Regulation of Mitochondrial Iron Import through Differential Turnover of Mitoferrin 1 and Mitoferrin 2

Mitoferrin 1 and mitoferrin 2 are homologous members of the mitochondrial solute carrier family. Mitoferrin 1 is required for mitochondrial iron delivery in developing erythrocytes. Here we show that mitoferrin 1 and mitoferrin 2 contribute to mitochondrial iron delivery in a variety of cells. Reductions in mitoferrin 1 and/or mitoferrin 2 levels by RNA interference result in decreased mitochondrial iron accumulation, heme synthesis, and iron-sulfur cluster synthesis. The ectopic expression of mitoferrin 1 in nonerythroid cells silenced for mitoferrin 2 or the expression of mitoferrin 2 in cells silenced for mitoferrin 1 restored heme synthesis to “baseline” levels. The ectopic expression of mitoferrin 2, however, did not support hemoglobinization in erythroid cells deficient in mitoferrin 1. Mitoferrin 2 could not restore heme synthesis in developing erythroid cells because of an inability of the protein to accumulate in mitochondria. The half-life of mitoferrin 1 was increased in developing erythroid cells, while the half-life of mitoferrin 2 did not change. These results suggest that mitochondrial iron accumulation is tightly regulated and that controlling mitoferrin levels within the mitochondrial membrane provides a mechanism to regulate mitochondrial iron levels.Iron is a required element for all eukaryotes, but iron can be toxic at high concentrations. Consequently, the cellular acquisition of iron is highly regulated, as is the concentration of free iron in biological fluids. The regulation of iron concentration is extended to cellular organelles that either store or utilize iron. Mitochondria utilize iron for the synthesis of heme and iron-sulfur (Fe-S) clusters. These prosthetic groups are used within the mitochondria and are exported for use by cytosolic and nuclear proteins. The mechanisms that regulate mitochondrial iron levels are not known, although it is clear that mitochondrial iron levels must be regulated. For example, the loss of function mutations in genes that encode enzymes required for Fe-S cluster synthesis or the Atm1 transporter that exports Fe-S clusters, results in excessive mitochondrial iron accumulation in yeast and humans (for a review, see reference 11).The mechanisms that regulate mitochondrial iron pools are not well defined. Mitochondrial iron pools might be regulated at the level of import. Mitoferrin 1 (Mfrn1) has been shown to be required for mitochondrial iron import in developing erythroid cells. A mutation in zebrafish Mfrn1 (frascati) or the deletion of mouse Mfrn1 leads to defects in hemoglobinization due to a deficit in mitochondrial iron uptake (17). The phenotype of frascati zebrafish is restricted to developing red blood cells; other cell types showed no evidence of a mitochondrial iron phenotype. Mfrn1 has a paralogue, Mfrn2, and both genes have homologues MRS3 and MRS4 in Saccharomyces cerevisiae. Yeast with deletions of MRS3 and MRS4 grows poorly under low iron conditions due to impaired mitochondrial iron acquisition (5, 10, 13, 23). In yeast, the expression of Mfrn1 or Mfrn2 in Δmrs3 Δmrs4 cells can correct the poor growth under low iron conditions. The expression of either mouse or zebrafish Mfrn1 as a transgene in frascati zebrafish corrected the hemoglobin deficiency in cells, but the expression of Mfrn2 did not (17). These observations raise three questions. (i) What is the role of Mfrn2 in mitochondrial iron metabolism? (ii) Is iron transport into mitochondria regulated? (iii) If Mfrn2 transports iron into the mitochondria of vertebrate cells, why doesn''t Mfrn2 rescue the mitochondrial defect in Mfrn1-deficient zebrafish?Here, we show that Mfrn1 and Mfrn2 can transport iron into the mammalian mitochondria of nonerythroid cells. The ectopic expression of either Mfrn1 or Mfrn2 can restore mitochondrial iron transport in cells silenced for Mfrn2 and -1, respectively, but ectopic expression has little effect on increasing mitochondrial iron levels above the baseline values. Mitochondrial iron levels do not increase over the baseline because the levels of Mfrns are regulated posttranslationally. Mfrn1 accumulates in the mitochondria of developing red blood cells as a result of an increased protein half-life. In contrast, Mfrn2 does not accumulate in developing red blood cells or other cells, as the half-life of Mfrn2 protein remains constant.     

