93 results on '"Mühlenhoff U"'
Search Results
2. Cloning and characterization of the psaE gene of the cyanobacterium Synechococcus sp. PCC 7002: characterization of a psaE mutant and overproduction of the protein in Escherichia coll.
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Zhao, J., Snyder, W. B., Mühlenhoff, U., Rhiel, E., Warren, P. V., Golbeck, J. H., and Bryant, D. A.
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ESCHERICHIA coli ,PROTEINS ,CYANOBACTERIA ,GENETIC transcription ,IMMUNOGLOBULINS - Abstract
The psaE gene, encoding a 7.5 kDa peripheral protein of the photosystem I complex, has been cloned and characterized from the cyanobacterium Synechococcus sp. PCC 7002. The gene is transcribed as an abundant monocistronic transcript of approximately 325 nt. The PsaE protein has been overproduced in Escherichia coli, purified to homogeneity, and used to raise polyclonal antibodies. Mutant strains, in which the psaE gene was insertionally inactivated by interposon mutagenesis, were constructed and characterized. Although the PS I complexes of these strains were similar to those of the wild type, the strains grew more slowly under conditions which favour cyclic electron transport and could not grow at all under photoheterotrophic conditions. The results suggest that PsaE plays a role in cyclic electron transport in cyanobacteria. [ABSTRACT FROM AUTHOR]
- Published
- 1993
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3. Requirements for the biogenesis of [2Fe-2S] proteins in the human and yeast cytosol.
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Braymer JJ, Stehling O, Stümpfig M, Rösser R, Spantgar F, Blinn CM, Mühlenhoff U, Pierik AJ, and Lill R
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- Humans, ATP-Binding Cassette Transporters metabolism, Mitochondrial Proteins metabolism, Cytosol metabolism, Iron-Sulfur Proteins metabolism, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism, Saccharomyces cerevisiae Proteins genetics, Glutathione metabolism, Mitochondria metabolism, Glutaredoxins metabolism, Glutaredoxins genetics
- Abstract
The biogenesis of iron-sulfur (Fe/S) proteins entails the synthesis and trafficking of Fe/S clusters, followed by their insertion into target apoproteins. In eukaryotes, the multiple steps of biogenesis are accomplished by complex protein machineries in both mitochondria and cytosol. The underlying biochemical pathways have been elucidated over the past decades, yet the mechanisms of cytosolic [2Fe-2S] protein assembly have remained ill-defined. Similarly, the precise site of glutathione (GSH) requirement in cytosolic and nuclear Fe/S protein biogenesis is unclear, as is the molecular role of the GSH-dependent cytosolic monothiol glutaredoxins (cGrxs). Here, we investigated these questions in human and yeast cells by various in vivo approaches. [2Fe-2S] cluster assembly of cytosolic target apoproteins required the mitochondrial ISC machinery, the mitochondrial transporter Atm1/ABCB7 and GSH, yet occurred independently of both the CIA system and cGrxs. This mechanism was strikingly different from the ISC-, Atm1/ABCB7-, GSH-, and CIA-dependent assembly of cytosolic-nuclear [4Fe-4S] proteins. One notable exception to this cytosolic [2Fe-2S] protein maturation pathway defined here was yeast Apd1 which used the CIA system via binding to the CIA targeting complex through its C-terminal tryptophan. cGrxs, although attributed as [2Fe-2S] cluster chaperones or trafficking proteins, were not essential in vivo for delivering [2Fe-2S] clusters to either CIA components or target apoproteins. Finally, the most critical GSH requirement was assigned to Atm1-dependent export, i.e. a step before GSH-dependent cGrxs function. Our findings extend the general model of eukaryotic Fe/S protein biogenesis by adding the molecular requirements for cytosolic [2Fe-2S] protein maturation., Competing Interests: Competing interests statement:The authors declare no competing interest.
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- 2024
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4. Hsp90 and metal-binding J-protein family chaperones are not critically involved in cellular iron-sulfur protein assembly and iron regulation in yeast.
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Carvalho FA, Mühlenhoff U, Braymer JJ, Root V, Stümpfig M, Oliveira CC, and Lill R
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- Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Iron metabolism, Mitochondrial Proteins metabolism, Molecular Chaperones metabolism, HSP90 Heat-Shock Proteins genetics, HSP90 Heat-Shock Proteins metabolism, Iron-Sulfur Proteins genetics, Iron-Sulfur Proteins metabolism, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism
- Abstract
Systematic studies have revealed interactions between components of the Hsp90 chaperone system and Fe/S protein biogenesis or iron regulation. In addition, two chloroplast-localized DnaJ-like proteins, DJA5 and DJA6, function as specific iron donors in plastidial Fe/S protein biogenesis. Here, we used Saccharomyces cerevisiae to study the impact of both the Hsp90 chaperone and the yeast DJA5-DJA6 homologs, the essential cytosolic Ydj1, and the mitochondrial Mdj1, on cellular iron-related processes. Despite severe phenotypes induced upon depletion of these crucial proteins, there was no critical in vivo impact on Fe/S protein biogenesis or iron regulation. Importantly, unlike the plant DJA5-DJA6 iron chaperones, Ydj1 and Mdj1 did not bind iron in vivo, suggesting that these proteins use zinc for function under normal physiological conditions., (© 2023 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)
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- 2023
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5. Functional spectrum and specificity of mitochondrial ferredoxins FDX1 and FDX2.
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Schulz V, Basu S, Freibert SA, Webert H, Boss L, Mühlenhoff U, Pierrel F, Essen LO, Warui DM, Booker SJ, Stehling O, and Lill R
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- Humans, Protein Isoforms metabolism, Cytochrome P-450 Enzyme System metabolism, Mitochondria metabolism, Ferredoxins, Iron-Sulfur Proteins metabolism
- Abstract
Ferredoxins comprise a large family of iron-sulfur (Fe-S) proteins that shuttle electrons in diverse biological processes. Human mitochondria contain two isoforms of [2Fe-2S] ferredoxins, FDX1 (aka adrenodoxin) and FDX2, with known functions in cytochrome P450-dependent steroid transformations and Fe-S protein biogenesis. Here, we show that only FDX2, but not FDX1, is involved in Fe-S protein maturation. Vice versa, FDX1 is specific not only for steroidogenesis, but also for heme a and lipoyl cofactor biosyntheses. In the latter pathway, FDX1 provides electrons to kickstart the radical chain reaction catalyzed by lipoyl synthase. We also identified lipoylation as a target of the toxic antitumor copper ionophore elesclomol. Finally, the striking target specificity of each ferredoxin was assigned to small conserved sequence motifs. Swapping these motifs changed the target specificity of these electron donors. Together, our findings identify new biochemical tasks of mitochondrial ferredoxins and provide structural insights into their functional specificity., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)
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- 2023
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6. Mitochondrial [2Fe-2S] ferredoxins: new functions for old dogs.
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Schulz V, Freibert SA, Boss L, Mühlenhoff U, Stehling O, and Lill R
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- Dogs, Animals, Humans, Adrenodoxin chemistry, Adrenodoxin metabolism, Saccharomyces cerevisiae metabolism, Cytochrome P-450 Enzyme System metabolism, Heme metabolism, Mammals metabolism, Ferredoxins metabolism, Iron-Sulfur Proteins metabolism
- Abstract
Ferredoxins (FDXs) comprise a large family of iron-sulfur proteins that shuttle electrons from NADPH and FDX reductases into diverse biological processes. This review focuses on the structure, function and specificity of mitochondrial [2Fe-2S] FDXs that are related to bacterial FDXs due to their endosymbiotic inheritance. Their classical function in cytochrome P450-dependent steroid transformations was identified around 1960, and is exemplified by mammalian FDX1 (aka adrenodoxin). Thirty years later the essential function in cellular Fe/S protein biogenesis was discovered for the yeast mitochondrial FDX Yah1 that is additionally crucial for the formation of haem a and ubiquinone CoQ
6 . In mammals, Fe/S protein biogenesis is exclusively performed by the FDX1 paralog FDX2, despite the high structural similarity of both proteins. Recently, additional and specific roles of human FDX1 in haem a and lipoyl cofactor biosyntheses were described. For lipoyl synthesis, FDX1 transfers electrons to the radical S-adenosyl methionine-dependent lipoyl synthase to kickstart its radical chain reaction. The high target specificity of the two mammalian FDXs is contained within small conserved sequence motifs, that upon swapping change the target selection of these electron donors., (© 2022 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)- Published
- 2023
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7. The iron-sulfur cluster assembly (ISC) protein Iba57 executes a tetrahydrofolate-independent function in mitochondrial [4Fe-4S] protein maturation.
