrial Morphology and Fundamental Parameters of the Mitochondrial Respiratory Chain Are Altered in Caenorhabditis elegans Strains Deficient in Mitochondrial Dynamics and Homeostasis Processes

Mitochondrial dysfunction has been linked to myriad human diseases and toxicant exposures, highlighting the need for assays capable of rapidly assessing mitochondrial health in vivo. Here, using the Seahorse XF^e24 Analyzer and the pharmacological inhibitors dicyclohexylcarbodiimide and oligomycin (ATP-synthase inhibitors), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (mitochondrial uncoupler) and sodium azide (cytochrome c oxidase inhibitor), we measured the fundamental parameters of mitochondrial respiratory chain function: basal oxygen consumption, ATP-linked respiration, maximal respiratory capacity, spare respiratory capacity and proton leak in the model organism Caenhorhabditis elegans. Since mutations in mitochondrial homeostasis genes cause mitochondrial dysfunction and have been linked to human disease, we measured mitochondrial respiratory function in mitochondrial fission (drp-1)-, fusion (fzo-1)-, mitophagy (pdr-1, pink-1)-, and electron transport chain complex III (isp-1)-deficient C. elegans. All showed altered function, but the nature of the alterations varied between the tested strains. We report increased basal oxygen consumption in drp-1; reduced maximal respiration in drp-1, fzo-1, and isp-1; reduced spare respiratory capacity in drp-1 and fzo-1; reduced proton leak in fzo-1 and isp-1; and increased proton leak in pink-1 nematodes. As mitochondrial morphology can play a role in mitochondrial energetics, we also quantified the mitochondrial aspect ratio for each mutant strain using a novel method, and for the first time report increased aspect ratios in pdr-1- and pink-1-deficient nematodes.     