Characterization of a nucleus located mollusc mitoferrin and its response to OsHV-1 infection

Mitoferrin genes as members of SLC25 family are conservatively existed across species, mainly locate on mitochondria and serve an important role in the regulation of whole cellular iron metabolism. Available iron withholding from pathogens presents an important host defense strategy, while the regulation role of mitoferrin against invading pathogens is largely unknown. In this study, a unique mollusc mitoferrin gene was identified in ark clams, named SbmiFn, that showed conserved three-dimensional structure with other mitoferrins, and its iron binding activity was verified by iron chelating assay. Besides cytoplasmic distribution, colocalization between SbmiFn and nuclei was observed by immunohistochemistry assay. Moreover, the response of SbmiFn to viral pathogen OsHV-1 was investigated. The results showed that nucleus located signal of SbmiFn was enhanced, the expressions of SbmiFn and ferritin were coordinately decreased, which might assist host against OsHV-1 replication as the increase of OsHV-1 copies were hardly detected after that. These results refreshed our knowledge on the sequence, structure and functional characteristics of mitoferrin subfamily, and would contribute to further comparative studies on iron metabolism.     

The yeast mitochondrial carrier proteins Mrs3p/Mrs4p mediate iron transport across the inner mitochondrial membrane

The yeast proteins Mrs3p and Mrs4p are two closely related members of the mitochondrial carrier family (MCF), which had previously been implicated in mitochondrial Fe²⁺ homeostasis. A vertebrate Mrs3/4 homologue named mitoferrin was shown to be essential for erythroid iron utilization and proposed to function as an essential mitochondrial iron importer. Indirect reporter assays in isolated yeast mitochondria indicated that the Mrs3/4 proteins are involved in mitochondrial Fe²⁺ utilization or transport under iron-limiting conditions. To have a more direct test for Mrs3/4p mediated iron uptake into mitochondria we studied iron (II) transport across yeast inner mitochondrial membrane vesicles (SMPs) using the iron-sensitive fluorophore PhenGreen SK (PGSK). Wild-type SMPs showed rapid uptake of Fe²⁺ which was driven by the external Fe²⁺ concentration and stimulated by acidic pH. SMPs from the double deletion strain mrs3/4Δ failed to show this rapid Fe²⁺ uptake, while SMPs from cells overproducing Mrs3/4p exhibited increased Fe²⁺ uptake rates. Cu²⁺ was transported at similar rates as Fe²⁺, while other divalent cations, such as Zn²⁺ and Cd²⁺ apparently did not serve as substrates for the Mrs3/4p transporters. We conclude that the carrier proteins Mrs3p and Mrs4p transport Fe²⁺ across the inner mitochondrial membrane. Their activity is dependent on the pH gradient and it is stimulated by iron shortage.     

Impairment of the proteasome is crucial for glucose-induced lifespan reduction in the mev-1 mutant of Caenorhabditis elegans

Hyperglycemia is a hallmark of diabetes that is associated with diabetic complications and a reduction of lifespan. Using the mev-1 mutant of the nematode Caenorhabditis elegans we here tried to identify molecular mechanisms underlying the lifespan reducing effects of glucose. The lowest glucose concentration tested (10 mM) caused a significant lifespan reduction at 37 °C and was used to assess effects on mitochondrial efficiency, formation of protein carbonyls and levels of methylglyoxal, a precursor of advanced glycation end products (AGEs). RNA-interference (RNAi) served the identification of targets for glucose-induced damage. Levels of protein carbonyls and AGEs remained unaffected by 10 mM glucose. Levels of reactive oxygen species inside mitochondria were increased but their scavenging by ascorbic acid did not influence lifespan reduction by glucose. Mitochondrial efficiency was reduced by glucose as concluded from a lowered P/O-ratio. A reduced lifespan of mev-1 that was unaffected by the addition of glucose resulted from RNAi of key players of mitochondrial unfolded protein response. Besides increased accumulation of misfolded proteins, reduced proteasomal degradation caused the same phenotype as was evidenced by RNAi for UBQ-1 or UBA-1. Accumulation of functionally impaired proteins, e.g. in mitochondria, underlies the lifespan reducing effects of glucose. Our study provides evidence for a crucial importance of the proteostasis network for lifespan regulation which is impaired by glucose.     

