GRIM-19 is essential for maintenance of mitochondrial membrane potential

GRIM-19 was found to copurify with complex I of mitochondrial respiratory chain and subsequently was demonstrated to be involved in complex I assembly and activity. To further understand its function in complex I, we dissected its functional domains by generating a number of deletion, truncation, and point mutants. The mitochondrial localization sequences were located at the N-terminus. Strikingly, deletion of residues 70-80, 90-100, or the whole C-terminal region (70-144) led to a loss of mitochondrial transmembrane potential (DeltaPsim). However, similar deletions of another two complex I subunits, NDUFA9 and NDUFS3, did not show such effect. We also found that deletion of the last 10 residues affected GRIM-19's ability to be assembled to complex I. We constructed a dominant-negative mutant containing the N-terminal 60 and the last C-terminal 10 residues, which could be assembled into complex I, but failed to maintain normal DeltaPsim. Cells overexpressing this mutant did not spontaneously undergo cell death, but were sensitized to apoptosis induced by cell death agents. Our results demonstrate that GRIM-19 is required for electron transfer activity of complex I, and disruption of DeltaPsim by GRIM-19 mutants enhances the cells' sensitivity to apoptotic stimuli.     

The yeast nuclear gene NAM2 is essential for mitochondrial DNA integrity and can cure a mitochondrial RNA-maturase deficiency

Dominant mutations in the yeast nuclear gene NAM2 cure the RNA splicing deficiency resulting from the inactivation of the bI4 maturase encoded by the fourth intron of the mitochondrial cytochrome b gene. This maturase is required to splice the fourth intron of this gene and to splice the fourth intron of the mitochondrial gene oxi3 encoding cytochrome oxidase subunit I. We have cloned the nuclear gene NAM2, which codes for two overlapping RNAs, 3.2 kb and 3.0 kb long, which are transcribed in the same direction but differ at their 5' ends. NAM2 compensating mutations probably result from point mutations in the structural gene. Integration of the cloned gene occurs at its homologous locus on the right arm of chromosome XII. Inactivation of the NAM2 gene either by transplacement with a deleted copy of the gene, or by disruption, is not lethal to the cell, but leads to the destruction of the mitochondrial genome with the production of 100% cytoplasmic petites.     

The control of mitochondrial respiration in yeast: A possible role of the outer mitochondrial membrane

Mitochondrial respiration in yeast (S. cerevisiae) is regulated by the level of glucose in the medium. Glucose is known to inhibit respiration by repressing key enzymes in the respiratory chain. We present evidence that the early events in this inhibition include the closure of VDAC channels, the primary pathway for metabolite flow across the outer membrane. Aluminum hydroxide is known to inhibit the closure of VDAC. Addition of aluminum acetylacetonate to yeast cells, which should elevate the aluminum hydroxide concentrations in the cytoplasm, caused the inhibition of cell respiration by glucose to be delayed for up to 100 min. No significant effect of aluminum was observed in cells grown on glycerol. Yeast cells lacking the VDAC gene were also unresponsive to the addition of aluminum salt in the presence of glucose. Therefore, the closure of VDAC channels may be an early step in the inhibition of the respiration of yeast by glucose.     

The major 45-kDa protein of the yeast mitochondrial outer membrane is not essential for cell growth or mitochondrial function

As part of an analysis of the function and assembly of the mitochondrial outer membrane, we have cloned and characterized the yeast gene encoding a 45-kDa polypeptide (OM45) which is a major constituent of this membrane. The nuclear gene was isolated by immunological screening of plaques of recombinant phage lambda gt11 containing fragments of yeast genomic DNA using an antibody against OM45. Determination of the nucleotide sequence of the DNA fragment isolated by this approach revealed a single open reading frame of 1179 base pairs which encodes a protein having a predicted molecular mass of 44.6-kDa. Disruption of the OM45 gene in haploid yeast cells eliminated the expression of OM45. The mutant strain showed no apparent defect in cell viability, growth, mitochondrial function, or mitochondrial protein import.     