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Mühlenhoff U, Weiler BD, Nadler F, Millar R, Kothe I, Freibert SA, Altegoer F, Bange G, and Lill R
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- Humans, Carrier Proteins metabolism, Folic Acid metabolism, Iron metabolism, Mitochondria metabolism, Mitochondrial Proteins genetics, Mitochondrial Proteins metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Tetrahydrofolates metabolism, Iron-Sulfur Proteins metabolism, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism
- Abstract
Mitochondria harbor the bacteria-inherited iron-sulfur cluster assembly (ISC) machinery to generate [2Fe-2S; iron-sulfur (Fe-S)] and [4Fe-4S] proteins. In yeast, assembly of [4Fe-4S] proteins specifically involves the ISC proteins Isa1, Isa2, Iba57, Bol3, and Nfu1. Functional defects in their human equivalents cause the multiple mitochondrial dysfunction syndromes, severe disorders with a broad clinical spectrum. The bacterial Iba57 ancestor YgfZ was described to require tetrahydrofolate (THF) for its function in the maturation of selected [4Fe-4S] proteins. Both YgfZ and Iba57 are structurally related to an enzyme family catalyzing THF-dependent one-carbon transfer reactions including GcvT of the glycine cleavage system. On this basis, a universally conserved folate requirement in ISC-dependent [4Fe-4S] protein biogenesis was proposed. To test this idea for mitochondrial Iba57, we performed genetic and biochemical studies in Saccharomyces cerevisiae, and we solved the crystal structure of Iba57 from the thermophilic fungus Chaetomium thermophilum. We provide three lines of evidence for the THF independence of the Iba57-catalyzed [4Fe-4S] protein assembly pathway. First, yeast mutants lacking folate show no defect in mitochondrial [4Fe-4S] protein maturation. Second, the 3D structure of Iba57 lacks many of the side-chain contacts to THF as defined in GcvT, and the THF-binding pocket is constricted. Third, mutations in conserved Iba57 residues that are essential for THF-dependent catalysis in GcvT do not impair Iba57 function in vivo, in contrast to an exchange of the invariant, surface-exposed cysteine residue. We conclude that mitochondrial Iba57, despite structural similarities to both YgfZ and THF-binding proteins, does not utilize folate for its function., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2022
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8. N-terminal tyrosine of ISCU2 triggers [2Fe-2S] cluster synthesis by ISCU2 dimerization.
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Freibert SA, Boniecki MT, Stümpfig C, Schulz V, Krapoth N, Winge DR, Mühlenhoff U, Stehling O, Cygler M, and Lill R
- Subjects
- Apoproteins, Carbon-Sulfur Lyases, Crystallography, X-Ray, Ferredoxins, HeLa Cells, Humans, Iron, Mitochondria, Mutant Proteins, Recombinant Proteins, Sulfur, Dimerization, Iron-Sulfur Proteins chemistry, Tyrosine chemistry
- Abstract
Synthesis of iron-sulfur (Fe/S) clusters in living cells requires scaffold proteins for both facile synthesis and subsequent transfer of clusters to target apoproteins. The human mitochondrial ISCU2 scaffold protein is part of the core ISC (iron-sulfur cluster assembly) complex that synthesizes a bridging [2Fe-2S] cluster on dimeric ISCU2. Initial iron and sulfur loading onto monomeric ISCU2 have been elucidated biochemically, yet subsequent [2Fe-2S] cluster formation and dimerization of ISCU2 is mechanistically ill-defined. Our structural, biochemical and cell biological experiments now identify a crucial function of the universally conserved N-terminal Tyr35 of ISCU2 for these late reactions. Mixing two, per se non-functional ISCU2 mutant proteins with oppositely charged Asp35 and Lys35 residues, both bound to different cysteine desulfurase complexes NFS1-ISD11-ACP, restores wild-type ISCU2 maturation demonstrating that ionic forces can replace native Tyr-Tyr interactions during dimerization-induced [2Fe-2S] cluster formation. Our studies define the essential mechanistic role of Tyr35 in the reaction cycle of de novo mitochondrial [2Fe-2S] cluster synthesis., (© 2021. The Author(s).)
- Published
- 2021
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9. Glutaredoxins with iron-sulphur clusters in eukaryotes - Structure, function and impact on disease.
- Author
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Berndt C, Christ L, Rouhier N, and Mühlenhoff U
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- Humans, Iron chemistry, Iron metabolism, Structure-Activity Relationship, Sulfur chemistry, Sulfur metabolism, Glutaredoxins chemistry, Glutaredoxins genetics, Glutaredoxins metabolism, Iron-Sulfur Proteins chemistry, Iron-Sulfur Proteins genetics, Iron-Sulfur Proteins metabolism, Plant Diseases, Plant Proteins chemistry, Plant Proteins genetics, Plant Proteins metabolism, Plants enzymology
- Abstract
Among the thioredoxin superfamily of proteins, the observation that numerous glutaredoxins bind iron-sulphur (Fe/S) clusters is one of the more recent and major developments concerning their functional properties. Glutaredoxins are present in most organisms. All members of the class II subfamily (including most monothiol glutaredoxins), but also some members of the class I (mostly dithiol glutaredoxins) and class III (land plant-specific monothiol or dithiol glutaredoxins) are Fe/S proteins. In glutaredoxins characterised so far, the [2Fe2S] cluster is coordinated by two active-site cysteine residues and two molecules of non-covalently bound glutathione in homo-dimeric complexes bridged by the cluster. In contrast to dithiol glutaredoxins, monothiol glutaredoxins possess no or very little oxidoreductase activity, but have emerged as important players in cellular iron metabolism. In this review we summarise the recent developments of the most prominent Fe/S glutaredoxins in eukaryotes, the mitochondrial single domain monothiol glutaredoxin 5, the chloroplastic single domain monothiol glutaredoxin S14 and S16, the nuclear/cytosolic multi-domain monothiol glutaredoxin 3, and the mitochondrial/cytosolic dithiol glutaredoxin 2., (Copyright © 2020 Elsevier B.V. All rights reserved.)
- Published
- 2021
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10. Glutaredoxins and iron-sulfur protein biogenesis at the interface of redox biology and iron metabolism.
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Mühlenhoff U, Braymer JJ, Christ S, Rietzschel N, Uzarska MA, Weiler BD, and Lill R
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- Oxidation-Reduction, Glutaredoxins metabolism, Iron metabolism, Iron-Sulfur Proteins metabolism, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism
- Abstract
The physiological roles of the intracellular iron and redox regulatory systems are intimately linked. Iron is an essential trace element for most organisms, yet elevated cellular iron levels are a potent generator and amplifier of reactive oxygen species and redox stress. Proteins binding iron or iron-sulfur (Fe/S) clusters, are particularly sensitive to oxidative damage and require protection from the cellular oxidative stress protection systems. In addition, key components of these systems, most prominently glutathione and monothiol glutaredoxins are involved in the biogenesis of cellular Fe/S proteins. In this review, we address the biochemical role of glutathione and glutaredoxins in cellular Fe/S protein assembly in eukaryotic cells. We also summarize the recent developments in the role of cytosolic glutaredoxins in iron metabolism, in particular the regulation of fungal iron homeostasis. Finally, we discuss recent insights into the interplay of the cellular thiol redox balance and oxygen with that of Fe/S protein biogenesis in eukaryotes.
- Published
- 2020
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11. Mitochondrial [4Fe-4S] protein assembly involves reductive [2Fe-2S] cluster fusion on ISCA1-ISCA2 by electron flow from ferredoxin FDX2.
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Weiler BD, Brück MC, Kothe I, Bill E, Lill R, and Mühlenhoff U
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- Aconitate Hydratase metabolism, Chaetomium, Humans, Iron-Sulfur Proteins metabolism, Mitochondria metabolism
- Abstract
The essential process of iron-sulfur (Fe/S) cluster assembly (ISC) in mitochondria occurs in three major phases. First, [2Fe-2S] clusters are synthesized on the scaffold protein ISCU2; second, these clusters are transferred to the monothiol glutaredoxin GLRX5 by an Hsp70 system followed by insertion into [2Fe-2S] apoproteins; third, [4Fe-4S] clusters are formed involving the ISC proteins ISCA1-ISCA2-IBA57 followed by target-specific apoprotein insertion. The third phase is poorly characterized biochemically, because previous in vitro assembly reactions involved artificial reductants and lacked at least one of the in vivo-identified ISC components. Here, we reconstituted the maturation of mitochondrial [4Fe-4S] aconitase without artificial reductants and verified the [2Fe-2S]-containing GLRX5 as cluster donor. The process required all components known from in vivo studies (i.e., ISCA1-ISCA2-IBA57), yet surprisingly also depended on mitochondrial ferredoxin FDX2 and its NADPH-coupled reductase FDXR. Electrons from FDX2 catalyze the reductive [2Fe-2S] cluster fusion on ISCA1-ISCA2 in an IBA57-dependent fashion. This previously unidentified electron transfer was occluded during previous in vivo studies due to the earlier FDX2 requirement for [2Fe-2S] cluster synthesis on ISCU2. The FDX2 function is specific, because neither FDX1, a mitochondrial ferredoxin involved in steroid production, nor other cellular reducing systems, supported maturation. In contrast to ISC factor-assisted [4Fe-4S] protein assembly, [2Fe-2S] cluster transfer from GLRX5 to [2Fe-2S] apoproteins occurred spontaneously within seconds, clearly distinguishing the mechanisms of [2Fe-2S] and [4Fe-4S] protein maturation. Our study defines the physiologically relevant mechanistic action of late-acting ISC factors in mitochondrial [4Fe-4S] cluster synthesis, trafficking, and apoprotein insertion., Competing Interests: The authors declare no competing interest.
- Published
- 2020
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12. Molecular basis for the distinct functions of redox-active and FeS-transfering glutaredoxins.
