Search

SURF1 deficiency (OMIM # 220110) causes Leigh syndrome (LS, OMIM # 256000), a mitochondrial disorder typified by stress-induced metabolic strokes, neurodevelopmental regression and progressive multisystem dysfunction. Here, we describe two novel surf1−/− zebrafish knockout models generated by CRISPR/Cas9 technology. While gross larval morphology, fertility, and survival into adulthood appeared unaffected, surf1−/− mutants manifested adult-onset ocular anomalies and decreased swimming activity, as well as classical biochemical hallmarks of human SURF1 disease, including reduced complex IV expression and enzymatic activity and increased tissue lactate. surf1−/− larvae also demonstrated oxidative stress and stressor hypersensitivity to the complex IV inhibitor, azide, which exacerbated their complex IV deficiency, reduced supercomplex formation, and induced acute neurodegeneration typical of LS including brain death, impaired neuromuscular responses, reduced swimming activity, and absent heartrate. Remarkably, prophylactic treatment of surf1−/− larvae with either cysteamine bitartrate or N-acetylcysteine, but not other antioxidants, significantly improved animal resiliency to stressor-induced brain death, swimming and neuromuscular dysfunction, and loss of heartbeat. Mechanistic analyses demonstrated cysteamine bitartrate pretreatment did not improve complex IV deficiency, ATP deficiency, or increased tissue lactate but did reduce oxidative stress and restore glutathione balance in surf1−/− animals. Overall, two novel surf1−/− zebrafish models recapitulate the gross neurodegenerative and biochemical hallmarks of LS, including azide stressor hypersensitivity that was associated with glutathione deficiency and ameliorated by cysteamine bitartrate or N-acetylcysteine therapy.

Rim2 is an unusual mitochondrial carrier protein capable of transporting both iron and pyrimidine nucleotides. Here we characterize two point mutations generated in the predicted substrate-binding site, finding that they yield disparate effects on iron and pyrimidine transport. The Rim2 (E248A) mutant was deficient in mitochondrial iron transport activity. By contrast, the Rim2 (K299A) mutant specifically abrogated pyrimidine nucleotide transport and exchange, while leaving iron transport activity largely unaffected. Strikingly, E248A preserved TTP/TTP homoexchange but interfered with TTP/TMP heteroexchange, perhaps because proton coupling was dependent on the E248 acidic residue. Rim2-dependent iron transport was unaffected by pyrimidine nucleotides. Rim2-dependent pyrimidine transport was competed by Zn2+ but not by Fe2+, Fe3+ or Cu2+. The iron and pyrimidine nucleotide transport processes displayed different salt requirements; pyrimidine transport was dependent on the salt content of the buffer whereas iron transport was salt independent. In mitochondria containing Rim2 (E248A), iron proteins were decreased, including aconitase (Fe-S), pyruvate dehydrogenase (lipoic acid containing) and cytochrome c (heme protein). Additionally, the rate of Fe-S cluster synthesis in isolated and intact mitochondria was decreased compared with the K299A mutant, consistent with the impairment of iron-dependent functions in that mutant. In summary, mitochondrial iron transport and pyrimidine transport by Rim2 occur separately and independently. Rim2 could be a bifunctional carrier protein.

Deep neural networks are trained so as to achieve a kind of the maximum overall accuracy through a learning process using given training data. Therefore, it is difficult to fix them to improve the accuracies of specific problematic classes or classes of interest that may be valuable to some users or applications. To address this issue, we propose the synaptic join method to tweak neural networks by adding certain additional synapses from the intermediate hidden layers to the output layer across layers and additionally training only these synapses, if necessary. To select the most effective synapses, the synaptic join method evaluates the performance of all the possible candidate synapses between the hidden neurons and output neurons based on the distribution of all the possible proper weights. The experimental results show that the proposed method can effectively improve the accuracies of specific classes in a controllable way.