Mmm2p, a mitochondrial outer membrane protein required for yeast mitochondrial shape and maintenance of mtDNA nucleoids

The mitochondrial outer membrane protein, Mmm1p, is required for normal mitochondrial shape in yeast. To identify new morphology proteins, we isolated mutations incompatible with the mmm1-1 mutant. One of these mutants, mmm2-1, is defective in a novel outer membrane protein. Lack of Mmm2p causes a defect in mitochondrial shape and loss of mitochondrial DNA (mtDNA) nucleoids. Like the Mmm1 protein (Aiken Hobbs, A.E., M. Srinivasan, J.M. McCaffery, and R.E. Jensen. 2001. J. Cell Biol. 152:401-410.), Mmm2p is located in dot-like particles on the mitochondrial surface, many of which are adjacent to mtDNA nucleoids. While some of the Mmm2p-containing spots colocalize with those containing Mmm1p, at least some of Mmm2p is separate from Mmm1p. Moreover, while Mmm2p and Mmm1p both appear to be part of large complexes, we find that Mmm2p and Mmm1p do not stably interact and appear to be members of two different structures. We speculate that Mmm2p and Mmm1p are components of independent machinery, whose dynamic interactions are required to maintain mitochondrial shape and mtDNA structure.     

TbPIF8, a Trypanosoma brucei protein related to the yeast Pif1 helicase, is essential for cell viability and mitochondrial genome maintenance

The trypanosome mitochondrial genome, kinetoplast DNA (kDNA), is a massive network of interlocked DNA rings, including several thousand minicircles and dozens of maxicircles. The unusual complexity of kDNA would indicate that numerous proteins must be involved in its condensation, replication, segregation and gene expression. During our investigation of trypanosome mitochondrial PIF1-like helicases, we found that TbPIF8 is the smallest and most divergent. It lacks some conserved helicase domains, thus implying that unlike other mitochondrial PIF1-like helicases, this protein may have no enzymatic activity. TbPIF8 is positioned on the distal face of kDNA disk and its localization patterns vary with different kDNA replication stages. Stem-loop RNAi of TbPIF8 arrests cell growth and causes defects in kDNA segregation. RNAi of TbPIF8 causes only limited kDNA shrinkage but the networks become disorganized. Electron microcopy of thin sections of TbPIF8-depleted cells shows heterogeneous electron densities in the kinetoplast disk. Although we do not yet know its exact function, we conclude that TbPIF8 is essential for cell viability and is important for maintenance of kDNA.     

MPI1, an essential gene encoding a mitochondrial membrane protein, is possibly involved in protein import into yeast mitochondria.

To identify components of the mitochondrial protein import pathway in yeast, we have adopted a positive selection procedure for isolating mutants disturbed in protein import. We have cloned and sequenced a gene, termed MPI1, that can rescue the genetic defect of one group of these mutants. MPI1 encodes a hydrophilic 48.8 kDa protein that is essential for cell viability. Mpi1p is a low abundance and constitutively expressed mitochondrial protein. Mpi1p is synthesized with a characteristic mitochondrial targeting sequence at its amino-terminus, which is most probably proteolytically removed during import. It is a membrane protein, oriented with its carboxy-terminus facing the intermembrane space. In cells depleted of Mpi1p activity, import of the precursor proteins that we tested thus far, is arrested. We speculate that the Mpi1 protein is a component of a proteinaceous import channel for translocation of precursor proteins across the mitochondrial inner membrane.     

In quiescence of fission yeast,autophagy and the proteasome collaborate for mitochondrial maintenance and longevity

Regulation of proliferation and quiescence in response to intra- or extracellular environmental signals are important for medicine and basic biology. Quiescence is relevant to tumorigenesis and tissue regeneration, and the maintenance of post-mitotic cells is important with regard to a number of senescence-related diseases such as neurodegeneration. We employ fission yeast, Schizosaccharomyces pombe, as a model to study quiescence and longevity as this lower eukaryote has a long chronological life span (over months) in quiescence that is induced by nitrogen starvation. We recently reported that autophagy and the proteasome cooperate in proper mitochondrial maintenance in the quiescent phase. Such cooperativity is not found in proliferating cells. In quiescence, the proteasome is required for normal mitochondrial functions; inactivation of the proteasome results in a large accumulation of reactive oxygen species (ROS), diminished mitochondrial function, and the elevation of proteins and compounds having anti-oxidant activities. Autophagy contributes to preventing the lethal accumulation of ROS by degrading mitochondria, the primary source of ROS. Our results indicate that the degradation of mitochondria by autophagy during proteasome dysfunction is a defense mechanism of quiescenct cells against the accumulation of ROS.     