Mitochondrial Protein Synthesis,Import, and Assembly

The mitochondrion is arguably the most complex organelle in the budding yeast cell cytoplasm. It is essential for viability as well as respiratory growth. Its innermost aqueous compartment, the matrix, is bounded by the highly structured inner membrane, which in turn is bounded by the intermembrane space and the outer membrane. Approximately 1000 proteins are present in these organelles, of which eight major constituents are coded and synthesized in the matrix. The import of mitochondrial proteins synthesized in the cytoplasm, and their direction to the correct soluble compartments, correct membranes, and correct membrane surfaces/topologies, involves multiple pathways and macromolecular machines. The targeting of some, but not all, cytoplasmically synthesized mitochondrial proteins begins with translation of messenger RNAs localized to the organelle. Most proteins then pass through the translocase of the outer membrane to the intermembrane space, where divergent pathways sort them to the outer membrane, inner membrane, and matrix or trap them in the intermembrane space. Roughly 25% of mitochondrial proteins participate in maintenance or expression of the organellar genome at the inner surface of the inner membrane, providing 7 membrane proteins whose synthesis nucleates the assembly of three respiratory complexes.TO think about how mitochondrial proteins are synthesized, imported, and assembled, it is useful to have a clear picture of the organellar structures that they, along with membrane lipids, compose and the functions that they carry out. As almost every schoolchild learns, mitochondria carry out oxidative phosphorylation, the controlled burning of nutrients coupled to ATP synthesis. Since Saccharomyces cerevisiae prefers to ferment sugars, respiration is a dispensable function and nonrespiring mutants are viable [although they cannot undergo meiosis (Jambhekar and Amon 2008)]. However, mitochondria themselves are not dispensable. A substantial fraction of intermediary metabolism occurs in mitochondria (Strathern et al. 1982), and at least one of these pathways, iron–sulfur cluster assembly, is essential for growth (Kispal et al. 2005). Thus, any mutation that prevents the biogenesis of mitochondria by, for example, preventing the import of protein constituents from the cytoplasm, is lethal (Baker and Schatz 1991).The mitochondria of S. cerevisiae are tubular structures at the cell cortex. While the number of distinct compartments can range from 1 to ∼50 depending upon conditions (Stevens 1981; Pon and Schatz 1991), continual fusion and fission events among them effectively form a single dynamic network (Nunnari et al. 1997). The outer membrane surrounds the tubules. The inner membrane has a boundary domain closely juxtaposed beneath the outer membrane and cristae domains that project internally from the boundary into the matrix (Figure 1A). The matrix is the aqueous compartment surrounded by the inner membrane. The aqueous intermembrane space lies between the membranes and is continuous with the space within cristae.Open in a separate windowFigure 1 Overview of mitochondrial structure in yeast. (A) Schematic of compartments comprising mitochondrial tubules. The outer membrane surrounds the organelle. The inner membrane surrounds the matrix and consists of two domains, the inner boundary membrane and the cristae membranes, which are joined at cristae junctions. The intermembrane space lies between the outer membrane and inner membrane. (B) Electron tomograph image of a highly contracted yeast mitochondrion observed en face (a) with the outer membrane (red) and (b) without the outer membrane. Reprinted by permission from John Wiley & Sons from Mannella et al. (2001).Inner membrane cristae are often depicted as baffles emanating from the boundary domain. However, electron tomography of mitochondria from several species, including yeast, shows that cristae actually emanate from the boundary membrane as narrow tubular structures at sites termed “crista junctions” and expand as they project into the matrix (Frey and Mannella 2000; Mannella et al. 2001) (Figure 1B). It seems clear that the boundary and cristae domains of the inner membrane have distinct compositions with respect to the respiratory complexes that are embedded preferentially in the cristae membrane domains, as well as other components (Vogel et al. 2006; Wurm and Jakobs 2006; Rabl et al. 2009; Suppanz et al. 2009; Zick et al. 2009; Davies et al. 2011).The outer and inner boundary membranes are connected at multiple contact sites, at least some of which are involved in protein translocation and may be transient (Pon and Schatz 1991). In addition, there appear to be firm contact sites, not directly involved with protein translocation, preferentially colocalized with crista junctions (Harner et al. 2011a).Overall, there appear to be ∼1000 distinct proteins in yeast mitochondria (Premsler et al. 2009). One series of proteomic studies on highly purified organelles identified 851 proteins thought to represent 85% of the total number of species (Sickmann et al. 2003; Reinders et al. 2006; Zahedi et al. 2006). Another study identified an additional 209 candidates (Prokisch et al. 2004). A computationally driven search for candidates involved in yeast mitochondrial function, coupled with experiments to assay respiratory function and maintenance of mitochondrial DNA (mtDNA), identified 109 novel candidates, although many of these may not be mitochondrial per se (Hess et al. 2009). Taking the boundary and cristae domains together, the inner membrane is the most protein-rich mitochondrial compartment, followed by the matrix (Daum et al. 1982).Only eight of the yeast mitochondrial proteins detected in proteomic studies are encoded by mtDNA and synthesized within the organelle. They are hydrophobic subunits of respiratory complexes III (bc₁ complex or ubiquinol-cytochrome c reductase), IV (cytochrome c oxidase), and V (ATP synthase), as well as a hydrophilic mitochondrial small subunit ribosomal protein. The remaining ∼99% of yeast mitochondrial proteins are encoded by nuclear genes, synthesized in cytoplasmic ribosomes, and imported into the organelle.An overview of known nuclearly encoded mitochondrial protein functions (Figure 2) reveals that ∼25% of them are involved directly in genome maintenance and expression of the eight major mitochondrial genes (Schmidt et al. 2010). The functions of ∼20% of the proteins are not known. Fifteen percent are involved in the well-known processes of energy metabolism. Protein translocation, folding, and turnover functions occupy ∼10% of mitochondrial proteins.Open in a separate windowFigure 2 Classification of identified mitochondrial proteins according to function. Reprinted by permission from Nature Publishing Group from Schmidt et al. (2010).The following discussion reviews our understanding of the biogenesis of mitochondria starting on the outside, the cytoplasm, and working inward through the mitochondrial compartments.     

The Yeast Gene, MDM20, Is Necessary for Mitochondrial Inheritance and Organization of the Actin Cytoskeleton

In Saccharomyces cerevisiae, the growing bud inherits a portion of the mitochondrial network from the mother cell soon after it emerges. Although this polarized transport of mitochondria is thought to require functions of the cytoskeleton, there are conflicting reports concerning the nature of the cytoskeletal element involved. Here we report the isolation of a yeast mutant, mdm20, in which both mitochondrial inheritance and actin cables (bundles of actin filaments) are disrupted. The MDM20 gene encodes a 93-kD polypeptide with no homology to other characterized proteins. Extra copies of TPM1, a gene encoding the actin filament–binding protein tropomyosin, suppress mitochondrial inheritance defects and partially restore actin cables in mdm20Δ cells. Synthetic lethality is also observed between mdm20 and tpm1 mutant strains. Overexpression of a second yeast tropomyosin, Tpm2p, rescues mutant phenotypes in the mdm20 strain to a lesser extent. Together, these results provide compelling evidence that mitochondrial inheritance in yeast is an actin-mediated process. MDM20 and TPM1 also exhibit the same pattern of genetic interactions; mutations in MDM20 are synthetically lethal with mutations in BEM2 and MYO2 but not SAC6. Although MDM20 and TPM1 are both required for the formation and/or stabilization of actin cables, mutations in these genes disrupt mitochondrial inheritance and nuclear segregation to different extents. Thus, Mdm20p and Tpm1p may act in vivo to establish molecular and functional heterogeneity of the actin cytoskeleton.     