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Trnka D, Engelke AD, Gellert M, Moseler A, Hossain MF, Lindenberg TT, Pedroletti L, Odermatt B, de Souza JV, Bronowska AK, Dick TP, Mühlenhoff U, Meyer AJ, Berndt C, and Lillig CH
- Subjects
- Animals, Catalytic Domain, Glutaredoxins chemistry, Humans, Iron-Sulfur Proteins chemistry, Oxidation-Reduction, Protein Binding, Protein Structure, Secondary, Signal Transduction physiology, Substrate Specificity, Glutaredoxins metabolism
- Abstract
Despite their very close structural similarity, CxxC/S-type (class I) glutaredoxins (Grxs) act as oxidoreductases, while CGFS-type (class II) Grxs act as FeS cluster transferases. Here we show that the key determinant of Grx function is a distinct loop structure adjacent to the active site. Engineering of a CxxC/S-type Grx with a CGFS-type loop switched its function from oxidoreductase to FeS transferase. Engineering of a CGFS-type Grx with a CxxC/S-type loop abolished FeS transferase activity and activated the oxidative half reaction of the oxidoreductase. The reductive half-reaction, requiring the interaction with a second GSH molecule, was enabled by switching additional residues in the active site. We explain how subtle structural differences, mostly depending on the structure of one particular loop, act in concert to determine Grx function.
- Published
- 2020
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13. Depletion of thiol reducing capacity impairs cytosolic but not mitochondrial iron-sulfur protein assembly machineries.
- Author
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Braymer JJ, Stümpfig M, Thelen S, Mühlenhoff U, and Lill R
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- Cell Nucleus metabolism, Cytosol metabolism, Genomic Instability, Glutaredoxins metabolism, Homeostasis, Iron metabolism, Iron-Sulfur Proteins genetics, Mitochondria metabolism, Oxidation-Reduction, Protein Transport, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, Sulfhydryl Compounds metabolism, Sulfur metabolism, Thioredoxins metabolism, Iron-Sulfur Proteins biosynthesis, Iron-Sulfur Proteins metabolism
- Abstract
Iron‑sulfur (Fe/S) clusters are versatile inorganic cofactors that play central roles in essential cellular functions, from respiration to genome stability. >30 proteins involved in Fe/S protein biogenesis in eukaryotes are known, many of which bind clusters via cysteine residues. This opens up the possibility that the thiol-reducing glutaredoxin and thioredoxin systems are required at both the Fe/S biogenesis and target protein level to counteract thiol oxidation. To address the possible interplay of thiol redox chemistry and Fe/S protein biogenesis, we have characterized the status of the mitochondrial (ISC) and cytosolic (CIA) Fe/S protein assembly machineries in Saccharomyces cerevisiae mutants in which the three partially redundant glutathione (Glr1) and thioredoxin (Trr1 and Trr2) oxidoreductases have been inactivated in either mitochondria, cytosol, or both compartments. Cells devoid of mitochondrial oxidoreductases maintained a functional mitochondrial ISC machinery and showed no altered iron homeostasis despite a non-functional complex II of the respiratory chain due to redox-specific defects. In cells that lack either cytosolic or total cellular thiol reducing capacity, both the ISC system and iron homeostasis were normal, yet cytosolic and nuclear Fe/S target proteins were not matured. This dysfunction could be attributed to a failure in the assembly of [4Fe‑4S] clusters in the CIA factor Nar1, even though Nar1 maintained robust protein levels and stable interactions with later-acting CIA components. Overall, our analysis has uncovered a hitherto unknown thiol-dependence of the CIA machinery and has demonstrated the surprisingly varying sensitivity of Fe/S proteins to thiol oxidation., (Copyright © 2018 Elsevier B.V. All rights reserved.)
- Published
- 2019
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14. Conserved functions of Arabidopsis mitochondrial late-acting maturation factors in the trafficking of iron‑sulfur clusters.
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Uzarska MA, Przybyla-Toscano J, Spantgar F, Zannini F, Lill R, Mühlenhoff U, and Rouhier N
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- Arabidopsis genetics, Arabidopsis Proteins genetics, Cloning, Molecular, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Evolution, Molecular, Iron metabolism, Mitochondria genetics, Mitochondria metabolism, Mitochondrial Proteins genetics, Saccharomyces cerevisiae genetics, Sulfur metabolism, Arabidopsis metabolism, Arabidopsis Proteins metabolism, Mitochondrial Proteins metabolism, Saccharomyces cerevisiae growth & development
- Abstract
Numerous proteins require iron‑sulfur (Fe-S) clusters as cofactors for their function. Their biogenesis is a multi-step process occurring in the cytosol and mitochondria of all eukaryotes and additionally in plastids of photosynthetic eukaryotes. A basic model of Fe-S protein maturation in mitochondria has been obtained based on studies achieved in mammals and yeast, yet some molecular details, especially of the late steps, still require investigation. In particular, the late-acting biogenesis factors in plant mitochondria are poorly understood. In this study, we expressed the factors belonging to NFU, BOLA, SUFA/ISCA and IBA57 families in the respective yeast mutant strains. Expression of the Arabidopsis mitochondrial orthologs was usually sufficient to rescue the growth defects observed on specific media and/or to restore the abundance or activity of the defective Fe-S or lipoic acid-dependent enzymes. These data demonstrate that the plant mitochondrial counterparts, including duplicated isoforms, likely retained their ancestral functions. In contrast, the SUFA1 and IBA57.2 plastidial isoforms cannot rescue the lysine and glutamate auxotrophies of the respective isa1-isa2Δ and iba57Δ strains or of the isa1-isa2-iba57Δ triple mutant when expressed in combination. This suggests a specialization of the yeast mitochondrial and plant plastidial factors in these late steps of Fe-S protein biogenesis, possibly reflecting substrate-specific interactions in these different compartments., (Copyright © 2018 Elsevier B.V. All rights reserved.)
- Published
- 2018
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15. Fe-S cluster coordination of the chromokinesin KIF4A alters its subcellular localization during mitosis.
- Author
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Ben-Shimon L, Paul VD, David-Kadoch G, Volpe M, Stümpfig M, Bill E, Mühlenhoff U, Lill R, and Ben-Aroya S
- Subjects
- Humans, Mitosis, DNA-Binding Proteins metabolism, Iron-Sulfur Proteins metabolism, Kinesins genetics, Kinesins metabolism, Nuclear Proteins metabolism
- Abstract
Fe-S clusters act as co-factors of proteins with diverse functions, for example, in DNA repair. Downregulation of the cytosolic iron-sulfur protein assembly (CIA) machinery promotes genomic instability through the inactivation of multiple DNA repair pathways. Furthermore, CIA deficiencies are associated with so far unexplained mitotic defects. Here, we show that CIA2B (also known as FAM96B) and MMS19, constituents of the CIA targeting complex involved in facilitating Fe-S cluster insertion into cytosolic and nuclear target proteins, colocalize with components of the mitotic machinery. Downregulation of CIA2B and MMS19 impairs the mitotic cycle. We identify the chromokinesin KIF4A as a mitotic component involved in these effects. KIF4A binds a Fe-S cluster in vitro through its conserved cysteine-rich domain. We demonstrate in vivo that this domain is required for the mitosis-related KIF4A localization and for the mitotic defects associated with KIF4A knockout. KIF4A is the first identified mitotic component carrying such a post-translational modification. These findings suggest that the lack of Fe-S clusters in KIF4A upon downregulation of the CIA targeting complex contributes to the mitotic defects., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)
- Published
- 2018
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16. Biochemical Reconstitution and Spectroscopic Analysis of Iron-Sulfur Proteins.
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Freibert SA, Weiler BD, Bill E, Pierik AJ, Mühlenhoff U, and Lill R
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- Animals, Circular Dichroism methods, Electron Spin Resonance Spectroscopy methods, Humans, Mitochondria chemistry, Spectrophotometry, Ultraviolet methods, Spectroscopy, Mossbauer methods, Iron-Sulfur Proteins chemistry
- Abstract
Iron-sulfur (Fe/S) proteins are involved in numerous key biological functions such as respiration, metabolic processes, protein translation, DNA synthesis, and DNA repair. The simplest types of Fe/S clusters include [2Fe-2S], [3Fe-4S], and [4Fe-4S] forms that sometimes are present in multiple copies. De novo assembly of Fe/S cofactors and their insertion into apoproteins in living cells requires complex proteinaceous machineries that are frequently highly conserved. In eukaryotes such as yeast and mammals, the mitochondrial iron-sulfur cluster assembly machinery and the cytosolic iron-sulfur protein assembly system consist of more than 30 components that cooperate in the generation of some 50 cellular Fe/S proteins. Both the mechanistic dissection of the intracellular Fe/S protein assembly pathways and the identification and characterization of Fe/S proteins rely on tool boxes of in vitro and in vivo methods. These cell biological, biochemical, and biophysical techniques help to determine the extent, stability, and type of bound Fe/S cluster. They also serve to distinguish bona fide Fe/S proteins from other metal-binding proteins containing similar cofactor coordination motifs. Here, we present a collection of in vitro methods that have proven useful for basic biochemical and biophysical characterization of Fe/S proteins. First, we describe the chemical assembly of [2Fe-2S] or [4Fe-4S] clusters on purified apoproteins. Then, we summarize a reconstitution system reproducing the de novo synthesis of a [2Fe-2S] cluster in mitochondria. Finally, we explain the use of UV-vis, CD, electron paramagnetic resonance, and Mössbauer spectroscopy for the routine characterization of Fe/S proteins., (© 2018 Elsevier Inc. All rights reserved.)
- Published
- 2018
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17. The heme synthesis defect of mutants impaired in mitochondrial iron-sulfur protein biogenesis is caused by reversible inhibition of ferrochelatase.
- Author
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Lange H, Mühlenhoff U, Denzel M, Kispal G, and Lill R
- Published
- 2017
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18. A novel de novo dominant mutation in ISCU associated with mitochondrial myopathy.