Yeast mitochondrial protein, Nfs1p, coordinately regulates iron-sulfur cluster proteins, cellular iron uptake, and iron distribution.

Nfs1p is the yeast homolog of the bacterial proteins NifS and IscS, enzymes that release sulfur from cysteine for iron-sulfur cluster assembly. Here we show that the yeast mitochondrial protein Nfs1p regulates cellular and mitochondrial iron homeostasis. A strain of Saccharomyces cerevisiae, MA14, with a missense NFS1 allele (I191S) was isolated in a screen for altered iron-dependent gene regulation. This mutant exhibited constitutive up-regulation of the genes of the cellular iron uptake system, mediated through effects on the Aft1p iron-regulatory protein. Iron accumulating in the mutant cells was retained in the mitochondrial matrix while, at the same time, iron-sulfur proteins were deficient. In this work, the yeast protein was localized to mitochondria, and the gene was shown to be essential for viability. Furthermore, Nfs1p in the MA14 mutant was found to be markedly decreased, suggesting that this low protein level produced the observed regulatory effects. This hypothesis was confirmed by experiments in which expression of wild-type Nfs1p from a regulated galactose-induced promoter was turned off, leading to recapitulation of the iron regulatory phenotypes characteristic of the MA14 mutant. These phenotypes include decreases in iron-sulfur protein activities coordinated with increases in cellular iron uptake and iron distribution to mitochondria.     

Mitochondria regulation in ferroptosis

Ferroptosis is recognized as a new form of regulated cell death which is initiated by severe lipid peroxidation relying on reactive oxygen species (ROS) generation and iron overload. This iron-dependent cell death manifests evident morphological, biochemical and genetic differences from other forms of regulated cell death, such as apoptosis, autophagy, necrosis and pyroptosis. Ferroptosis was primarily characterized by condensed mitochondrial membrane densities and smaller volume than normal mitochondria, as well as the diminished or vanished of mitochondria crista and outer membrane ruptured. Mitochondria take the center role in iron metabolism, as well as substance and energy metabolism as it’s the major organelle in iron utilization, catabolic and anabolic pathways. Interference of key regulators of mitochondrial lipid metabolism (e.g., ASCF2 and CS), iron homeostasis (e.g., ferritin, mitoferrin1/2 and NEET proteins), glutamine metabolism and other signaling pathways make a difference to ferroptotic sensitivity. Targeted induction of ferroptosis was also considered as a potential therapeutic strategy to some oxidative stress diseases, including neurodegenerative disorders, ischemia-reperfusion injury, traumatic spinal cord injury. However, the pertinence between mitochondria and ferroptosis is still in dispute. Here we systematic elucidate the morphological characteristics and metabolic regulation of mitochondria in the regulation of ferroptosis.     

Mitochondrial quality control during inheritance is associated with lifespan and mother-daughter age asymmetry in budding yeast

Fluorescence loss in photobleaching experiments and analysis of mitochondrial function using superoxide and redox potential biosensors revealed that mitochondria within individual yeast cells are physically and functionally distinct. Mitochondria that are retained in mother cells during yeast cell division have a significantly more oxidizing redox potential and higher superoxide levels compared to mitochondria in buds. Retention of mitochondria with more oxidizing redox potential in mother cells occurs to the same extent in young and older cells and can account for the age-associated decline in total cellular mitochondrial redox potential in yeast as they age from 0 to 5 generations. Deletion of Mmr1p, a member of the DSL1 family of tethering proteins that localizes to mitochondria at the bud tip and is required for normal mitochondrial inheritance, produces defects in mitochondrial quality control and heterogeneity in replicative lifespan (RLS). Long-lived mmr1Δ cells exhibit prolonged RLS, reduced mean generation times, more reducing mitochondrial redox potential and lower mitochondrial superoxide levels compared to wild-type cells. Short-lived mmr1Δ cells exhibit the opposite phenotypes. Moreover, short-lived cells give rise exclusively to short-lived cells, while the majority of daughters of long-lived cells are long lived. These findings support the model that the mitochondrial inheritance machinery promotes retention of lower-functioning mitochondria in mother cells and that this process contributes to both mother-daughter age asymmetry and age-associated declines in cellular fitness.     