Mutations in FBXL4, Encoding a Mitochondrial Protein,Cause Early-Onset Mitochondrial Encephalomyopathy

Whole-exome sequencing and autozygosity mapping studies, independently performed in subjects with defective combined mitochondrial OXPHOS-enzyme deficiencies, identified a total of nine disease-segregating FBXL4 mutations in seven unrelated mitochondrial disease families, composed of six singletons and three siblings. All subjects manifested early-onset lactic acidemia, hypotonia, and developmental delay caused by severe encephalomyopathy consistently associated with progressive cerebral atrophy and variable involvement of the white matter, deep gray nuclei, and brainstem structures. A wide range of other multisystem features were variably seen, including dysmorphism, skeletal abnormalities, poor growth, gastrointestinal dysmotility, renal tubular acidosis, seizures, and episodic metabolic failure. Mitochondrial respiratory chain deficiency was present in muscle or fibroblasts of all tested individuals, together with markedly reduced oxygen consumption rate and hyperfragmentation of the mitochondrial network in cultured cells. In muscle and fibroblasts from several subjects, substantially decreased mtDNA content was observed. FBXL4 is a member of the F-box family of proteins, some of which are involved in phosphorylation-dependent ubiquitination and/or G protein receptor coupling. We also demonstrate that FBXL4 is targeted to mitochondria and localizes in the intermembrane space, where it participates in an approximately 400 kDa protein complex. These data strongly support a role for FBXL4 in controlling bioenergetic homeostasis and mtDNA maintenance. FBXL4 mutations are a recurrent cause of mitochondrial encephalomyopathy onset in early infancy.     

Assembly of the Mitochondrial Membrane System: Nuclear Suppression of a Cytochrome b Mutation in Yeast Mitochondrial DNA

In a previous study, a mitochondrial mutant expressing a specific enzymatic deficiency in co-enzyme QH₂-cytochrome c reductase was described (Tzagoloff, Foury and Akai 1976). Analysis of the mitochondrially translated proteins revealed the absence in the mutant of the mitochondrial product corresponding to cytochrome b and the presence of a new low molecular weight product. The premature chain-termination mutant was used to obtain suppressor mutants with wild-type properties. One such revertant strain was analyzed genetically and biochemically. The revertant was determined to have a second mutation in a nuclear gene that is capable of partially suppressing the original mitochondrial cytochrome b mutation. Genetic data indicate that the nuclear mutation is recessive and is probably in a gene coding for a protein involved in the mitochondrial translation machinery.     

Functional Characterization of drim2, the Drosophila melanogaster Homolog of the Yeast Mitochondrial Deoxynucleotide Transporter

The CG18317 gene (drim2) is the Drosophila melanogaster homolog of the Saccharomyces cerevisiae Rim2 gene, which encodes a pyrimidine (deoxy)nucleotide carrier. Here, we tested if the drim2 gene also encodes for a deoxynucleotide transporter in the fruit fly. The protein was localized to mitochondria. Drosophila S2R⁺ cells, silenced for drim2 expression, contained markedly reduced pools of both purine and pyrimidine dNTPs in mitochondria, whereas cytosolic pools were unaffected. In vivo drim2 homozygous knock-out was lethal at the larval stage, preceded by the following: (i) impaired locomotor behavior; (ii) decreased rates of oxygen consumption, and (iii) depletion of mtDNA. We conclude that the Drosophila mitochondrial carrier dRIM2 transports all DNA precursors and is essential to maintain mitochondrial function.     