The cysteine desulfurase Nfs1/Isd11 uses the amino acid cysteine as the substrate and its activity is absolutely required for contributing persulfide sulfur to the essential process of iron-sulfur (Fe-S) cluster assembly in mitochondria. Here we describe a novel regulatory process involving phosphorylation of Nfs1 in mitochondria. Phosphorylation enhanced cysteine desulfurase activity, while dephosphorylation decreased its activity. Nfs1 phosphopeptides were identified, and the corresponding phosphosite mutants showed impaired persulfide formation. Nfs1 pull down from mitochondria recovered an associated kinase activity, and Yck2, a kinase present in the pull down, was able to phosphorylate Nfs1 in vitro and stimulate cysteine desulfurase activity. Yck2 exhibited an eclipsed distribution in the mitochondrial matrix, although other cellular localizations have been previously described. Mitochondria lacking the Yck2 protein kinase (∆yck2) showed less phosphorylating activity for Nfs1. Compared with wild-type mitochondria, ∆yck2 mitochondria revealed slower persulfide formation on Nfs1 consistent with a role of Yck2 in regulating mitochondrial cysteine desulfurase activity. We propose that Nfs1 phosphorylation may provide a means of rapid adaptation to increased metabolic demand for sulfur and Fe-S clusters within mitochondria.

Cysteine desulfurases generate a covalent persulfide intermediate from cysteine, and this activated form of sulfur is essential for the synthesis of iron-sulfur (Fe-S) clusters. In yeast mitochondria, there is a complete machinery for Fe-S cluster synthesis, including a cysteine desulfurase, Nfs1p. Here we show that following supplementation of isolated mitochondria with [(35)S]cysteine, a radiolabeled persulfide could be detected on Nfs1p. The persulfide persisted under conditions that did not permit Fe-S cluster formation, such as nucleotide and/or iron depletion of mitochondria. By contrast, under permissive conditions, the radiolabeled Nfs1p persulfide was greatly reduced and radiolabeled aconitase was formed, indicating transfer of persulfide to downstream Fe-S cluster recipients. Nfs1p in mitochondria was found to be relatively more resistant to inactivation by N-ethylmaleimide (NEM) as compared with a prokaryotic cysteine desulfurase. Mitochondria treated with NEM (1 mM) formed the persulfide on Nfs1p but failed to generate Fe-S clusters on aconitase, likely due to inactivation of downstream recipient(s) of the Nfs1p persulfide. Thus the Nfs1p-bound persulfide as described here represents a precursor en route to Fe-S cluster synthesis in mitochondria.

Comparison and identification of mitochondrial matrix proteins from wild-type and cysteine desulfurase-defective (nfs1-14, carrying a hypomorphic allele of NFS1) yeast strains, using two-dimensional gel electrophoresis coupled to mass spectrometry analyses, revealed large changes in the amounts of various proteins. Protein spots that were specifically increased in the nfs1-14 mutant included subunits of lipoamide-containing enzyme complexes: Kgd2, Lat1, and Gcv3, subunits of the mitochondrial alpha-ketoglutarate dehydrogenase, pyruvate dehydrogenase, and glycine cleavage system complexes, respectively. Moreover the increased protein spots corresponded to lipoamide-deficient forms in the nfs1-14 mutant. The increased proteins migrated as separate, cathode-shifted spots, consistent with gain of a lysine charge due to lack of lipoamide addition. Lack of lipoylation of these proteins was further validated using an antibody specific for lipoamide-containing proteins. In addition, this antibody revealed a fourth lipoamide-containing protein, probably corresponding to the E2 component of the branched-chain keto acid dehydrogenase complex. Like the lipoamide-containing forms of Kgd2, Lat1, and Gcv3, this protein also showed decreased lipoic acid reactivity in the nfs1-14 mutant. Cysteine desulfurases, such as yeast NFS1, are required for sulfur addition to iron-sulfur clusters and other sulfur-requiring processes. The results demonstrate that Nfs1 protein is required for the proper post-translational modification of the lipoamide-containing mitochondrial subproteome in yeast and pave the road toward a thorough understanding of its precise role in lipoic acid synthesis.

Monocarboxylate transporters (MCTs) are a family of highly homologous membrane proteins that mediate the 1:1 transport of a proton and a lactate ion. In chicken, MCT3 is preferentially expressed in the retinal pigment epithelium (RPE). We have isolated the mouse MCT3 cDNA and gene and characterized the pattern of tissue expression. MCT3 is a single copy gene with a 1.8-kb transcript that encodes a protein with a predicted molecular mass of 51.5 kDa. Based on Northern hybridization analysis, MCT3 transcript was expressed in only two tissues: RPE and choroid plexus epithelium (CPE). The choroid plexus forms a barrier between the cerebrospinal fluid and fenestrated capillaries, similar to the organization of the RPE and choroidal vessels. Immunohistochemical staining demonstrated that MCT3 was restricted to the basolateral membranes of both epithelia but was more abundant in RPE than CPE. Differences in the level of protein expression were confirmed by Western blot analysis. The cloning of MCT3 identifies a specific transporter that could regulate lactate levels in fluid-bathing neuronal tissues.