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Legati A, Reyes A, Ceccatelli Berti C, Stehling O, Marchet S, Lamperti C, Ferrari A, Robinson AJ, Mühlenhoff U, Lill R, Zeviani M, Goffrini P, and Ghezzi D
- Subjects
- Amino Acid Sequence, Biomarkers, Biopsy, Computational Biology methods, Electroencephalography, Electromyography, Fibroblasts metabolism, Heterozygote, High-Throughput Nucleotide Sequencing, Humans, Iron-Sulfur Proteins chemistry, Magnetic Resonance Imaging, Male, Models, Molecular, Muscle, Skeletal pathology, Pedigree, Phenotype, Sequence Analysis, DNA, Structure-Activity Relationship, Young Adult, Genes, Dominant, Iron-Sulfur Proteins genetics, Mitochondrial Myopathies diagnosis, Mitochondrial Myopathies genetics, Mutation
- Abstract
Background: Hereditary myopathy with lactic acidosis and myopathy with deficiency of succinate dehydrogenase and aconitase are variants of a recessive disorder characterised by childhood-onset early fatigue, dyspnoea and palpitations on trivial exercise. The disease is non-progressive, but life-threatening episodes of widespread weakness, metabolic acidosis and rhabdomyolysis may occur. So far, this disease has been molecularly defined only in Swedish patients, all homozygous for a deep intronic splicing affecting mutation in ISCU encoding a scaffold protein for the assembly of iron-sulfur (Fe-S) clusters. A single Scandinavian family was identified with a different mutation, a missense change in compound heterozygosity with the common intronic mutation. The aim of the study was to identify the genetic defect in our proband., Methods: A next-generation sequencing (NGS) approach was carried out on an Italian male who presented in childhood with ptosis, severe muscle weakness and exercise intolerance. His disease was slowly progressive, with partial recovery between episodes. Patient's specimens and yeast models were investigated., Results: Histochemical and biochemical analyses on muscle biopsy showed multiple defects affecting mitochondrial respiratory chain complexes. We identified a single heterozygous mutation p.Gly96Val in ISCU , which was absent in DNA from his parents indicating a possible de novo dominant effect in the patient. Patient fibroblasts showed normal levels of ISCU protein and a few variably affected Fe-S cluster-dependent enzymes. Yeast studies confirmed both pathogenicity and dominance of the identified missense mutation., Conclusion: We describe the first heterozygous dominant mutation in ISCU which results in a phenotype reminiscent of the recessive disease previously reported., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)
- Published
- 2017
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19. Structure and functional dynamics of the mitochondrial Fe/S cluster synthesis complex.
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Boniecki MT, Freibert SA, Mühlenhoff U, Lill R, and Cygler M
- Subjects
- Acyl Carrier Protein chemistry, Acyl Carrier Protein genetics, Acyl Carrier Protein metabolism, Amino Acid Sequence, Amino Acid Substitution, Carbon-Sulfur Lyases chemistry, Carbon-Sulfur Lyases genetics, Carbon-Sulfur Lyases metabolism, Chaetomium chemistry, Chaetomium genetics, Crystallography, X-Ray, Fungal Proteins chemistry, Fungal Proteins genetics, Fungal Proteins metabolism, Humans, Iron-Binding Proteins chemistry, Iron-Binding Proteins genetics, Iron-Binding Proteins metabolism, Iron-Regulatory Proteins chemistry, Iron-Regulatory Proteins genetics, Iron-Regulatory Proteins metabolism, Iron-Sulfur Proteins genetics, Mitochondrial Proteins chemistry, Mitochondrial Proteins genetics, Mitochondrial Proteins metabolism, Models, Molecular, Molecular Dynamics Simulation, Multiprotein Complexes chemistry, Multiprotein Complexes metabolism, Mutagenesis, Site-Directed, Protein Conformation, Protein Multimerization, Protein Stability, Saccharomyces cerevisiae Proteins chemistry, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism, Scattering, Small Angle, Sequence Homology, Amino Acid, Static Electricity, X-Ray Diffraction, Frataxin, Iron-Sulfur Proteins chemistry, Iron-Sulfur Proteins metabolism, Mitochondria metabolism
- Abstract
Iron-sulfur (Fe/S) clusters are essential protein cofactors crucial for many cellular functions including DNA maintenance, protein translation, and energy conversion. De novo Fe/S cluster synthesis occurs on the mitochondrial scaffold protein ISCU and requires cysteine desulfurase NFS1, ferredoxin, frataxin, and the small factors ISD11 and ACP (acyl carrier protein). Both the mechanism of Fe/S cluster synthesis and function of ISD11-ACP are poorly understood. Here, we present crystal structures of three different NFS1-ISD11-ACP complexes with and without ISCU, and we use SAXS analyses to define the 3D architecture of the complete mitochondrial Fe/S cluster biosynthetic complex. Our structural and biochemical studies provide mechanistic insights into Fe/S cluster synthesis at the catalytic center defined by the active-site Cys of NFS1 and conserved Cys, Asp, and His residues of ISCU. We assign specific regulatory rather than catalytic roles to ISD11-ACP that link Fe/S cluster synthesis with mitochondrial lipid synthesis and cellular energy status.
- Published
- 2017
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20. Specialized function of yeast Isa1 and Isa2 proteins in the maturation of mitochondrial [4Fe-4S] proteins.
- Author
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Mühlenhoff U, Richter N, Pines O, Pierik AJ, and Lill R
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- 2017
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21. Mitochondrial Bol1 and Bol3 function as assembly factors for specific iron-sulfur proteins.
- Author
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Uzarska MA, Nasta V, Weiler BD, Spantgar F, Ciofi-Baffoni S, Saviello MR, Gonnelli L, Mühlenhoff U, Banci L, and Lill R
- Subjects
- Glutaredoxins metabolism, Protein Multimerization, Saccharomyces cerevisiae Proteins metabolism, Iron-Sulfur Proteins metabolism, Mitochondria metabolism, Mitochondrial Proteins metabolism, Saccharomyces cerevisiae metabolism
- Abstract
Assembly of mitochondrial iron-sulfur (Fe/S) proteins is a key process of cells, and defects cause many rare diseases. In the first phase of this pathway, ten Fe/S cluster (ISC) assembly components synthesize and insert [2Fe-2S] clusters. The second phase is dedicated to the assembly of [4Fe-4S] proteins, yet this part is poorly understood. Here, we characterize the BOLA family proteins Bol1 and Bol3 as specific mitochondrial ISC assembly factors that facilitate [4Fe-4S] cluster insertion into a subset of mitochondrial proteins such as lipoate synthase and succinate dehydrogenase. Bol1-Bol3 perform largely overlapping functions, yet cannot replace the ISC protein Nfu1 that also participates in this phase of Fe/S protein biogenesis. Bol1 and Bol3 form dimeric complexes with both monothiol glutaredoxin Grx5 and Nfu1. Complex formation differentially influences the stability of the Grx5-Bol-shared Fe/S clusters. Our findings provide the biochemical basis for explaining the pathological phenotypes of patients with mutations in BOLA3., Competing Interests: The authors declare that no competing interests exist.
- Published
- 2016
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22. Defects in Mitochondrial Iron-Sulfur Cluster Assembly Induce Cysteine S-Polythiolation on Iron-Sulfur Apoproteins.
- Author
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Christ S, Leichert LI, Willms A, Lill R, and Mühlenhoff U
- Subjects
- Apoproteins chemistry, Cytosol metabolism, Escherichia coli genetics, Iron metabolism, Iron-Sulfur Proteins chemistry, Ligands, Mitochondria metabolism, Models, Molecular, Saccharomyces cerevisiae genetics, Sulfhydryl Compounds chemistry, Sulfur metabolism, Apoproteins metabolism, Cysteine metabolism, Iron-Sulfur Proteins metabolism, Sulfhydryl Compounds metabolism
- Abstract
Aims: Mitochondria play a central role in the maturation of proteins with iron-sulfur (Fe/S) clusters. During their biogenesis, the apoforms of Fe/S proteins expose unprotected Fe/S cluster-coordinating cysteine side chains, rendering them vulnerable to oxidative modifications that interfere with subsequent Fe/S cluster insertion. Whether and how cells protect these delicate cysteine residues are unknown., Results: In this study, we show that sulfhydryl groups of Fe/S cluster-coordinating cysteine residues of mitochondrial Fe/S apoproteins acquire cyclic S-polythiol modifications. These adducts are the result of persulfide addition, followed by a subsequent oxidation step. These modifications not only accumulate upon defects in the early stages of the mitochondrial Fe/S cluster assembly system but are also found in wild-type cells under normal growth conditions. They are, however, not found on Fe/S apoproteins in the cytosol., Innovation and Conclusion: Our work describes a novel in vivo chemical modification of cysteine side chains in mitochondrial Fe/S apoproteins. These cyclic S-polythiolation adducts are resistant to oxidation, yet can be removed by reductive cleavage, suggesting that they serve as a reversible protection device for cysteine ligands sensitive to oxidative modification. Antioxid. Redox Signal. 25, 28-40.
- Published
- 2016
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23. The mitochondrial monothiol glutaredoxin S15 is essential for iron-sulfur protein maturation in Arabidopsis thaliana.