Staphylococcus aureus Sepsis Induces Early Renal Mitochondrial DNA Repair and Mitochondrial Biogenesis in Mice

Acute kidney injury (AKI) contributes to the high morbidity and mortality of multi-system organ failure in sepsis. However, recovery of renal function after sepsis-induced AKI suggests active repair of energy-producing pathways. Here, we tested the hypothesis in mice that Staphyloccocus aureus sepsis damages mitochondrial DNA (mtDNA) in the kidney and activates mtDNA repair and mitochondrial biogenesis. Sepsis was induced in wild-type C57Bl/6J and Cox-8 Gfp-tagged mitochondrial-reporter mice via intraperitoneal fibrin clots embedded with S. aureus. Kidneys from surviving mice were harvested at time zero (control), 24, or 48 hours after infection and evaluated for renal inflammation, oxidative stress markers, mtDNA content, and mitochondrial biogenesis markers, and OGG1 and UDG mitochondrial DNA repair enzymes. We examined the kidneys of the mitochondrial reporter mice for changes in staining density and distribution. S. aureus sepsis induced sharp amplification of renal Tnf, Il-10, and Ngal mRNAs with decreased renal mtDNA content and increased tubular and glomerular cell death and accumulation of protein carbonyls and 8-OHdG. Subsequently, mtDNA repair and mitochondrial biogenesis was evidenced by elevated OGG1 levels and significant increases in NRF-1, NRF-2, and mtTFA expression. Overall, renal mitochondrial mass, tracked by citrate synthase mRNA and protein, increased in parallel with changes in mitochondrial GFP-fluorescence especially in proximal tubules in the renal cortex and medulla. Sub-lethal S. aureus sepsis thus induces widespread renal mitochondrial damage that triggers the induction of the renal mtDNA repair protein, OGG1, and mitochondrial biogenesis as a conspicuous resolution mechanism after systemic bacterial infection.     

Mitochondrial fission/fusion dynamics and apoptosis

Mitochondria play an important role in the progression of apoptosis through the release of pro-apoptotic factors, such as cytochrome c, from the mitochondrial intermembrane space. During this process, mitochondrial networks are dramatically reorganised from long filamentous interconnected tubules into small punctate spheres. Whether remodelling of mitochondrial networks is necessary for apoptosis-associated cytochrome c release, or merely an accompanying process, has been a subject of debate. Here we discuss evidence for and against the role of mitochondrial fragmentation in the progression of apoptosis and highlight recent advances which indicate that mitochondrial fission is not a critical requirement for apoptosis-associated cytochrome c release. We also discuss an emerging role for Bcl-2 family members as regulators of mitochondrial fission and fusion dynamics, independent of the role of this family in the regulation of apoptosis.     

Mutation of the Mitochondrial Tyrosyl-tRNA Synthetase Gene, YARS2, Causes Myopathy, Lactic Acidosis, and Sideroblastic Anemia—MLASA Syndrome

Mitochondrial respiratory chain disorders are a heterogeneous group of disorders in which the underlying genetic defect is often unknown. We have identified a pathogenic mutation (c.156C>G [p.F52L]) in YARS2, located at chromosome 12p11.21, by using genome-wide SNP-based homozygosity analysis of a family with affected members displaying myopathy, lactic acidosis, and sideroblastic anemia (MLASA). We subsequently identified the same mutation in another unrelated MLASA patient. The YARS2 gene product, mitochondrial tyrosyl-tRNA synthetase (YARS2), was present at lower levels in skeletal muscle whereas fibroblasts were relatively normal. Complex I, III, and IV were dysfunctional as indicated by enzyme analysis, immunoblotting, and immunohistochemistry. A mitochondrial protein-synthesis assay showed reduced levels of respiratory chain subunits in myotubes generated from patient cell lines. A tRNA aminoacylation assay revealed that mutant YARS2 was still active; however, enzyme kinetics were abnormal compared to the wild-type protein. We propose that the reduced aminoacylation activity of mutant YARS2 enzyme leads to decreased mitochondrial protein synthesis, resulting in mitochondrial respiratory chain dysfunction. MLASA has previously been associated with PUS1 mutations; hence, the YARS2 mutation reported here is an alternative cause of MLASA.     