Lactate transport across cell membranes is mediated by a family of proton-coupled monocarboxylate transporters (MCTs). The retinal pigment epithelium (RPE) expresses a unique member of this family, MCT3. A portion of the human MCT3 gene was cloned by polymerase chain reaction using primers designed from rat RPE MCT3 cDNA sequence. The human genomic sequence was used to design primers to clone human MCT3 cDNA and to identify a bacterial artificial chromosome clone containing the human MCT3 gene. The human MCT3 cDNA contained a 1512-nucleotide open reading frame with a deduced amino sequence 85% identical to rat MCT3. Comparison of the cDNA and genomic sequences revealed that the MCT3 gene was composed of five exons distributed over 5 kb of DNA. The exon-intron borders were conserved between the human and the chicken MCT3 genes. Using radiation hybrid mapping, the MCT3 gene was mapped to chromosome 22 between markers WI11639 and SGC30687. A search of chromosome 22 in the Sanger Centre database confirmed the location of the human MCT3 gene at 22q12.3-q13.2.

MCT3 is a monocarboxylate transporter that is specifically expressed on the basolateral membrane of retinal pigment epithelial cells (RPE). In these studies the temporal expression of MCT3 during ocular development was examined using Northern blot analysis. A 2.2 kb transcript (MCT3b) was detected in RPE by embryonic day 7 (E7) and was present throughout embryonic development. A 2.45 kb transcript (MCT3a) was expressed at low levels before E11 but its expression increased between E11 and E17. Using 5'-RACE (rapid amplification of cDNA ends) it has demonstrated that MCT3a and MCT3b mRNA had distinct 5'-untranslated sequences but shared the same translation start site. To determine the exon-intron structure and to understand the elements that control the tissue specific and developmental expression of MCT3, the MCT3 gene was cloned and sequenced from a chicken genomic library. The MCT3 gene is distributed over 8 kb of DNA and is composed of 6 exons. Coding sequences for MCT3 are found on exon 2 through exon 5. Comparison of the 5'-RACE sequence with the genomic sequence reveals that the two 5'-untranslated regions of the mRNAs are encoded by distinct exons, 1a and 1b, which are alternatively spliced to exon 2. These data suggest that two forms of MCT3 mRNAs could be generated by two distinct promoters that may be regulated in response to changes in the metabolic activity of the retina during development.

The retinal pigment epithelium (RPE) forms the outer blood-retinal barrier and regulates the movement of nutrients, water, and ions between the choroidal blood supply and the retina. The transport properties of the RPE maintain retinal adhesion and regulate the pH and osmolarity in the space surrounding the photoreceptor cell outer segments. In this report we identify two monocarboxylate transporters, MCT1 and MCT3, expressed in rat RPE. On the basis of Northern and Western blot analyses, MCT1 is expressed in both the neural retina and the RPE, whereas the expression of MCT3 is restricted to the RPE. Using indirect immunolocalization we show that the two transporters are polarized to distinct membrane domains. MCT1 antibody labels the apical surface and the apical processes of the RPE. A polyclonal antibody produced against the carboxy terminus of rat MCT3 labels only the basolateral membrane of the RPE. The demonstration of MCT1 on the apical membrane and MCT3 on the basal membrane identifies specific proteins involved in the discriminate and critical regulation of water and lactate transport from the retina to the choroid.

Frataxin is a conserved mitochondrial protein, and deficiency underlies the neurodegenerative disease Friedreich's ataxia. Frataxin interacts with the core machinery for Fe–S cluster assembly in mitochondria. Recently we reported that in frataxin-deleted yeast strains, a spontaneously occurring mutation in one of two genes encoding redundant Isu scaffold proteins, bypassed the mutant phenotypes. In the present study we created strains expressing a single scaffold protein, either Isu1 or the bypass mutant M107I Isu1. Our results show that in the frataxin-deletion strain expressing the bypass mutant Isu1, cell growth, Fe–S cluster protein activities, haem proteins and iron homoeostasis were restored to normal or close to normal. The bypass effects were not mediated by changes in Isu1 expression level. The persulfide-forming activity of the cysteine desulfurase was diminished in the frataxin deletion (∆yfh1 ISU1) and was improved by expression of the bypass Isu1 (∆yfh1 M107I ISU1). The addition of purified bypass M107I Isu1 protein to a ∆yfh1 lysate conferred similar enhancement of cysteine desulfurase as did frataxin, suggesting that this effect contributed to the bypass mechanism. Fe–S cluster-forming activity in isolated mitochondria was stimulated by the bypass Isu1, albeit at a lower rate. The rescuing effects of the bypass Isu1 point to ways that the core defects in Friedreich's ataxia mitochondria can be restored.