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Moseler A, Aller I, Wagner S, Nietzel T, Przybyla-Toscano J, Mühlenhoff U, Lill R, Berndt C, Rouhier N, Schwarzländer M, and Meyer AJ
- Subjects
- Arabidopsis growth & development, Genetic Complementation Test, Arabidopsis metabolism, Glutaredoxins metabolism, Iron-Sulfur Proteins metabolism, Mitochondria metabolism
- Abstract
The iron-sulfur cluster (ISC) is an ancient and essential cofactor of many proteins involved in electron transfer and metabolic reactions. In Arabidopsis, three pathways exist for the maturation of iron-sulfur proteins in the cytosol, plastids, and mitochondria. We functionally characterized the role of mitochondrial glutaredoxin S15 (GRXS15) in biogenesis of ISC containing aconitase through a combination of genetic, physiological, and biochemical approaches. Two Arabidopsis T-DNA insertion mutants were identified as null mutants with early embryonic lethal phenotypes that could be rescued by GRXS15. Furthermore, we showed that recombinant GRXS15 is able to coordinate and transfer an ISC and that this coordination depends on reduced glutathione (GSH). We found the Arabidopsis GRXS15 able to complement growth defects based on disturbed ISC protein assembly of a yeast Δgrx5 mutant. Modeling of GRXS15 onto the crystal structures of related nonplant proteins highlighted amino acid residues that after mutation diminished GSH and subsequently ISC coordination, as well as the ability to rescue the yeast mutant. When used for plant complementation, one of these mutant variants, GRXS15K83/A, led to severe developmental delay and a pronounced decrease in aconitase activity by approximately 65%. These results indicate that mitochondrial GRXS15 is an essential protein in Arabidopsis, required for full activity of iron-sulfur proteins.
- Published
- 2015
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24. The deca-GX3 proteins Yae1-Lto1 function as adaptors recruiting the ABC protein Rli1 for iron-sulfur cluster insertion.
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Paul VD, Mühlenhoff U, Stümpfig M, Seebacher J, Kugler KG, Renicke C, Taxis C, Gavin AC, Pierik AJ, and Lill R
- Subjects
- Carrier Proteins genetics, Genetic Complementation Test, Humans, Intracellular Signaling Peptides and Proteins, Protein Binding, Protein Interaction Mapping, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, ATP-Binding Cassette Transporters metabolism, Carrier Proteins metabolism, Iron metabolism, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism, Sulfur metabolism
- Abstract
Cytosolic and nuclear iron-sulfur (Fe-S) proteins are involved in many essential pathways including translation and DNA maintenance. Their maturation requires the cytosolic Fe-S protein assembly (CIA) machinery. To identify new CIA proteins we employed systematic protein interaction approaches and discovered the essential proteins Yae1 and Lto1 as binding partners of the CIA targeting complex. Depletion of Yae1 or Lto1 results in defective Fe-S maturation of the ribosome-associated ABC protein Rli1, but surprisingly no other tested targets. Yae1 and Lto1 facilitate Fe-S cluster assembly on Rli1 in a chain of binding events. Lto1 uses its conserved C-terminal tryptophan for binding the CIA targeting complex, the deca-GX3 motifs in both Yae1 and Lto1 facilitate their complex formation, and Yae1 recruits Rli1. Human YAE1D1 and the cancer-related ORAOV1 can replace their yeast counterparts demonstrating evolutionary conservation. Collectively, the Yae1-Lto1 complex functions as a target-specific adaptor that recruits apo-Rli1 to the generic CIA machinery.
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- 2015
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25. Compartmentalization of iron between mitochondria and the cytosol and its regulation.
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Mühlenhoff U, Hoffmann B, Richter N, Rietzschel N, Spantgar F, Stehling O, Uzarska MA, and Lill R
- Subjects
- Biological Transport physiology, Heme biosynthesis, Homeostasis, Iron-Sulfur Proteins biosynthesis, Membrane Transport Proteins metabolism, Mitochondrial Proteins metabolism, Cell Compartmentation physiology, Cytosol metabolism, Iron metabolism, Mitochondria metabolism, Saccharomyces cerevisiae metabolism
- Abstract
Iron is essential for life. Its coordinated distribution between intracellular compartments and the adaptation of iron uptake to intracellular demands are central for a balanced iron homeostasis. Mitochondria take center stage in cellular iron metabolism as they harbor the two major iron-utilizing pathways, the synthesis of heme and the biogenesis of iron-sulfur (Fe/S) proteins. Consistent with this central role, mitochondria are also critical regulators of cellular iron homeostasis. They directly influence cellular iron uptake and the status of iron-utilizing metabolic processes through iron-dependent co-factors or by control of gene expression. For all these aspects of cellular iron metabolism, the uptake of iron into mitochondria is critical. During the last decade, considerable progress has been made with respect to the functional characterization of mitochondrial iron acquisition and the identification of transporters involved. The model organism Saccharomyces cerevisiae has been especially useful for the elucidation of this process. Here, we summarize the recent advances in the mechanism of mitochondrial iron transport and the impact of mitochondria on the regulation of cellular iron homeostasis., (Copyright © 2015 Elsevier GmbH. All rights reserved.)
- Published
- 2015
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26. The role of mitochondria and the CIA machinery in the maturation of cytosolic and nuclear iron-sulfur proteins.
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Lill R, Dutkiewicz R, Freibert SA, Heidenreich T, Mascarenhas J, Netz DJ, Paul VD, Pierik AJ, Richter N, Stümpfig M, Srinivasan V, Stehling O, and Mühlenhoff U
- Subjects
- Cell Nucleus metabolism, Humans, Membrane Transport Proteins metabolism, Protein Transport physiology, ATP-Binding Cassette Transporters metabolism, Cytosol metabolism, Iron-Sulfur Proteins metabolism, Mitochondria metabolism, Mitochondrial Proteins metabolism
- Abstract
Mitochondria have been derived from alpha-bacterial endosymbionts during the evolution of eukaryotes. Numerous bacterial functions have been maintained inside the organelles including fatty acid degradation, citric acid cycle, oxidative phosphorylation, and the synthesis of heme or lipoic acid cofactors. Additionally, mitochondria have inherited the bacterial iron-sulfur cluster assembly (ISC) machinery. Many of the ISC components are essential for cell viability because they generate a still unknown, sulfur-containing compound for the assembly of cytosolic and nuclear Fe/S proteins that perform important functions in, e.g., protein translation, DNA synthesis and repair, and chromosome segregation. The sulfur-containing compound is exported by the mitochondrial ABC transporter Atm1 (human ABCB7) and utilized by components of the cytosolic iron-sulfur protein assembly (CIA) machinery. An appealing minimal model for the striking compartmentation of eukaryotic Fe/S protein biogenesis is provided by organisms that contain mitosomes instead of mitochondria. Mitosomes have been derived from mitochondria by reductive evolution, during which they have lost virtually all classical mitochondrial tasks. Nevertheless, mitosomes harbor all core ISC components which presumably have been maintained for assisting the maturation of cytosolic-nuclear Fe/S proteins. The current review is centered around the Atm1 export process. We present an overview on the mitochondrial requirements for the export reaction, summarize recent insights into the 3D structure and potential mechanism of Atm1, and explain how the CIA machinery uses the mitochondrial export product for the assembly of cytosolic and nuclear Fe/S proteins., (Copyright © 2015 Elsevier GmbH. All rights reserved.)
- Published
- 2015
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27. The basic leucine zipper stress response regulator Yap5 senses high-iron conditions by coordination of [2Fe-2S] clusters.
- Author
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Rietzschel N, Pierik AJ, Bill E, Lill R, and Mühlenhoff U
- Subjects
- Animals, Cation Transport Proteins metabolism, Cysteine metabolism, Gene Expression Regulation, Fungal genetics, Saccharomyces cerevisiae genetics, Basic-Leucine Zipper Transcription Factors metabolism, Iron metabolism, Iron-Sulfur Proteins metabolism, Leucine Zippers genetics, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism, Sulfur metabolism
- Abstract
Iron is an essential, yet at elevated concentrations toxic trace element. To date, the mechanisms of iron sensing by eukaryotic iron-responsive transcription factors are poorly understood. The Saccharomyces cerevisiae transcription factor Yap5, a member of the Yap family of bZIP stress response regulators, administrates the adaptive response to high-iron conditions. Despite the central role of the iron-sensing process for cell viability, the molecule perceived by Yap5 and the underlying regulatory mechanisms are unknown. Here, we show that Yap5 senses high-iron conditions by two Fe/S clusters bound to its activator domain (Yap5-AD). The more stable iron-regulatory Fe/S cluster at the N-terminal cysteine-rich domain (n-CRD) of Yap5 is detected in vivo and in vitro. The second cluster coordinated by the C-terminal CRD can only be shown after chemical reconstitution, since it is bound in a labile fashion. Both clusters are of the [2Fe-2S] type as characterized by UV/visible (UV/Vis), circular dichroism, electron paramagnetic resonance (EPR), and Mössbauer spectroscopy. Fe/S cluster binding to Yap5-AD induces a conformational change that may activate transcription. The cluster-binding motif of the n-CRD domain is highly conserved in HapX-like transcription factors of pathogenic fungi and thus may represent a general sensor module common to many eukaryotic stress response regulators., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)
- Published
- 2015
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28. The role of mitochondria in cytosolic-nuclear iron–sulfur protein biogenesis and in cellular iron regulation.
- Author
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Lill R, Srinivasan V, and Mühlenhoff U
- Subjects
- ATP-Binding Cassette Transporters metabolism, Biological Transport, Cell Nucleus metabolism, Cytosol metabolism, Glutathione metabolism, Homeostasis, Oxidoreductases metabolism, Protein Binding, Iron metabolism, Iron-Sulfur Proteins metabolism, Mitochondria metabolism
- Abstract
Mitochondria are indispensable in eukaryotes because of their function in the maturation of cytosolic and nuclear iron–sulfur proteins that are essential for DNA synthesis and repair, tRNA modification, and protein translation. The mitochondrial Fe/S cluster assembly machinery not only generates the organelle's iron–sulfur proteins, but also extra-mitochondrial ones. Biogenesis of the latter proteins requires the mitochondrial ABC transporter Atm1 that exports a sulfur-containing compound in a glutathione-dependent fashion. The process is further assisted by the cytosolic iron–sulfur protein assembly machinery. Here, we discuss the knowns and unknowns of the mitochondrial export process that is also crucial for signaling the cellular iron status to the regulatory systems involved in the maintenance of cellular iron homeostasis.