The Mitochondrial Genome of Raphanus sativus and Gene Evolution of Cruciferous Mitochondrial Types

To explore the mitochondrial genes of the Cruciferae family, the mitochondrial genome of Raphanus sativus (sat) was sequenced and annotated. The circular mitochondrial genome of sat is 239,723 bp and includes 33 protein-coding genes, three rRNA genes and 17 tRNA genes. The mitochondrial genome also contains a pair of large repeat sequences 5.9 kb in length, which may mediate genome reorga-nization into two sub-genomic circles, with predicted sizes of 124.8 kb and 115.0 kb, respectively. Furthermore, gene evolution of mitochondrial genomes within the Cruciferae family was analyzed using sat mitochondrial type (mitotype), together with six other re-ported mitotypes. The cruciferous mitochondrial genomes have maintained almost the same set of functional genes. Compared with Cycas taitungensis (a representative gymnosperm), the mitochondrial genomes of the Cruciferae have lost nine protein-coding genes and seven mitochondrial-like tRNA genes, but acquired six chloroplast-like tRNAs. Among the Cruciferae, to maintain the same set of genes that are necessary for mitochondrial function, the exons of the genes have changed at the lowest rates, as indicated by the numbers of single nucleotide polymorphisms. The open reading frames (ORFs) of unknown function in the cruciferous genomes are not conserved. Evolutionary events, such as mutations, genome reorganizations and sequence insertions or deletions (indels), have resulted in the non- conserved ORFs in the cruciferous mitochondrial genomes, which is becoming significantly different among mitotypes. This work represents the first phylogenic explanation of the evolution of genes of known function in the Cruciferae family. It revealed significant variation in ORFs and the causes of such variation.     

Prohibitin Family Members Interact Genetically with Mitochondrial Inheritance Components in Saccharomyces cerevisiae

Phb2p, a homolog of the tumor suppressor protein prohibitin, was identified in a genetic screen for suppressors of the loss of Mdm12p, a mitochondrial outer membrane protein required for normal mitochondrial morphology and inheritance in Saccharomyces cerevisiae. Phb2p and its homolog, prohibitin (Phb1p), were localized to the mitochondrial inner membrane and characterized as integral membrane proteins which depend on each other for their stability. In otherwise wild-type genetic backgrounds, null mutations in PHB1 and PHB2 did not confer any obvious phenotypes. However, loss of function of either PHB1 or PHB2 in cells with mitochondrial DNA deleted led to altered mitochondrial morphology, and phb1 or phb2 mutations were synthetically lethal when combined with a mutation in any of three mitochondrial inheritance components of the mitochondrial outer membrane, Mdm12p, Mdm10p, and Mmm1p. These results provide the first evidence of a role for prohibitin in mitochondrial inheritance and in the regulation of mitochondrial morphology.     

Mitochondrial genome of the eurasian otterLutra lutra (Mammalia, Carnivora, Mustelidae)

We determined the complete mitochondrial genome of the Eurasian otterLutra lutra, which is an endangered species in Korea. The circle genome (16,536 bp in size) consists of 13 protein-coding, 22 tRNA, and 2 rRNA genes, and a control region, as found in other metazoan animals. Out of the 37 genes, 28 are encoded on the H-strand, and the nine (ND6 and 8 tRNA genes) on the L-strand. Three overlaps among the 13 protein-coding genes were found: ATP8-ATP6, ND4L-ND4, and ND5-ND6. A control region (1090 bp) including the origin of H-strand replication (OH), TAS (a conserved motif TACAT-16bp-ATGTA) and CSB (CSB-1, CSB-2. and CSB-3) was observed between tRNA-Pro and tRNA-Phe genes, and O_L, with 36 highly conserved nucleotides between tRNA-Asn (N) and tRNA-Cys (C) within a cluster of five tRNA genes (WANCY), as typically found in vertebrates. The other important characteristics of theL. lutra mitochondrial genome were described in detail. In addition, a maximum likelihood and Bayesian trees of 9 mustelid species and 1 outgroup were reconstructed based on the nucleotide sequences of 11 protein-coding genes excluding ATP8 and ND6. It showed that Lutrinae formed a monophyletic group with Mustelinae that is not monophyletic. Within the subfamily Lutrinae,L. lutra andEnhydra lutris were grouped together and thenLontra canadentis placed as a sister of the clade. The present result is the first complete mitochondrial genome sequence reported from the genusLutra, and is applicable to molecular phylogenetic, phylogeographic, conservation biological studies for mustelid members. In particular, exploration of sequence variations of the control region may be helpful for analyzing inter-and intra-species variations in the genusLutra.     

miR-181c Regulates the Mitochondrial Genome,Bioenergetics, and Propensity for Heart Failure In Vivo