Polyethylenimine-mediated impairment of mitochondrial membrane potential, respiration and membrane integrity: implications for nucleic acid delivery and gene therapy

The 25 kDa branched polyethylenimine (PEI) is a highly efficient synthetic polycation used in transfection protocols, but also triggers mitochondrial-mediated apoptotic cell death processes where the mechanistic issues are poorly understood. We now demonstrate that PEI in a concentration- and time-dependent manner can affect functions (membrane potential, swelling and respiration) and ultrastructural integrity of freshly isolated rat liver mitochondria. The threshold concentration for detection of PEI-mediated impairment of rat liver mitochondrial functions is 3 μg/mL, however, lower PEI levels still exert some effects on mitochondrial morphology and respiration, and these may be related to the inherent membrane perturbing properties of this polycation. The PEI-mediated mitochondrial swelling phase is biphasic, with a fast decaying initial period (most prominent from 4 μg/mL PEI) followed by a slower, linear swelling response. The slow phase is presumably the result of a time-dependent transition permeability opening in mitochondria initially resistant to swelling/depolarization, but may further be related to PEI-induced nanoscale structural defects and/or formation of pores in the outer membrane. Respiration assessments further suggested that PEI in the presence of exogenous ADP behaves in a similar fashion to a slow-acting inhibitory compound. PEI further shows an uncoupling property that is detectable at low respiration rates. The relevance of these findings to PEI-mediated initiation of intrinsic apoptotic pathway is discussed.     

The mitochondrial inner membrane protein Lpe10p, a homologue of Mrs2p, is essential for magnesium homeostasis and group II intron splicing in yeast

The yeast ORF YPL060w/LPE10 encodes a homologue of the mitochondrial protein Mrs2p. These two proteins are 32% identical, and have two transmembrane domains in their C-terminal regions and a putative magnesium transporter signature, Y/F-G-M-N, at the end of one of these domains. Data presented here indicate that Lpe10p is inserted into the inner mitochondrial membrane with both termini oriented towards the matrix space. Disruption of the LPE10 gene results in a growth defect on non-fermentable substrates (petite phenotype) and a marked defect in group II intron splicing. The fact that in intron-less strains lpe10 disruptants also exhibit a petite phenotype indicates that functions other than RNA splicing are affected by the absence of Lpe10p. In the mitochondria, concentrations of magnesium, but not of several other divalent metal ions, are increased when Lpe10p is overexpressed and reduced when it is absent. Magnesium concentrations are raised to normal levels and growth on non-fermentable substrates is partially restored by the expression of CorA, the bacterial magnesium transporter, in the lpe10 disruptant. These features are similar to those previously reported for Mrs2p, suggesting that Lpe10p and Mrs2p are functional homologues. However, they cannot easily substitute for each other. Their roles in magnesium homeostasis and, possibly as a secondary effect, in RNA splicing are discussed.     

Mouse homologue of coq7/clk-1, longevity gene in Caenorhabditis elegans, is essential for coenzyme Q synthesis, maintenance of mitochondrial integrity, and neurogenesis.

coq7/clk-1 was isolated from a long-lived mutant of Caenorhabditis elegans, which showed sluggish behavior and an extended life span. Mouse coq7 is homologous to Saccharomyces cerevisiae coq7/cat5 that is required for biosynthesis of coenzyme Q (CoQ), an essential cofactor in mitochondrial respiration. Here we generated COQ7-deficient mice to investigate the biological role of COQ7 in mammals. COQ7-deficient mouse embryos failed to survive beyond embryonic day 10.5, exhibiting small-sized body and delayed embryogenesis. Morphological studies showed that COQ7-deficient neuroepithelial cells failed to show the radial arrangement in the developing cerebral wall, aborting neurogenesis at E10.5. Electron microscopic analysis further showed the enlarged mitochondria with vesicular cristae and enlarged lysosomes filled with disrupted membranes, which is consistent with mitochondriopathy. Biochemical analysis demonstrated that COQ7-deficient embryos failed to synthesize CoQ(9), but instead yielded demethoxyubiquinone 9 (DMQ(9)). Cultured embryonic cells from COQ7-deficient mice were rescued by adding bovine fetal serum in vitro, but exhibited slowed cell proliferation, which resembled to the phenotype of clk-1 with delayed cell divisions. The result implied the essential role of coq7 in CoQ synthesis, maintenance of mitochondrial integrity, and neurogenesis in mice.     

