Frataxin and mitochondrial carrier proteins, Mrs3p and Mrs4p, cooperate in providing iron for heme synthesis

Frataxin is a conserved mitochondrial protein implicated in cellular iron metabolism. Deletion of the yeast frataxin homolog (YFH1) was combined with deletions of MRS3 and MRS4, mitochondrial carrier proteins implicated in iron homeostasis. As previously reported, the Deltayfh1 mutant accumulated iron in mitochondria, whereas the triple mutant (DeltaDeltaDelta) did not. When wild-type, Deltamrs3/4, Deltayfh1, and DeltaDeltaDelta strains were incubated anaerobically, all strains were devoid of heme and protected from iron and oxygen toxicity. The cultures were then shifted to air for a short time (4-5 h) or a longer time (15 h), and the evolving mutant phenotypes were analyzed (heme-dependent growth, total heme, cytochromes, heme proteins, and iron levels). A picture emerges from these data of defective heme formation in the mutants, with a markedly more severe defect in the DeltaDeltaDelta than in the individual Deltamrs3/4 or Deltayfh1 mutants (a "synthetic" defect in the genetic sense). The defect(s) in heme formation could be traced to lack of iron. Using a real time assay of heme biosynthesis, porphyrin precursor and iron were presented to permeabilized cells, and the appearance and disappearance of fluorescent porphyrins were followed. The Mrs3/4p carriers were required for rapid iron transport into mitochondria for heme synthesis, whereas there was also evidence for an alternative slower system. A different role for Yfh1p was observed under conditions of low mitochondrial iron and aerobic growth (revealed in the DeltaDeltaDelta), acting to protect bioavailable iron within mitochondria and to facilitate its use for heme synthesis.     

Protein transport machineries for precursor translocation across the inner mitochondrial membrane

The mitochondrial inner membrane has a central function for the energy metabolism of the cell. The respiratory chain generates a proton gradient across the inner mitochondrial membrane, which is used to produce ATP by the F1Fo-ATPase. To maintain the electrochemical gradient, the inner membrane represents an efficient permeability barrier for small molecules. Nevertheless, metabolites as well as polypeptide chains need to be transported across the inner membrane while the electrochemical gradient is retained. While specialized metabolite carrier proteins mediate the transport of small molecules, dedicated protein translocation machineries in the inner mitochondrial membrane (so called TIM complexes) transport precursor proteins across the inner membrane. Here we describe the organization of the TIM complexes and discuss the current models as to how they mediate the posttranslational import of proteins across and into the inner mitochondrial membrane.     

The proton leak across the mitochondrial inner membrane

The proton conductance of the mitochondrial inner membrane increases at high protonmotive force in isolated mitochondria and in mitochondria in situ in rat hepatocytes. Quantitative analysis of its importance shows that about 20-30% of the oxygen consumption by resting hepatocytes is used to drive a heat-producing cycle of proton pumping by the respiratory chain and proton leak back to the matrix. The flux control coefficient of the proton leak pathway over respiration rate varies between 0.9 and zero in mitochondria depending on the rate of respiration, and has a value of about 0.2 in hepatocytes. Changes in the proton leak pathway in situ will therefore change respiration rate. Mitochondria isolated from hypothyroid animals have decreased proton leak pathway, causing slower state 4 respiration rates. Hepatocytes from hypothyroid rats also have decreased proton leak pathway, and this accounts for about 30% of the decrease in hepatocyte respiration rate. Mitochondrial proton leak may be a significant contributor to standard metabolic rate in vivo.     

The bacterial magnesium transporter CorA can functionally substitute for its putative homologue Mrs2p in the yeast inner mitochondrial membrane.

The yeast nuclear gene MRS2 encodes a protein of 54 kDa, the presence of which has been shown to be essential for the splicing of group II intron RNA in mitochondria and, independently, for the maintenance of a functional respiratory system. Here we show that the MRS2 gene product (Mrs2p) is an integral protein of the inner mitochondrial membrane. It appears to be inserted into this membrane by virtue of two neighboring membrane spanning domains in its carboxyl-terminal half. A large amino-terminal and a shorter carboxyl-terminal part are likely to be exposed to the matrix space. Structural features and a short sequence motif indicate that Mrs2p may be related to the bacterial CorA Mg2+ transporter. In fact, overexpression of the CorA gene in yeast partially suppresses the pet- phenotype of an mrs2 disrupted yeast strain. Disruption of the MRS2 gene leads to a significant decrease in total magnesium content of mitochondria which is compensated for by the overexpression of the CorA gene. Mutants lacking or overproducing Mrs2p exhibit phenotypes consistent with the involvement of Mrs2p in mitochondrial Mg2+ homeostasis.     