MTABC3, a novel mitochondrial ATP-binding cassette protein involved in iron homeostasis

Atm1p, a mitochondrial half-type ATP-binding cassette (ABC) protein in Saccharomyces cerevisiae, transports a precursor of the iron-sulfur (Fe/S) cluster from mitochondria to the cytosol. We have identified a novel half-type human ABC protein, designating it MTABC3 (mammalian mitochondrial ABC protein 3). MTABC3 mRNA is ubiquitously expressed in all of the rat and human tissues examined. MTABC3 protein is shown to be present in the mitochondria, as assessed by immunoblot analysis and confocal microscopic analysis of subcellular fractions of Chinese hamster ovary cells stably expressing MTABC3. Accumulation of iron in the mitochondria, mitochondrial DNA damage, and respiratory dysfunction in the yeast ATM1 mutant strain (atm1-1 mutant cells) were almost fully reversed by expressing MTABC3 in these mutant cells. These results indicate that MTABC3 is a novel ortholog of the yeast and suggest an important role in mitochondrial function. Interestingly, the human MTABC3 gene has been mapped to chromosome 2q36, a region within the candidate locus for lethal neonatal metabolic syndrome, a disorder of the mitochondrial function associated with iron metabolism, indicating that MTABC3 is a candidate gene for this disorder.     

Ancestral roles of eukaryotic frataxin: mitochondrial frataxin function and heterologous expression of hydrogenosomal Trichomonas homologues in trypanosomes

Frataxin is a small conserved mitochondrial protein; in humans, mutations affecting frataxin expression or function result in Friedreich's ataxia. Much of the current understanding of frataxin function comes from informative studies with yeast models, but considerable debates remain with regard to the primary functions of this ubiquitous protein. We exploit the tractable reverse genetics of Trypanosoma brucei in order to specifically consider the importance of frataxin in an early branching lineage. Using inducible RNAi, we show that frataxin is essential in T. brucei and that its loss results in reduced activity of the marker Fe–S cluster-containing enzyme aconitase in both the mitochondrion and cytosol. Activities of mitochondrial succinate dehydrogenase and fumarase also decreased, but the concentration of reactive oxygen species increased. Trypanosomes lacking frataxin also exhibited a low mitochondrial membrane potential and reduced oxygen consumption. Crucially, however, iron did not accumulate in frataxin-depleted mitochondria, and as T. brucei frataxin does not form large complexes, it suggests that it plays no role in iron storage. Interestingly, RNAi phenotypes were ameliorated by expression of frataxin homologues from hydrogenosomes of another divergent protist Trichomonas vaginalis . Collectively, the data suggest trypanosome frataxin functions primarily only in Fe–S cluster biogenesis and protection from reactive oxygen species.     

Adrenodoxin reductase homolog (Arh1p) of yeast mitochondria required for iron homeostasis

Arh1p is an essential mitochondrial protein of yeast with reductase activity. Here we show that this protein is involved in iron metabolism. A yeast strain was constructed in which the open reading frame was placed under the control of a galactose-regulated promoter. Protein expression was induced by galactose and repressed to undetectable levels in the absence of galactose, although cells grew quite well in the absence of inducer. Under noninducing conditions, cellular iron uptake was dysregulated, exhibiting a failure to repress in response to medium iron. Iron trafficking within the cell was also disturbed. Exposure of Arh1p-depleted cells to increasing iron concentrations during growth led to drastic increases in mitochondrial iron, indicating a loss of homeostatic control. Activity of aconitase, a prototype Fe-S protein, was deficient at all concentrations of mitochondrial iron, although the protein level was unaltered. Heme protein deficiencies were exacerbated in the iron-loaded mitochondria, suggesting a toxic side effect of accumulated iron. Finally, a time course correlated the cellular depletion of Arh1p with the coordinated appearance of various mutant phenotypes including dysregulated cellular iron uptake, deficiency of Fe-S protein activities in mitochondria and cytoplasm, and deficiency of hemoproteins. Thus, Arh1p is required for control of cellular and mitochondrial iron levels and for the activities of Fe-S cluster proteins.     