The polyubiquitin gene is essential for meiosis in fission yeast

We isolated a novel sporulation-deficient mutant of Schizosaccharomyces pombe. The mutant did not have a mitotic growth defect but aborted meiosis at the first or the second division with condensed chromosomes that failed to separate, abnormal spindle(s), and disintegrated spindle pole bodies (SPBs). During the first division, the centromeres were pulled to near the spindle poles but condensed divalent chromosomes remained at the center. The failure to proceed to anaphase was also observed during a time-lapse recording of a SPB protein tagged with green fluorescent protein. The polyubiquitin gene ubi4(+), which encoded eight ubiquitins fused in tandem, complemented this mutant. The mutation, an A to G substitution, was identified within the ubi4(+) gene at the ATG initiation codon. Disruption of the ubi4(+) gene produced the same phenotypes. The ubi4(+) mRNA was strongly induced for meiosis. However, ubiquitin increases only slightly, suggesting that the role of the polyubiquitin gene is to supply ubiquitin that is consumed by unidentified mechanisms. Before the ubi4 mutant cells entered meiosis, ubiquitin was greatly decreased indicating that shortage of ubiquitin caused abortion of meiosis. This work provides insights for the role of polyubiquitin gene and importance of ubiquitination in SPB integrity at the meiotic divisions.     

Impaired cardiac mitochondrial membrane potential and respiration in copper-deficient rats

Cardiac mitochondrial respiration, ATP synthase activity, and membrane potential and intactness were evaluated in copper-deficient rats. In the presence of NADH, both copper-deficient and copper-adequate mitochondria had very low oxygen consumption rates, indicating membrane intactness. However copper-deficient mitochondria had significantly lower oxygen consumption rates with NADH than did copper-adequate mitochondria. Copper-deficient mitochondria had significantly lower membrane potential than did copper-adequate mitochondria using fluorescent dyes. Copper-deficient mitochondria had significantly lower state 3 oxygen consumption rates and were less sensitive to inhibition by oligomycin, an ATP synthase inhibitor. Copper-deficient and copper-adequate mitochondria responded similiarly to CCCP. No difference was observed in mitochondrial ATPase activity between copper-deficient and copper-adequate rats using submitochondrial particles. We conclude that cardiac mitochondrial respiration is compromised in copper-deficient rats, and may be related to an altered ATP synthase complex and/or a decreased mitochondrial membrane potential.     

DHTKD1 is essential for mitochondrial biogenesis and function maintenance

Maintaining the functional integrity of mitochondria is crucial for cell function, signal transduction and overall cell activities. Mitochondrial dysfunctions may alter energy metabolism and in many cases are associated with neurological diseases. Recent studies have reported that mutations in dehydrogenase E1 and transketolase domain-containing 1 (DHTKD1), a mitochondrial protein encoding gene, could cause neurological abnormalities. However, the function of DHTKD1 in mitochondria remains unknown. Here, we report a strong correlation of DHTKD1 expression level with ATP production, revealing the fact that DHTKD1 plays a critical role in energy production in mitochondria. Moreover, suppression of DHTKD1 leads to impaired mitochondrial biogenesis and increased reactive oxygen species (ROS), thus leading to retarded cell growth and increased cell apoptosis. These findings demonstrate that DHTKD1 contributes to mitochondrial biogenesis and function maintenance.     

Loss of mitochondrial membrane potential is associated with increase in mitochondrial volume: physiological role in neurones

Mitochondrial volume homeostasis is a housekeeping cellular function, thought to help regulate oxidative capacity, apoptosis, and mechanical signaling. The volume is mainly regulated by potassium flux into and out of the matrix and controlled by the electrochemical potential. Mitochondrial depolarization will therefore affect this flux but studies showing how have not been consistent, and it is unclear what mitochondrial volume changes also occur. The aim of the present study was to investigate mitochondrial volume changes in permeabilized neurons under various bioenergetic conditions using deconvolution confocal microscopy. Under control conditions, mitochondria in situ appeared rod-shaped with mean length, surface area, and volume values of 2.29+/-0.10 microm, 1.41+/-0.10 microm2, and 0.062+/-0.006 microm3, respectively (n=42). Valinomycin, a K+-selective ionophore, increased mitochondrial volume by 63+/-22%, although surface area was almost unchanged because mitochondrial shape became more spherical. Pinacidil, an opener of mitochondrial ATP-dependent channels, produced similar effects, although some mitochondria were insensitive to its action. Mitochondrial depolarization with the protonophore FCCP, or with respiratory chain inhibitors antimycin and sodium azide was associated with a considerable increase in mitochondrial volume (by 75%-140%). Effects of mitochondrial modulators were also studied in intact neurones. Tracking of single mitochondria showed that during 65+/-2% of their time, mitochondria were motile with an average velocity of 0.19+/-0.01 microm/s. Antimycin, azide, and FCCP induced mitochondrial swelling and significantly decreased mitochondrial motility. In the presence of pinacidil, swollen mitochondria had reduced their motility, although mitochondria with normal volume stayed motile. These data show that mitochondrial depolarization was followed by significant swelling, which, in turn, impaired mitochondrial trafficking.     