- Published
- 2014
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29. Functional reconstitution of mitochondrial Fe/S cluster synthesis on Isu1 reveals the involvement of ferredoxin.
- Author
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Webert H, Freibert SA, Gallo A, Heidenreich T, Linne U, Amlacher S, Hurt E, Mühlenhoff U, Banci L, and Lill R
- Subjects
- Adrenodoxin chemistry, Biocatalysis, Chaetomium, Escherichia coli, Ferredoxin-NADP Reductase metabolism, Ferredoxins metabolism, Humans, Membrane Proteins metabolism, Mitochondria metabolism, Mitochondrial Proteins chemistry, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins chemistry, Sulfurtransferases metabolism, Adrenodoxin metabolism, Iron-Sulfur Proteins biosynthesis, Mitochondrial Proteins metabolism, Saccharomyces cerevisiae Proteins metabolism
- Abstract
Maturation of iron-sulphur (Fe/S) proteins involves complex biosynthetic machinery. In vivo synthesis of [2Fe-2S] clusters on the mitochondrial scaffold protein Isu1 requires the cysteine desulphurase complex Nfs1-Isd11, frataxin, ferredoxin Yah1 and its reductase Arh1. The roles of Yah1-Arh1 have remained enigmatic, because they are not required for in vitro Fe/S cluster assembly. Here, we reconstitute [2Fe-2S] cluster synthesis on Isu1 in a reaction depending on Nfs1-Isd11, frataxin, Yah1, Arh1 and NADPH. Unlike in the bacterial system, frataxin is an essential part of Fe/S cluster biosynthesis and is required simultaneously and stoichiometrically to Yah1. Reduced but not oxidized Yah1 tightly interacts with apo-Isu1 indicating a dynamic interaction between Yah1-apo-Isu1. Nuclear magnetic resonance structural studies identify the Yah1-apo-Isu1 interaction surface and suggest a pathway for electron flow from reduced ferredoxin to Isu1. Together, our study defines the molecular function of the ferredoxin Yah1 and its human orthologue FDX2 in mitochondrial Fe/S cluster synthesis.
- Published
- 2014
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30. The Janus transcription factor HapX controls fungal adaptation to both iron starvation and iron excess.
- Author
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Gsaller F, Hortschansky P, Beattie SR, Klammer V, Tuppatsch K, Lechner BE, Rietzschel N, Werner ER, Vogan AA, Chung D, Mühlenhoff U, Kato M, Cramer RA, Brakhage AA, and Haas H
- Subjects
- Aspergillosis genetics, Aspergillosis virology, Blotting, Western, Chromatin Immunoprecipitation, Fungal Proteins genetics, Homeostasis, Immunoprecipitation, Starvation, Surface Plasmon Resonance, Transcription Factors genetics, Vacuoles metabolism, Virulence, Adaptation, Physiological, Aspergillosis metabolism, Aspergillus fumigatus pathogenicity, Fungal Proteins metabolism, Gene Expression Regulation, Fungal, Iron metabolism, Transcription Factors metabolism
- Abstract
Balance of physiological levels of iron is essential for every organism. In Aspergillus fumigatus and other fungal pathogens, the transcription factor HapX mediates adaptation to iron limitation and consequently virulence by repressing iron consumption and activating iron uptake. Here, we demonstrate that HapX is also essential for iron resistance via activating vacuolar iron storage. We identified HapX protein domains that are essential for HapX functions during either iron starvation or high-iron conditions. The evolutionary conservation of these domains indicates their wide-spread role in iron sensing. We further demonstrate that a HapX homodimer and the CCAAT-binding complex (CBC) cooperatively bind an evolutionary conserved DNA motif in a target promoter. The latter reveals the mode of discrimination between general CBC and specific HapX/CBC target genes. Collectively, our study uncovers a novel regulatory mechanism mediating both iron resistance and adaptation to iron starvation by the same transcription factor complex with activating and repressing functions depending on ambient iron availability., (© 2014 The Authors. Published under the terms of the CC BY 4.0 license.)
- Published
- 2014
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31. The mitochondrial carrier Rim2 co-imports pyrimidine nucleotides and iron.
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Froschauer EM, Rietzschel N, Hassler MR, Binder M, Schweyen RJ, Lill R, Mühlenhoff U, and Wiesenberger G
- Subjects
- Binding Sites, Biological Transport, Cation Transport Proteins deficiency, Cation Transport Proteins genetics, Cations, Divalent, Heme biosynthesis, Mitochondria genetics, Mitochondrial Proteins deficiency, Mitochondrial Proteins genetics, Mutation, Nucleotide Transport Proteins genetics, Oxidation-Reduction, Protein Binding, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, Iron metabolism, Mitochondria metabolism, Mitochondrial Membranes metabolism, Nucleotide Transport Proteins metabolism, Pyrimidine Nucleotides metabolism, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism
- Abstract
Mitochondrial iron uptake is of key importance both for organelle function and cellular iron homoeostasis. The mitochondrial carrier family members Mrs3 and Mrs4 (homologues of vertebrate mitoferrin) function in organellar iron supply, yet other low efficiency transporters may exist. In Saccharomyces cerevisiae, overexpression of RIM2 (MRS12) encoding a mitochondrial pyrimidine nucleotide transporter can overcome the iron-related phenotypes of strains lacking both MRS3 and MRS4. In the present study we show by in vitro transport studies that Rim2 mediates the transport of iron and other divalent metal ions across the mitochondrial inner membrane in a pyrimidine nucleotide-dependent fashion. Mutations in the proposed substrate-binding site of Rim2 prevent both pyrimidine nucleotide and divalent ion transport. These results document that Rim2 catalyses the co-import of pyrimidine nucleotides and divalent metal ions including ferrous iron. The deletion of RIM2 alone has no significant effect on mitochondrial iron supply, Fe-S protein maturation and haem synthesis. However, RIM2 deletion in mrs3/4Δ cells aggravates their Fe-S protein maturation defect. We conclude that under normal physiological conditions Rim2 does not play a significant role in mitochondrial iron acquisition, yet, in the absence of the main iron transporters Mrs3 and Mrs4, this carrier can supply the mitochondrial matrix with iron in a pyrimidine-nucleotide-dependent fashion.
- Published
- 2013
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32. Crucial function of vertebrate glutaredoxin 3 (PICOT) in iron homeostasis and hemoglobin maturation.
- Author
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Haunhorst P, Hanschmann EM, Bräutigam L, Stehling O, Hoffmann B, Mühlenhoff U, Lill R, Berndt C, and Lillig CH
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Carrier Proteins genetics, Embryo, Nonmammalian embryology, Embryo, Nonmammalian metabolism, Glutaredoxins genetics, Glutaredoxins metabolism, HeLa Cells, Humans, Iron Regulatory Protein 1 metabolism, Iron Regulatory Protein 2 metabolism, Microscopy, Fluorescence, Molecular Sequence Data, RNA Interference, Sequence Homology, Amino Acid, Zebrafish embryology, Zebrafish genetics, Zebrafish metabolism, Zebrafish Proteins genetics, Zebrafish Proteins metabolism, Carrier Proteins metabolism, Hemoglobins metabolism, Homeostasis, Iron metabolism
- Abstract
The mechanisms by which eukaryotic cells handle and distribute the essential micronutrient iron within the cytosol and other cellular compartments are only beginning to emerge. The yeast monothiol multidomain glutaredoxins (Grx) 3 and 4 are essential for both transcriptional iron regulation and intracellular iron distribution. Despite the fact that the mechanisms of iron metabolism differ drastically in fungi and higher eukaryotes, the glutaredoxins are conserved, yet their precise function in vertebrates has remained elusive. Here we demonstrate a crucial role of the vertebrate-specific monothiol multidomain Grx3 (PICOT) in cellular iron homeostasis. During zebrafish embryonic development, depletion of Grx3 severely impairs the maturation of hemoglobin, the major iron-consuming process. Silencing of human Grx3 expression in HeLa cells decreases the activities of several cytosolic Fe/S proteins, for example, iron-regulatory protein 1, a major component of posttranscriptional iron regulation. As a consequence, Grx3-depleted cells show decreased levels of ferritin and increased levels of transferrin receptor, features characteristic of cellular iron starvation. Apparently, Grx3-deficient cells are unable to efficiently use iron, despite unimpaired cellular iron uptake. These data suggest an evolutionarily conserved role of cytosolic monothiol multidomain glutaredoxins in cellular iron metabolism pathways, including the biogenesis of Fe/S proteins and hemoglobin maturation.
- Published
- 2013
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33. The mitochondrial Hsp70 chaperone Ssq1 facilitates Fe/S cluster transfer from Isu1 to Grx5 by complex formation.