A specific role of the yeast mitochondrial carriers MRS3/4p in mitochondrial iron acquisition under iron-limiting conditions

The yeast genes MRS3 and MRS4 encode two members of the mitochondrial carrier family with high sequence similarity. To elucidate their function we utilized genome-wide expression profiling and found that both deletion and overexpression of MRS3/4 lead to up-regulation of several genes of the "iron regulon." We therefore analyzed the two major iron-utilizing processes, heme formation and Fe/S protein biosynthesis in vivo, in organello (intact mitochondria), and in vitro (mitochondrial extracts). Radiolabeling of yeast cells with 55Fe revealed a clear correlation between MRS3/4 expression levels and the efficiency of these biosynthetic reactions indicating a role of the carriers in utilization and/or transport of iron in vivo. Similar effects on both heme formation and Fe/S protein biosynthesis were seen in organello using mitochondria isolated from cells grown under iron-limiting conditions. The correlation between MRS3/4 expression levels and the efficiency of the two iron-utilizing processes was lost upon detergent lysis of mitochondria. As no significant changes in the mitochondrial membrane potential were observed upon overexpression or deletion of MRS3/4, our results suggest that Mrs3/4p carriers are directly involved in mitochondrial iron uptake. Mrs3/4p function in mitochondrial iron transport becomes evident under iron-limiting conditions only, indicating that the two carriers do not represent the sole system for mitochondrial iron acquisition.     

The yeast dynamin-like protein, Mgm1p, functions on the mitochondrial outer membrane to mediate mitochondrial inheritance

The mdm17 mutation causes temperature-dependent defects in mitochondrial inheritance, mitochondrial morphology, and the maintenance of mitochondrial DNA in the yeast Saccharomyces cerevisiae. Defects in mitochondrial transmission to daughter buds and changes in mitochondrial morphology were apparent within 30 min after shifting cells to 37 degrees C, while loss of the mitochondrial genome occurred after 4-24 h at the elevated temperature. The mdm17 lesion mapped to MGM1, a gene encoding a dynamin-like GTPase previously implicated in mitochondrial genome maintenance, and the cloned MGM1 gene complements all of the mdm17 mutant phenotypes. Cells with an mgm1-null mutation displayed aberrant mitochondrial inheritance and morphology. A version of mgm1 mutated in a conserved residue in the putative GTP-binding site was unable to complement any of the mutant defects. It also caused aberrant mitochondrial distribution and morphology when expressed at high levels in cells that also contained a wild-type copy of the gene. Mgm1p was localized to the mitochondrial outer membrane and fractionated as a component of a high molecular weight complex. These results indicate that Mgm1p is a mitochondrial inheritance and morphology component that functions on the mitochondrial surface.     

The dynamic dimerization of the yeast ADP/ATP carrier in the inner mitochondrial membrane is affected by conserved cysteine residues

The ADP/ATP carrier (AAC) that facilitates the translocation of ATP made in mitochondria is inserted at the inner mitochondrial membrane by the TIM10-TIM22 protein import system. Here we addressed the state of the AAC precursor during insertion (stage IV of import) and identified residues of the carrier important for dimerization. By a combination of (i) import of a mix of His-tagged and untagged versions of AAC either 35S-labeled or unlabeled, (ii) import of a tandem covalent dimer AAC into wild-type mitochondria, and (iii) import of monomeric AAC into mitochondria expressing only the tandem covalent dimer AAC, we found that the stage IV intermediate is a monomer, and this stage is probably the rate-limiting step of insertion in the membrane. Subsequent dimerization occurs extremely rapidly (within less than a minute). The incoming monomer dimerizes with monomeric endogenous AAC suggesting that the AAC dimer is very dynamic. Conserved Cys residues were found not to affect insertion significantly, but they are crucial for the dimerization process to obtain a functional carrier.     

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.     