The nuclear gene MRS2 is essential for the excision of group II introns from yeast mitochondrial transcripts in vivo.

RNA splicing defects in mitochondrial intron mutants can be suppressed by a high dosage of several proteins encoded by nuclear genes. In this study we report on the isolation, nucleotide sequence, and possible functions of the nuclear MRS2 gene. When present on high copy number plasmids, the MRS2 gene acts as a suppressor of various mitochondrial intron mutations, suggesting that the MRS2 protein functions as a splicing factor. This notion is supported by the observations that disruption of the single chromosomal copy of the MRS2 gene causes (i) a pet- phenotype and (ii) a block in mitochondrial RNA splicing of all four mitochondrial group II introns, some of which are efficiently self-splicing in vitro. In contrast, the five group I introns monitored here are excised from pre-mRNA in a MRS2-disrupted background although at reduced rates. So far the MRS2 gene product is unique in that it is essential for splicing of all four group II introns, but relatively unimportant for splicing of group I introns. In strains devoid of any mitochondrial introns the MRS2 gene disruption still causes a pet- phenotype and cytochrome deficiency, although the standard pattern of mitochondrial translation products is produced. Therefore, apart from RNA splicing, the absence of the MRS2 protein may disturb the assembly of mitochondrial membrane complexes.     

MAS1, a gene essential for yeast mitochondrial assembly, encodes a subunit of the mitochondrial processing protease.

We have previously described a yeast mutant (mas1) that accumulates mitochondrial precursor proteins at high temperature and is deficient in the activity of a matrix-localized protease which cleaves presequences from mitochondrial precursor proteins. We have now cloned and sequenced the wild-type MAS1 gene and found that it encodes a subunit of the mitochondrial processing protease, that it is essential for cell viability and that the protein product participates in its own cleavage during import into mitochondria. The MAS1 protein is thus the first genetically defined component of the mitochondrial protein import pathway.     

The role of a conserved dodecamer sequence in yeast mitochondrial gene expression

All mRNAs on the yeast mitochondrial genome terminate at a conserved dodecamer sequence 5'-AAUAAUAUUCUU-3'. We have characterized two mutants with altered dodecamers. One contains a deletion of the dodecamer at the end of the var1 gene, and the other contains two adjacent transversions in the dodecamer at the end of the reading frame of fit1, a gene within the omega+ allele of the 21S rRNA gene. In each mutant, expression of the respective gene is blocked completely. A dominant nuclear suppressor, SUV3-1, was isolated that suppresses the var1 deletion but is without effect on the fit1 dodecamer mutations. Unexpectedly, however, we found that SUV3-1 blocks expression of the wild-type fit1 allele by blocking processing at its dodecamer. SUV3-1 has pleiotropic effects on mitochondrial gene expression, affecting RNA processing, RNA stability, and translation. Our results suggest that RNA metabolism and translation may be part of a multicomponent complex within mitochondria.     

Fast kinase domain-containing protein 3 is a mitochondrial protein essential for cellular respiration

Fas-activated serine/threonine phosphoprotein (FAST) is the founding member of the FAST kinase domain-containing protein (FASTKD) family that includes FASTKD1-5. FAST is a sensor of mitochondrial stress that modulates protein translation to promote the survival of cells exposed to adverse conditions. Mutations in FASTKD2 have been linked to a mitochondrial encephalomyopathy that is associated with reduced cytochrome c oxidase activity, an essential component of the mitochondrial electron transport chain. We have confirmed the mitochondrial localization of FASTKD2 and shown that all FASTKD family members are found in mitochondria. Although human and mouse FASTKD1-5 genes are expressed ubiquitously, some of them are most abundantly expressed in mitochondria-enriched tissues. We have found that RNA interference-mediated knockdown of FASTKD3 severely blunts basal and stress-induced mitochondrial oxygen consumption without disrupting the assembly of respiratory chain complexes. Tandem affinity purification reveals that FASTKD3 interacts with components of mitochondrial respiratory and translation machineries. Our results introduce FASTKD3 as an essential component of mitochondrial respiration that may modulate energy balance in cells exposed to adverse conditions by functionally coupling mitochondrial protein synthesis to respiration.