- Author
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Uzarska MA, Dutkiewicz R, Freibert SA, Lill R, and Mühlenhoff U
- Subjects
- Adenosine Diphosphate metabolism, Adenosine Triphosphate metabolism, Binding Sites, Cytosol metabolism, Electrophoresis, Polyacrylamide Gel, Glutaredoxins genetics, HSP70 Heat-Shock Proteins genetics, Immunoprecipitation, Iron-Sulfur Proteins genetics, Mitochondrial Proteins genetics, Molecular Chaperones genetics, Molecular Chaperones metabolism, Mutation, Protein Binding, Saccharomyces cerevisiae Proteins genetics, Glutaredoxins metabolism, HSP70 Heat-Shock Proteins metabolism, Iron-Sulfur Proteins metabolism, Mitochondria metabolism, Mitochondrial Proteins metabolism, Saccharomyces cerevisiae Proteins metabolism
- Abstract
The mitochondrial Hsp70 chaperone Ssq1 plays a dedicated role in the maturation of iron-sulfur (Fe/S) proteins, an essential process of mitochondria. Similar to its bacterial orthologue HscA, Ssq1 binds to the scaffold protein Isu1, thereby facilitating dissociation of the newly synthesized Fe/S cluster on Isu1 and its transfer to target apoproteins. Here we use in vivo and in vitro approaches to show that Ssq1 also interacts with the monothiol glutaredoxin 5 (Grx5) at a binding site different from that of Isu1. Grx5 binding does not stimulate the ATPase activity of Ssq1 and is most pronounced for the ADP-bound form of Ssq1, which interacts with Isu1 most tightly. The vicinity of Isu1 and Grx5 on the Hsp70 chaperone facilitates rapid Fe/S cluster transfer from Isu1 to Grx5. Grx5 and its bound Fe/S cluster are required for maturation of all cellular Fe/S proteins, regardless of the type of bound Fe/S cofactor and subcellular localization. Hence Grx5 functions as a late-acting component of the core Fe/S cluster (ISC) assembly machinery linking the Fe/S cluster synthesis reaction on Isu1 with late assembly steps involving Fe/S cluster targeting to dedicated apoproteins.
- Published
- 2013
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34. The role of mitochondria in cellular iron-sulfur protein biogenesis and iron metabolism.
- Author
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Lill R, Hoffmann B, Molik S, Pierik AJ, Rietzschel N, Stehling O, Uzarska MA, Webert H, Wilbrecht C, and Mühlenhoff U
- Subjects
- Animals, Fungi metabolism, Gene Expression Regulation, Heme biosynthesis, Homeostasis physiology, Humans, Ion Transport physiology, Iron Deficiencies, Iron-Binding Proteins genetics, Iron-Binding Proteins metabolism, Iron-Sulfur Proteins genetics, Mitochondrial Proteins genetics, Molecular Chaperones genetics, Molecular Chaperones metabolism, Oxidation-Reduction, Frataxin, Iron metabolism, Iron-Sulfur Proteins metabolism, Mitochondria metabolism, Mitochondrial Proteins metabolism
- Abstract
Mitochondria play a key role in iron metabolism in that they synthesize heme, assemble iron-sulfur (Fe/S) proteins, and participate in cellular iron regulation. Here, we review the latter two topics and their intimate connection. The mitochondrial Fe/S cluster (ISC) assembly machinery consists of 17 proteins that operate in three major steps of the maturation process. First, the cysteine desulfurase complex Nfs1-Isd11 as the sulfur donor cooperates with ferredoxin-ferredoxin reductase acting as an electron transfer chain, and frataxin to synthesize an [2Fe-2S] cluster on the scaffold protein Isu1. Second, the cluster is released from Isu1 and transferred toward apoproteins with the help of a dedicated Hsp70 chaperone system and the glutaredoxin Grx5. Finally, various specialized ISC components assist in the generation of [4Fe-4S] clusters and cluster insertion into specific target apoproteins. Functional defects of the core ISC assembly machinery are signaled to cytosolic or nuclear iron regulatory systems resulting in increased cellular iron acquisition and mitochondrial iron accumulation. In fungi, regulation is achieved by iron-responsive transcription factors controlling the expression of genes involved in iron uptake and intracellular distribution. They are assisted by cytosolic multidomain glutaredoxins which use a bound Fe/S cluster as iron sensor and additionally perform an essential role in intracellular iron delivery to target metalloproteins. In mammalian cells, the iron regulatory proteins IRP1, an Fe/S protein, and IRP2 act in a post-transcriptional fashion to adjust the cellular needs for iron. Thus, Fe/S protein biogenesis and cellular iron metabolism are tightly linked to coordinate iron supply and utilization. This article is part of a Special Issue entitled: Cell Biology of Metals., (Copyright © 2012 Elsevier B.V. All rights reserved.)
- Published
- 2012
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35. The human mitochondrial ISCA1, ISCA2, and IBA57 proteins are required for [4Fe-4S] protein maturation.
- Author
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Sheftel AD, Wilbrecht C, Stehling O, Niggemeyer B, Elsässer HP, Mühlenhoff U, and Lill R
- Subjects
- Cytosol metabolism, HeLa Cells, Homeostasis, Humans, Iron metabolism, Iron-Sulfur Proteins antagonists & inhibitors, Iron-Sulfur Proteins genetics, Microscopy, Electron, Transmission, Mitochondria metabolism, Mitochondria pathology, Mitochondrial Proteins antagonists & inhibitors, Mitochondrial Proteins genetics, Protein Multimerization, Protein Processing, Post-Translational, RNA Interference, RNA, Small Interfering genetics, Signal Transduction, Iron-Sulfur Proteins chemistry, Iron-Sulfur Proteins metabolism, Mitochondrial Proteins metabolism
- Abstract
Members of the bacterial and mitochondrial iron-sulfur cluster (ISC) assembly machinery include the so-called A-type ISC proteins, which support the assembly of a subset of Fe/S apoproteins. The human genome encodes two A-type proteins, termed ISCA1 and ISCA2, which are related to Saccharomyces cerevisiae Isa1 and Isa2, respectively. An additional protein, Iba57, physically interacts with Isa1 and Isa2 in yeast. To test the cellular role of human ISCA1, ISCA2, and IBA57, HeLa cells were depleted for any of these proteins by RNA interference technology. Depleted cells contained massively swollen and enlarged mitochondria that were virtually devoid of cristae membranes, demonstrating the importance of these proteins for mitochondrial biogenesis. The activities of mitochondrial [4Fe-4S] proteins, including aconitase, respiratory complex I, and lipoic acid synthase, were diminished following depletion of the three proteins. In contrast, the mitochondrial [2Fe-2S] enzyme ferrochelatase and cellular heme content were unaffected. We further provide evidence against a localization and direct Fe/S protein maturation function of ISCA1 and ISCA2 in the cytosol. Taken together, our data suggest that ISCA1, ISCA2, and IBA57 are specifically involved in the maturation of mitochondrial [4Fe-4S] proteins functioning late in the ISC assembly pathway.
- Published
- 2012
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36. Specialized function of yeast Isa1 and Isa2 proteins in the maturation of mitochondrial [4Fe-4S] proteins.
- Author
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Mühlenhoff U, Richter N, Pines O, Pierik AJ, and Lill R
- Subjects
- DNA-Binding Proteins genetics, Iron metabolism, Iron-Sulfur Proteins genetics, Mitochondrial Proteins genetics, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, Sulfur metabolism, Transcription Factors genetics, DNA-Binding Proteins metabolism, Iron-Sulfur Proteins metabolism, Mitochondrial Proteins metabolism, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism, Transcription Factors metabolism
- Abstract
Most eukaryotes contain iron-sulfur cluster (ISC) assembly proteins related to Saccharomyces cerevisiae Isa1 and Isa2. We show here that Isa1 but not Isa2 can be functionally replaced by the bacterial relatives IscA, SufA, and ErpA. The specific function of these "A-type" ISC proteins within the framework of mitochondrial and bacterial Fe/S protein biogenesis is still unresolved. In a comprehensive in vivo analysis, we show that S. cerevisiae Isa1 and Isa2 form a complex that is required for maturation of mitochondrial [4Fe-4S] proteins, including aconitase and homoaconitase. In contrast, Isa1-Isa2 were dispensable for the generation of mitochondrial [2Fe-2S] proteins and cytosolic [4Fe-4S] proteins. Targeting of bacterial [2Fe-2S] and [4Fe-4S] ferredoxins to yeast mitochondria further supported this specificity. Isa1 and Isa2 proteins are shown to bind iron in vivo, yet the Isa1-Isa2-bound iron was not needed as a donor for de novo assembly of the [2Fe-2S] cluster on the general Fe/S scaffold proteins Isu1-Isu2. Upon depletion of the ISC assembly factor Iba57, which specifically interacts with Isa1 and Isa2, or in the absence of the major mitochondrial [4Fe-4S] protein aconitase, iron accumulated on the Isa proteins. These results suggest that the iron bound to the Isa proteins is required for the de novo synthesis of [4Fe-4S] clusters in mitochondria and for their insertion into apoproteins in a reaction mediated by Iba57. Taken together, these findings define Isa1, Isa2, and Iba57 as a specialized, late-acting ISC assembly subsystem that is specifically dedicated to the maturation of mitochondrial [4Fe-4S] proteins.
- Published
- 2011
- Full Text
- View/download PDF
37. Coenzyme Q biosynthesis: Coq6 is required for the C5-hydroxylation reaction and substrate analogs rescue Coq6 deficiency.