Expression and function of heterologous forms of malate dehydrogenase in yeast.

The structure of the tricarboxylic acid cycle enzyme malate dehydrogenase is highly conserved in various organisms. To test the extent of functional conservation, the rat mitochondrial enzyme and the enzyme from Escherichia coli were expressed in a strain of Saccharomyces cerevisiae containing a disruption of the chromosomal MDH1 gene encoding yeast mitochondrial malate dehydrogenase. The authentic precursor form of the rat enzyme, expressed using a yeast promoter and a multicopy plasmid, was found to be efficiently targeted to yeast mitochondria and processed to a mature active form in vivo. Mitochondrial levels of the polypeptide and malate dehydrogenase activity were found to be similar to those for MDH1 in wild-type yeast cells. Efficient expression of the E. coli mdh gene was obtained with multicopy plasmids carrying gene fusions encoding either a mature form of the procaryotic enzyme or a precursor form with the amino terminal mitochondrial targeting sequence from yeast MDH1. Very low levels of mitochondrial import and processing of the precursor form were obtained in vivo and activity could be demonstrated for only the expressed precursor fusion protein. Results of in vitro import experiments suggest that the percursor form of the E. coli protein associates with yeast mitochondria but is not efficiently internalized. Respiratory rates measured for isolated yeast mitochondria containing the mammalian or procaryotic enzyme were, respectively, 83 and 62% of normal, suggesting efficient delivery of NADH to the respiratory chain. However, expression of the heterologous enzymes did not result in full complementation of growth phenotypes associated with disruption of the yeast MDH1 gene.     

Mitoferrin modulates iron toxicity in a Drosophila model of Friedreich׳s ataxia

Friedreich׳s ataxia is the most important recessive ataxia in the Caucasian population. Loss of frataxin expression affects the production of iron–sulfur clusters and, therefore, mitochondrial energy production. One of the pathological consequences is an increase of iron transport into the mitochondrial compartment leading to a toxic accumulation of reactive iron. However, the mechanism underlying this inappropriate mitochondrial iron accumulation is still unknown. Control and frataxin-deficient flies were fed with an iron diet in order to mimic an iron overload and used to assess various cellular as well as mitochondrial functions. We showed that frataxin-deficient flies were hypersensitive toward dietary iron and developed an iron-dependent decay of mitochondrial functions. In the fly model exhibiting only partial frataxin loss, we demonstrated that the inability to activate ferritin translation and the enhancement of mitochondrial iron uptake via mitoferrin upregulation were likely the key molecular events behind the iron-induced phenotype. Both defects were observed during the normal process of aging, confirming their importance in the progression of the pathology. In an effort to further assess the importance of these mechanisms, we carried out genetic interaction studies. We showed that mitoferrin downregulation improved many of the frataxin-deficient conditions, including nervous system degeneration, whereas mitoferrin overexpression exacerbated most of them. Taken together, this study demonstrates the crucial role of mitoferrin dysfunction in the etiology of Friedreich׳s ataxia and provides evidence that impairment of mitochondrial iron transport could be an effective treatment of the disease.     