Mrs3p, Mrs4p, and frataxin provide iron for Fe-S cluster synthesis in mitochondria

Yeast Mrs3p and Mrs4p are evolutionarily conserved mitochondrial carrier proteins that transport iron into mitochondria under some conditions. Yeast frataxin (Yfh1p), the homolog of the human protein implicated in Friedreich ataxia, is involved in iron homeostasis. However, its precise functions are controversial. Anaerobically grown triple mutant cells (Deltamrs3/4/Deltayfh1) displayed a severe growth defect corrected by in vivo iron supplementation. Because anaerobically grown cells do not synthesize heme, and they do not experience oxidative stress, this growth defect was most likely due to Fe-S cluster deficiency. Fe-S cluster formation was assessed in anaerobically grown cells shifted to air for a brief period. In isolated mitochondria, Fe-S clusters were detected on newly imported yeast ferredoxin precursor and on endogenous aconitase by means of [35S]cysteine labeling and native gel separation. New cluster formation was dependent on iron addition to mitochondria, and the iron concentration dependence was shifted dramatically upward in the Deltamrs3/4 mutant, indicating a role of Mrs3/4p in iron transport. The frataxin mutant strain lacked protein import capacity because of low mitochondrial membrane potential, although this was partially restored by growth in the presence of high iron. Under these conditions, a kinetic defect in new Fe-S cluster formation was still noted. Import of frataxin into frataxin-minus isolated mitochondria promptly corrected the Fe-S cluster assembly defect without further iron addition. These findings show that Mrs3/4p transports iron into mitochondria, whereas frataxin makes iron already within mitochondria available for Fe-S cluster synthesis.     

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.     

The N-terminal cleavable extension of plant carrier proteins is responsible for efficient insertion into the inner mitochondrial membrane

A subset of mitochondrial carrier proteins from plants contain a cleavable N-terminal extension. We have used a reconstituted protein import assay system into intermembrane space-depleted mitochondria to study the role of the cleavable extension in the carrier import pathway. Insertion of carrier proteins into the inner membrane can be stimulated by the addition of a soluble intermembrane space fraction isolated from plant mitochondria. Greater stimulation of import of the adenine nucleotide carrier (ANT) and phosphate carrier (Pic), which contain N-terminal cleavable extensions, was observed compared to the import of the oxoglutarate malate carrier (OMT), which does not contain a cleavable extension. Removal of the N-terminal cleavable extension from ANT and Pic resulted in loss of stimulation of insertion into the inner membrane. Conversely, addition of the N-terminal extension from ANT or Pic to OMT resulted in significantly enhanced insertion into the inner membrane. The polytopic inner membrane proteins TIM17 and TIM23 that are imported via the carrier import pathway contain no cleavable extension, displayed high-level stimulation of insertion into the inner membrane by addition of the intermembrane space fraction. Addition of the N-terminal cleavable extension from carrier proteins to TIM23 enhanced insertion of TIM23 into the inner membrane even in the absence of the soluble intermembrane space fraction. Together, these results demonstrate that the cleavable N-terminal extensions present on carrier proteins from plants are required for efficient insertion into the inner mitochondrial membrane, and that they can stimulate insertion of any carrier-like protein into the inner membrane.     

Mechanism of glutamate-aspartate translocation across the mitochondrial inner membrane.

In order to study the mechanism of the glutamate-aspartate translocator, rat liver mitochondria were loaded with either glutamate or aspartate. In the presence of ascorbate plus tetramethyl-p-phenylenediamine as an electron donor at the third energy conservation site, exchange of external glutamate for matrix aspartate is highly favored over the reverse exchange. In the absence of an energy source, although the asymmetry of the exchange rates is much smaller, it is still observable. Further studies have shown that the proton uptake accompanying influx of glutamate in exchange for aspartate efflux occurs by protonation of a group on the carrier (pK = 7.9) at the external side of the inner mitochondrial membrane, followed by deprotonation at the matrix surface. It is postulated that glutamate binds to the protonated form of the carrier and aspartate to the deprotonated form. Because of the alkaline pK, aspartate efflux is inhibited with increased matrix [H⁺] due to limitation of the availability of deprotonated carrier for aspartate binding. For the reverse exchange, aspartate uptake is inhibited by increasing external [H⁺]. Thus the rate of aspartate uptake by mitochondria is apparently impeded both by a proton motive force (Δp) unfavorable to entry of ions with net negative charge as well as by the small proportion of deprotonated carrier at the outer surface of the membrane. This conclusion is further illustrated by inhibition of the aspartate-aspartate exchange with increased [H⁺] and by addition of an energy source. The glutamate-glutamate exchange, however, showed a slight stimulation by increased [H⁺] and was unaffected by the energy state.The model initially proposed for the carrier, in which a neutral glutamate-carrier complex exchanges for a negatively charged aspartate-carrier complex, is tested further. Glutamate uptake was noncompetitively inhibited by external aspartate, which indicates that aspartate and glutamate bind to separate forms of the carrier. Intramitochrondrial glutamate at a concentration of 18 mm, however, had no effect on aspartate efflux. Arrhenius plots for the glutamate-aspartate and aspartate-glutamate exchanges were linear over the range of temperatures tested (1–35 °C and 5–25 °C, respectively) and provided an average value of 14.3 kcal/mol for the energy of activation. In addition, there appear to be two pools, exchangeable and nonexchangeable, of matrix aspartate available to the translocator, since extramitochondrial radiolabeled aspartate can equilibrate only with unlabeled matrix aspartate at low aspartate loading (1–2 nmol of aspartate/mg of protein). The physiological significance of the data is discussed.     