International audience; Mitochondria transport and utilize iron for the synthesis of haem and Fe-S clusters. Although many proteins are known to be involved in these processes, additional proteins are likely to participate. To test this hypothesis, in the present study we used a genetic screen looking for yeast mutants that are synthetically lethal with the mitochondrial iron carriers Mrs3 and Mrs4. Several genes were identified, including an isolate mutated for Yfh1, the yeast frataxin homologue. All such triple mutants were complemented by increased expression of Rim2, another mitochondrial carrier protein. Rim2 overexpression was able to enhance haem and Fe-S cluster synthesis in wild-type or Δmrs3/Δmrs4 backgrounds. Conversely Rim2 depletion impaired haem and Fe-S cluster synthesis in wild-type or Δmrs3/Δmrs4 backgrounds, indicating a unique requirement for this mitochondrial transporter for these processes. Rim2 was previously shown to mediate pyrimidine exchange in and out of vesicles. In the present study we found that isolated mitochondria lacking Rim2 exhibited concordant iron defects and pyrimidine transport defects, although the connection between these two functions is not explained. When organellar membranes were ruptured to bypass iron transport, haem synthesis from added iron and porphyrin was still markedly deficient in Rim2-depleted mitochondrial lysate. The results indicate that Rim2 is a pyrimidine exchanger with an additional unique function in promoting mitochondrial iron utilization.

Frataxin is a conserved mitochondrial protein deficient in patients with Friedreich's ataxia. Frataxin has been implicated in control of iron homoeostasis and Fe–S cluster assembly. In yeast or human mitochondria, frataxin interacts with components of the Fe–S cluster synthesis machinery, including the cysteine desulfurase Nfs1, accessory protein Isd11 and scaffold protein Isu. In the present paper, we report that a single amino acid substitution (methionine to isoleucine) at position 107 in the mature form of Isu1 restored many deficient functions in Δyfh1 or frataxin-depleted yeast cells. Iron homoeostasis was improved such that soluble/usable mitochondrial iron was increased and accumulation of insoluble/non-usable iron within mitochondria was largely prevented. Cytochromes were returned to normal and haem synthesis was restored. In mitochondria carrying the mutant Isu1 and no frataxin, Fe–S cluster enzyme activities were improved. The efficiency of new Fe–S cluster synthesis in isolated mitochondria was markedly increased compared with frataxin-negative cells, although the response to added iron was minimal. The M107I substitution in the highly conserved Isu scaffold protein is typically found in bacterial orthologues, suggesting that a unique feature of the bacterial Fe–S cluster machinery may be involved. The mechanism by which the mutant Isu bypasses the absence of frataxin remains to be determined, but could be related to direct effects on Fe–S cluster assembly and/or indirect effects on mitochondrial iron availability.

Cysteine desulfurases perform pyridoxal phosphate (PLP)-dependent desulfuration of cysteine. The key steps of the enzymatic cycle include substrate binding to PLP, formation of a covalent persulfide intermediate at the active site cysteine, and transfer of sulfur to recipients for use in various metabolic pathways. In Saccharomyces cerevisiae, the cysteine desulfurase Nfs1p and an accessory protein, Isd11p, are found primarily in mitochondria, and both are essential for cell viability. Although cysteine desulfurases are conserved from bacteria to humans, Isd11p is found only in eukaryotes and not in prokaryotes. Here we show that Isd11p activates Nfs1p. The enzyme without Isd11p was inactive and did not form the [(35)S]persulfide intermediate from the substrate [(35)S]cysteine. Addition of Isd11p to inactive Nfs1p induced formation of the persulfide. Remarkably, in a two-step assay, [(35)S]cysteine could be bound to the inactive Nfs1p in a PLP-dependent manner, and the enzyme could be subsequently induced to form the persulfide by addition of Isd11p. A mutant form of Isd11p with the (15)LYK(17) motif changed to (15)AAA(17) was able to bind but failed to activate Nfs1p, thus separating these two functions of Isd11p. Finally, compared with Nfs1p with or without the bound Isd11p mutant, the Nfs1p·Isd11p complex was more resistant to inactivation by an alkylating agent. On the basis of these novel findings, we propose that interaction of Isd11p with Nfs1p activates the enzyme by inducing a conformational change, thereby promoting formation of the persulfide intermediate at the active site cysteine. Such a conformational change may protect the active site cysteine from alkylating agents.