过表达 PINK1改善SCA3/MJD转基因果蝇模型的线粒体功能

脊髓小脑性共济失调3型（SCA3/MJD）,是一种因致病基因MJD1编码区内CAG异常重复扩增所致的常染色体显性遗传迟发性神经退行性疾病. 已知PINK1蛋白可通过抗氧化稳定线粒体,阻止帕金森疾病的发生,但其在SCA3/MJD中的作用尚不清楚. 本文旨在探索过表达PINK1对SCA3/MJD转基因果蝇模型的保护作用.本研究利用Mhc-Gal4启动子表达致病蛋白质片段（MJDtr-Q78）获得SCA3/MJD果蝇模型,分别运用过表达PINK1和RNA干扰PINK1研究其在SCA3/MJD果蝇模型中的功能.结果显示,疾病模型组翅膀异常率增高,线粒体呈过度融合状态,ATP值降低;PINK1 RNA干扰组翅膀异常率明显增高,线粒体呈显著过度融合状态,ATP值明显降低;PINK1过表达组翅膀异常率明显降低,线粒体清晰、完整,ATP值明显升高.本文的结果提示, 过表达PINK1对SCA3/MJD转基因果蝇模型起保护作用,而RNA干扰PINK1表达加重SCA3/MJD转基因果蝇模型病情.PINK1在SCA3/MJD果蝇模型中的功能可能通过改善细胞内线粒体功能实现.     

Functional characterization of AtATM1, AtATM2, and AtATM3, a subfamily of Arabidopsis half-molecule ATP-binding cassette transporters implicated in iron homeostasis

The functional capabilities of one of the smallest subfamilies of ATP-binding cassette transporters from Arabidopsis thaliana, the AtATMs, are described. Designated AtATM1, AtAATM2, and AtATM3, these half-molecule ABC proteins are homologous to the yeast mitochondrial membrane protein ATM1 (ScATM1), which is clearly implicated in the export of mitochondrially synthesized iron/sulfur clusters. Yeast ATM1-deficient (atm1) mutants grow very slowly (have a petite phenotype), are respiration-deficient, accumulate toxic levels of iron in their mitochondria, and show enhanced compensatory high affinity iron uptake. Of the three Arabidopsis ATMs, AtATM3 bears the closest functional resemblance to ScATM1. Heterologously expressed AtATM3 is not only able to complement the yeast atm1 petite phenotype but is also able to suppress the constitutively high capacity for high affinity iron uptake associated with loss of the chromosomal copy of ScATM1, abrogate intra-mitochondrial iron hyperaccumulation, and restore mitochondrial respiratory function and cytochrome c levels. By comparison, AtATM1 only weakly suppresses the atm1 phenotype, and AtATM2 exerts little or no suppressive action but instead is toxic when expressed in this system. The differences between AtATM3 and AtATM1 are maintained after exchanging their target peptides, and these proteins as well as AtATM2 colocalize with the mitochondrial fluor MitoTracker Red when expressed in yeast as GFP fusions. Although its toxicity when heterologously expressed in yeast, except when fused with GFP, precludes the functional analysis of native AtATM2, a common function, mitochondrial export of Fe/S clusters or their precursors for the assembly of cytosolic Fe/S proteins, is inferred for AtATM3 and AtATM1.     

A C. elegans Model for Mitochondrial Fatty Acid Synthase II: The Longevity-Associated Gene W09H1.5/mecr-1 Encodes a 2-trans-Enoyl-Thioester Reductase

Our recognition of the mitochondria as being important sites of fatty acid biosynthesis is continuously unfolding, especially in light of new data becoming available on compromised fatty acid synthase type 2 (FASII) in mammals. For example, perturbed regulation of murine 17β-HSD8 encoding a component of the mitochondrial FASII enzyme 3-oxoacyl-thioester reductase is implicated in polycystic kidney disease. In addition, over-expression in mice of the Mecr gene coding for 2-trans-enoyl-thioester reductase, also of mitochondrial FASII, leads to impaired heart function. However, mouse knockouts for mitochondrial FASII have hitherto not been reported and, hence, there is a need to develop alternate metazoan models such as nematodes or fruit flies. Here, the identification of Caenorhabditis elegans W09H1.5/MECR-1 as a 2-trans-enoyl-thioester reductase of mitochondrial FASII is reported. To identify MECR-1, Saccharomyces cerevisiae etr1Δ mutant cells were employed that are devoid of mitochondrial 2-trans-enoyl-thioester reductase Etr1p. These yeast mutants fail to synthesize sufficient levels of lipoic acid or form cytochrome complexes, and cannot respire or grow on non-fermentable carbon sources. A mutant yeast strain ectopically expressing nematode mecr-1 was shown to contain reductase activity and resemble the self-complemented mutant strain for these phenotype characteristics. Since MECR-1 was not intentionally targeted for compartmentalization using a yeast mitochondrial leader sequence, this inferred that the protein represented a physiologically functional mitochondrial 2-trans-enoyl-thioester reductase. In accordance with published findings, RNAi-mediated knockdown of mecr-1 in C. elegans resulted in life span extension, presumably due to mitochondrial dysfunction. Moreover, old mecr-1(RNAi) worms had better internal organ appearance and were more mobile than control worms, indicating a reduced physiological age. This is the first report on RNAi work dedicated specifically to curtailing mitochondrial FASII in metazoans. The availability of affected survivors will help to position C. elegans as an excellent model for future pursuits in the emerging field of mitochondrial FASII research.     