The inner membrane protein Mdm33 controls mitochondrial morphology in yeast

Mitochondrial distribution and morphology depend on MDM33, a Saccharomyces cerevisiae gene encoding a novel protein of the mitochondrial inner membrane. Cells lacking Mdm33 contain ring-shaped, mostly interconnected mitochondria, which are able to form large hollow spheres. On the ultrastructural level, these aberrant organelles display extremely elongated stretches of outer and inner membranes enclosing a very narrow matrix space. Dilated parts of Delta mdm33 mitochondria contain well-developed cristae. Overexpression of Mdm33 leads to growth arrest, aggregation of mitochondria, and generation of aberrant inner membrane structures, including septa, inner membrane fragments, and loss of inner membrane cristae. The MDM33 gene is required for the formation of net-like mitochondria in mutants lacking components of the outer membrane fission machinery, and mitochondrial fusion is required for the formation of extended ring-like mitochondria in cells lacking the MDM33 gene. The Mdm33 protein assembles into an oligomeric complex in the inner membrane where it performs homotypic protein-protein interactions. Our results indicate that Mdm33 plays a distinct role in the mitochondrial inner membrane to control mitochondrial morphology. We propose that Mdm33 is involved in fission of the mitochondrial inner membrane.     

A yeast mitochondrial ATPase inhibitor interacts with three proteins that are easy to dissociate from the mitochondrial inner membrane

A mitochondrial ATPase inhibitor is a 7.4 kDa protein that regulates the catalytic activity of ATP synthase (F(1)F(o)-ATPase). In the present study, we examined the binding sites of the inhibitor on the mitochondrial membrane using chemical cross-linkers, disuccinimidyl suberate (DSS) and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). Most of the inhibitors were recovered from the inner membrane fraction of mitochondria, indicating that the inhibitor binds to the membrane. Seven different cross-linked products that reacted with the antibody against the inhibitor were detected. The apparent molecular masses of the products were 61, 58, 47, 41, 28, 27, and 26 kDa. The 61 and 58 kDa products were attributed to the inhibitor+alpha and inhibitor+beta adducts on immunoblotting. The proteins cross-linked to the inhibitor in the 28, 27, and 26 kDa products were distinguished from subunit 4 (23 kDa), oligomycin sensitivity conferring protein (21 kDa), and subunit d (20 kDa) of F(1)F(o)-ATPase by analysis of the cross-linked products of mutant mitochondria in which the three proteins were replaced by hemagglutinin-tagged versions. The 28, 27, and 26 kDa products could be gradually dissociated from the mitochondrial membrane by increasing the salt concentration. These results shows that the endogenous inhibitor binds not only to the catalytic part of the enzyme, but also to the 19-21 kDa proteins that loosely associate with the mitochondrial inner membrane.     

Identification of a novel member of yeast mitochondrial Hsp70-associated motor and chaperone proteins that facilitates protein translocation across the inner membrane

Here, we report the identification of yeast 15-kD Tim15/Zim17, a new member of mitochondrial Hsp70 (mtHsp70)-associated motor and chaperone (MMC) proteins. The 15-kD MMC protein is a peripheral inner membrane protein with a zinc-finger motif. Depletion of the 15-kD protein led to impaired import of presequence-containing proteins into the matrix in vivo and in vitro. Overexpression of the 15-kD protein rescued the functional defects of mtHsp70 in ssc1-3 cells, and a fusion protein containing the 15-kD protein physically interacts with purified mtHsp70. Tim15/Zim17 therefore cooperates with mtHsp70 to facilitate import of presequence-containing proteins into the matrix.