In a forward genetic screen for interaction with mitochondrial iron carrier proteins in Saccharomyces cerevisiae, a hypomorphic mutation of the essential DRE2 gene was found to confer lethality when combined with Delta mrs3 and Delta mrs4. The dre2 mutant or Dre2-depleted cells were deficient in cytosolic Fe/S cluster protein activities while maintaining mitochondrial Fe/S clusters. The Dre2 amino acid sequence was evolutionarily conserved, and cysteine motifs (CX(2)CXC and twin CX(2)C) in human and yeast proteins were perfectly aligned. The human Dre2 homolog (implicated in blocking apoptosis and called CIAPIN1 or anamorsin) was able to complement the nonviability of a Deltadre2 deletion strain. The Dre2 protein with triple hemagglutinin tag was located in the cytoplasm and in the mitochondrial intermembrane space. Yeast Dre2 overexpressed and purified from bacteria was brown and exhibited signature absorption and electron paramagnetic resonance spectra, indicating the presence of both [2Fe-2S] and [4Fe-4S] clusters. Thus, Dre2 is an essential conserved Fe/S cluster protein implicated in extramitochondrial Fe/S cluster assembly, similar to other components of the so-called CIA (cytoplasmic Fe/S cluster assembly) pathway although partially localized to the mitochondrial intermembrane space.

Purpose To evaluate the expression and subcellular distribution of proton-coupled monocarboxylate transporters (MCTs) in human RPE in vivo and determine whether ARPE-19 cells retain the ability to express and differentially polarize these transporters. Methods Total RNA was prepared from human donor eyes and from ARPE-19 cell cultures. Expression of MCT transcripts was evaluated by RT-PCR amplification. Expression of MCT proteins in human RPE and ARPE-19 cells was evaluated by immunolocalization and Western blot analysis with isoform-specific anti-peptide antibodies. Results The expression of MCTs in human RPE was investigated by immunofluorescence analysis on frozen sections of human donor eyes. MCT1 antibody labeled the apical membrane of the RPE intensely, whereas MCT3 labeling was restricted to the basolateral membrane. MCT4 was detected in the neural retina but not in the RPE. ARPE-19 cells constitutively expressed MCT1 and MCT4 mRNAs. Expression of MCT3 mRNA increased over time as ARPE-19 cells established a differentiated phenotype. Western blot analysis revealed that ARPE-19 cells expressed high levels of MCT1 and MCT4 but very little MCT3 protein. Sections of differentiated ARPE-19 cells were labeled with MCT1, MCT4, and glucose transporter-1 antibodies. MCT1 was polarized to the apical membrane and MCT4 to the basolateral membrane, whereas GLUT1 was expressed in both membrane domains. CD147, which is necessary for targeting MCTs to the plasma membrane, was detected in the apical and basolateral membranes of human RPE in situ and ARPE-19 cells. Conclusions These studies demonstrate for the first time that human RPE expresses two proton-coupled monocarboxylate transporters: MCT1 in the apical membrane and MCT3 in the basolateral membrane. The coordinated activities of these two transporters could facilitate the flux of lactate from the retina to the choroid. ARPE-19 cells express two MCT isoforms, polarized to different membrane domains: MCT1 to the apical membrane and MCT4 to the basolateral membrane. The polarized expression of MCTs in ARPE-19 demonstrates that these cells retain the cellular machinery necessary for transepithelial transport of lactate.

The retinal pigment epithelium transports lactate between two tissue compartments, the interphotoreceptor matrix and the choriocapillaris. In this report we describe a 2.45-kb cDNA isolated from a chick cDNA RPE library that encodes a membrane protein found only in RPE cells. The deduced protein has 542 amino acids with twelve putative membrane spanning domains. The cDNA has been designated MCT3 based on its 45% identity in amino acid sequence and structural similarity with the monocarboxylate transporters MCT1 and MCT2. Stable transfectants (pCl-neo/MCT3), made in a rat thyroid epithelial cell line (FRTL-5), express MCT3 RNA. Transfectants had enhanced pyruvate uptake (used as a measure of lactate uptake) which was proton-dependent and inhibited by α-cyano-4-hydroxycinnamate. In summary, MCT3's unique expression in RPE cells, multiple potential phosphorylation sites, and basolateral distribution suggest that MCT3 may regulate lactate levels in the interphotoreceptor space.