Two modes of mitochondrial dysfunction lead independently to lifespan extension in Caenorhabditis elegans

In Caenorhabditis elegans, longevity is increased by a partial loss‐of‐function mutation in the mitochondrial complex III subunit gene isp‐1. Longevity is also increased by RNAi against the expression of a variety of mitochondrial respiratory chain genes, including isp‐1, but it is unknown whether the isp‐1(qm150) mutation and the RNAi treatments trigger the same underlying mechanisms of longevity. We have identified nuo‐6(qm200), a mutation in a conserved subunit of mitochondrial complex I (NUDFB4). The mutation reduces the function of complex I and, like isp‐1(qm150), results in low oxygen consumption, slow growth, slow behavior, and increased lifespan. We have compared the phenotypes of nuo‐6(qm200) to those of nuo‐6(RNAi) and found them to be distinct in crucial ways, including patterns of growth and fertility, behavioral rates, oxygen consumption, ATP levels, autophagy, and resistance to paraquat, as well as expression of superoxide dismutases, mitochondrial heat‐shock proteins, and other gene expression markers. RNAi treatments appear to generate a stress and autophagy response, while the genomic mutation alters electron transport and reactive oxygen species metabolism. For many phenotypes, we also compared isp‐1(qm150) to isp‐1(RNAi) and found the same pattern of differences. Most importantly, we found that, while the lifespan of nuo‐6, isp‐1 double mutants is not greater than that of the single mutants, the lifespan increase induced by nuo‐6(RNAi) is fully additive to that induced by isp‐1(qm150), and the increase induced by isp‐1(RNAi) is fully additive to that induced by nuo‐6(qm200). Our results demonstrate that distinct and separable aspects of mitochondrial biology affect lifespan independently.     

Deletion of the mitochondrial carrier genes MRS3 and MRS4 suppresses mitochondrial iron accumulation in a yeast frataxin-deficient strain

The mitochondrial solute carriers Mrs3p and Mrs4p were originally isolated as multicopy suppressors of intron splicing defects. We show here that MRS4 is co-regulated with the iron regulon genes, and up-regulated in a strain deficient for Yfh1p, the yeast homologue of human frataxin. Using in vivo 55Fe cell radiolabeling we show that in glucose-grown cells mitochondrial iron accumulation is 5-15 times higher in deltaYFH1 than in wild-type strain. However, although in a deltaYFH1deltaMRS3deltaMRS4 strain, the intracellular 55Fe content is extremely high, the mitochondrial iron concentration is decreased to almost wild-type levels. Moreover, deltaYFH1deltaMRS3deltaMRS4 cells grown in high iron media do not lose their mitochondrial genome. Conversely, a deltaYFH1 strain overexpressing MRS4 has an increased mitochondrial iron content and no mitochondrial genome. Therefore, MRS4 is required for mitochondrial iron accumulation in deltaYFH1 cells. Expression of the iron regulon and intracellular 55Fe content are higher in a deltaMRS3deltaMRS4 strain than in the wild type. Nevertheless, the mitochondrial 55Fe content, a balance between iron uptake and exit, is decreased by a factor of two. Moreover, 55Fe incorporation into heme by ferrochelatase is increased in an MRS4-overexpressing strain. The function of MRS4 in iron import into mitochondria is discussed.