MicroRNAs (miRNAs) are small non-coding RNAs, which inhibit the stability and/or translation of a mRNA. miRNAs have been found to play a powerful role in various cardiovascular diseases. Recently, we have demonstrated that a microRNA (miR-181c) can be encoded in the nucleus, processed to the mature form in the cytosol, translocated into the mitochondria, and ultimately can regulate mitochondrial gene expression. However the in vivo impact of miR-181c is unknown. Here we report an in-vivo method for administration of miR-181c in rats, which leads to reduced exercise capacity and signs of heart failure, by targeting the 3′-end of mt-COX1 (cytochrome c oxidase subunit 1). We cloned miR-181c and packaged it in lipid-based nanoparticles for systemic delivery. The plasmid DNA complexed nanovector shows no apparent toxicity. We find that the mRNA levels of mitochondrial complex IV genes in the heart, but not any other mitochondrial genes, are significantly altered with miR-181c overexpression, suggesting selective mitochondrial complex IV remodeling due to miR-181c targeting mt-COX1. Isolated heart mitochondrial studies showed significantly altered O₂-consumption, ROS production, matrix calcium, and mitochondrial membrane potential in miR-181c-treated animals. For the first time, this study shows that miRNA delivered to the heart in-vivo can lead to cardiac dysfunction by regulating mitochondrial genes.     

Phosphatidylserine Decarboxylase 1 (Psd1) Promotes Mitochondrial Fusion by Regulating the Biophysical Properties of the Mitochondrial Membrane and Alternative Topogenesis of Mitochondrial Genome Maintenance Protein 1 (Mgm1)

Non–bilayer-forming lipids such as cardiolipin, phosphatidic acid, and phosphatidylethanolamine (PE) are proposed to generate negative membrane curvature, promoting membrane fusion. However, the mechanism by which lipids regulate mitochondrial fusion remains poorly understood. Here, we show that mitochondrial-localized Psd1, the key yeast enzyme that synthesizes PE, is required for proper mitochondrial morphology and fusion. Yeast cells lacking Psd1 exhibit fragmented and aggregated mitochondria with impaired mitochondrial fusion during mating. More importantly, we demonstrate that a reduction in PE reduces the rate of lipid mixing during fusion of liposomes with lipid compositions reflecting the mitochondrial membrane. This suggests that the mitochondrial fusion defect in the Δpsd1 strain could be due to the altered biophysical properties of the mitochondrial membrane, resulting in reduced fusion kinetics. The Δpsd1 strain also has impaired mitochondrial activity such as oxidative phosphorylation and reduced mitochondrial ATP levels which are due to a reduction in mitochondrial PE. The loss of Psd1 also impairs the biogenesis of s-Mgm1, a protein essential for mitochondrial fusion, further exacerbating the mitochondrial fusion defect of the Δpsd1 strain. Increasing s-Mgm1 levels in Δpsd1 cells markedly reduced mitochondrial aggregation. Our results demonstrate that mitochondrial PE regulates mitochondrial fusion by regulating the biophysical properties of the mitochondrial membrane and by enhancing the biogenesis of s-Mgm1. While several proteins are required to orchestrate the intricate process of membrane fusion, we propose that specific phospholipids of the mitochondrial membrane promote fusion by enhancing lipid mixing kinetics and by regulating the action of profusion proteins.     

The Dynamin-related GTPase,Dnm1p,Controls Mitochondrial Morphology in Yeast

The Saccharomyces cerevisiae Dnm1 protein is structurally related to dynamin, a GTPase required for membrane scission during endocytosis. Here we show that Dnm1p is essential for the maintenance of mitochondrial morphology. Disruption of the DNM1 gene causes the wild-type network of tubular mitochondrial membranes to collapse to one side of the cell but does not affect the morphology or distribution of other cytoplasmic organelles. Dnm1 proteins containing point mutations in the predicted GTP-binding domain or completely lacking the GTP-binding domain fail to rescue mitochondrial morphology defects in a dnm1 mutant and induce dominant mitochondrial morphology defects in wild-type cells. Indirect immunofluorescence reveals that Dnm1p is distributed in punctate structures at the cell cortex that colocalize with the mitochondrial compartment. These Dnm1p-containing structures remain associated with the spherical mitochondria found in an mdm10 mutant strain. In addition, a portion of Dnm1p cofractionates with mitochondrial membranes during differential sedimentation and sucrose gradient fractionation of wild-type cells. Our results demonstrate that Dnm1p is required for the cortical distribution of the mitochondrial network in yeast, a novel function for a dynamin-related protein.