- Author
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Ozeir M, Mühlenhoff U, Webert H, Lill R, Fontecave M, and Pierrel F
- Subjects
- Adrenodoxin metabolism, Chromatography, High Pressure Liquid, Ferredoxin-NADP Reductase metabolism, Hydroxybenzoates chemistry, Hydroxybenzoates pharmacology, Hydroxylation, Membrane Proteins metabolism, Mutation, Parabens chemistry, Parabens pharmacology, Saccharomyces cerevisiae drug effects, Saccharomyces cerevisiae Proteins metabolism, Ubiquinone deficiency, Ubiquinone genetics, Ubiquinone metabolism, Vanillic Acid chemistry, Vanillic Acid pharmacology, Saccharomyces cerevisiae enzymology, Ubiquinone biosynthesis
- Abstract
Coenzyme Q (Q), an essential component of eukaryotic cells, is synthesized by several enzymes from the precursor 4-hydroxybenzoic acid. Mutations in six of the Q biosynthesis genes cause diseases that can sometimes be ameliorated by oral Q supplementation. We establish here that Coq6, a predicted flavin-dependent monooxygenase, is involved exclusively in the C5-hydroxylation reaction. In an unusual way, the ferredoxin Yah1 and the ferredoxin reductase Arh1 may be the in vivo source of electrons for Coq6. We also show that hydroxylated analogs of 4-hydroxybenzoic acid, such as vanillic acid or 3,4-dihydroxybenzoic acid, restore Q biosynthesis and respiration in a Saccharomyces cerevisiae coq6 mutant. Our results demonstrate that appropriate analogs of 4-hydroxybenzoic acid can bypass a deficient Q biosynthetic enzyme and might be considered for the treatment of some primary Q deficiencies., (Copyright © 2011 Elsevier Ltd. All rights reserved.)
- Published
- 2011
- Full Text
- View/download PDF
38. The oxidative stress response in yeast cells involves changes in the stability of Aft1 regulon mRNAs.
- Author
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Castells-Roca L, Mühlenhoff U, Lill R, Herrero E, and Bellí G
- Subjects
- Ceruloplasmin metabolism, Hydrogen Peroxide toxicity, Iron metabolism, Membrane Transport Proteins metabolism, Oxidants toxicity, Gene Expression Regulation, Fungal, Oxidative Stress, RNA Stability, Regulon, Saccharomyces cerevisiae drug effects, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins metabolism, Transcription Factors metabolism
- Abstract
Saccharomyces cerevisiae can import iron through a high-affinity system consisting of the Ftr1/Fet3-mediated reductive pathway and the siderophore-mediated non-reductive one. Expression of components of the high-affinity system is controlled by the Aft1 transcriptional factor. In this study we show that, upon oxidative stress, Aft1 is transitorily internalized into the nucleus, followed by transcription activation of components of its regulon. In these conditions, the mRNA levels of the genes of the non-reductive pathway become increased, while those of FTR1 and FET3 remain low because of destabilization of the mRNAs. Consequently, the respective protein levels also remain low. Such mRNA destabilization is mediated by the general 5'-3' mRNA decay pathway and is independent of the RNA binding protein Cth2. Yeast cells are hypersensitive to peroxides in growth conditions where only the high-affinity reductive pathway is functional for iron assimilation. On the contrary, peroxide does not affect growth when iron uptake occurs exclusively through the non-reductive pathway. This reinforces the idea that upon oxidative stress S. cerevisiae cells redirect iron assimilation through the non-reductive pathway to minimize oxidative damage by the ferrous ions, which are formed during iron import through the Ftr1/Fet3 complexes., (© 2011 Blackwell Publishing Ltd.)
- Published
- 2011
- Full Text
- View/download PDF
39. The multidomain thioredoxin-monothiol glutaredoxins represent a distinct functional group.
- Author
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Hoffmann B, Uzarska MA, Berndt C, Godoy JR, Haunhorst P, Lillig CH, Lill R, and Mühlenhoff U
- Subjects
- Catalytic Domain, Glutaredoxins genetics, Glutaredoxins metabolism, Protein Structure, Tertiary, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins genetics, Schizosaccharomyces genetics, Schizosaccharomyces metabolism, Schizosaccharomyces pombe Proteins genetics, Thioredoxins genetics, Thioredoxins metabolism, Saccharomyces cerevisiae Proteins metabolism, Schizosaccharomyces pombe Proteins metabolism
- Abstract
Monothiol glutaredoxins (Grxs) with a noncanonical CGFS active site are found in all kingdoms of life. They include members with a single domain and thioredoxin-Grx fusion proteins. In Saccharomyces cerevisiae, the multidomain Grx3 and Grx4 play an essential role in intracellular iron trafficking. This crucial task is mediated by an essential Fe/S cofactor. This study shows that this unique physiological role cannot be executed by single domain Grxs, because the thioredoxin domain is indispensable for function in vivo. Mutational analysis revealed that a CPxS active site motif is fully compatible with Fe/S cluster binding on Grx4, while a dithiol active site results in cofactor destabilization and a moderate impairment of in vivo function. These requirements for Fe/S cofactor stabilization on Grx4 are virtually the opposite of those previously reported for single domain Grxs. Grx4 functions as iron sensor for the iron-sensing transcription factor Aft1 in S. cerevisiae. We found that Aft1 binds to a conserved binding site at the C-terminus of Grx4. This interaction is essential for the regulation of Aft1. Collectively, our analysis demonstrates that the multidomain monothiol Grxs form a unique protein family distinct from that of the single domain Grxs.
- Published
- 2011
- Full Text
- View/download PDF
40. EGFR mutation detection in NSCLC--assessment of diagnostic application and recommendations of the German Panel for Mutation Testing in NSCLC.
- Author
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Penzel R, Sers C, Chen Y, Lehmann-Mühlenhoff U, Merkelbach-Bruse S, Jung A, Kirchner T, Büttner R, Kreipe HH, Petersen I, Dietel M, and Schirmacher P
- Subjects
- Exons genetics, Genetic Testing methods, Germany, Health Planning Guidelines, Humans, Single-Blind Method, Carcinoma, Non-Small-Cell Lung diagnosis, Carcinoma, Non-Small-Cell Lung genetics, Early Detection of Cancer methods, ErbB Receptors genetics, Lung Neoplasms diagnosis, Lung Neoplasms genetics, Mutation genetics
- Abstract
EGFR mutation testing in non-small cell lung cancer (NSCLC) is a novel and important molecular pathological diagnostic assay that is predictive of response to anti-epidermal growth factor receptor (EGFR) therapy. A comprehensive compilation of a large number of EGFR mutation analyses of the German Panel for Mutation Analyses in NSCLC demonstrates (a) a higher than previously reported mutation frequency outside the conventionally tested exons 19 and 21 and (b) an overall superiority of sequencing based assays over mutation-specific PCR. The implications for future diagnostic EGFR mutation testing are discussed.
- Published
- 2011
- Full Text
- View/download PDF
41. Cytosolic monothiol glutaredoxins function in intracellular iron sensing and trafficking via their bound iron-sulfur cluster.
- Author
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Mühlenhoff U, Molik S, Godoy JR, Uzarska MA, Richter N, Seubert A, Zhang Y, Stubbe J, Pierrel F, Herrero E, Lillig CH, and Lill R
- Subjects
- Biological Transport, Iron-Sulfur Proteins, Oxidation-Reduction, Oxidoreductases metabolism, Proteins metabolism, Saccharomyces cerevisiae Proteins metabolism, Sulfur metabolism, Yeasts metabolism, Cytosol metabolism, Glutaredoxins metabolism, Iron metabolism
- Abstract
Iron is an essential nutrient for cells. It is unknown how iron, after its import into the cytosol, is specifically delivered to iron-dependent processes in various cellular compartments. Here, we identify an essential function of the conserved cytosolic monothiol glutaredoxins Grx3 and Grx4 in intracellular iron trafficking and sensing. Depletion of Grx3/4 specifically impaired all iron-requiring reactions in the cytosol, mitochondria, and nucleus, including the synthesis of Fe/S clusters, heme, and di-iron centers. These defects were caused by impairment of iron insertion into proteins and iron transfer to mitochondria, indicating that intracellular iron is not bioavailable, despite highly elevated cytosolic levels. The crucial task of Grx3/4 is mediated by a bridging, glutathione-containing Fe/S center that functions both as an iron sensor and in intracellular iron delivery. Collectively, our study uncovers an important role of monothiol glutaredoxins in cellular iron metabolism, with a surprising connection to cellular redox and sulfur metabolisms., (Copyright © 2010 Elsevier Inc. All rights reserved.)
- Published
- 2010
- Full Text
- View/download PDF
42. Tah18 transfers electrons to Dre2 in cytosolic iron-sulfur protein biogenesis.
- Author
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Netz DJ, Stümpfig M, Doré C, Mühlenhoff U, Pierik AJ, and Lill R
- Subjects
- Cytosol chemistry, Electron Transport, Flavoproteins genetics, Flavoproteins metabolism, Humans, Intracellular Signaling Peptides and Proteins chemistry, Intracellular Signaling Peptides and Proteins metabolism, Iron-Sulfur Proteins chemistry, Mitochondrial Proteins metabolism, NADP metabolism, Nuclear Proteins biosynthesis, Nuclear Proteins metabolism, Oxidoreductases deficiency, Oxidoreductases genetics, Saccharomyces cerevisiae cytology, Saccharomyces cerevisiae Proteins chemistry, Saccharomyces cerevisiae Proteins genetics, Sulfurtransferases metabolism, Time Factors, Cytosol metabolism, Electrons, Iron-Sulfur Proteins biosynthesis, Iron-Sulfur Proteins metabolism, Oxidoreductases metabolism, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins biosynthesis, Saccharomyces cerevisiae Proteins metabolism
- Abstract
Cytosolic and nuclear iron-sulfur (Fe-S) proteins play key roles in processes such as ribosome maturation, transcription and DNA repair-replication. For biosynthesis of their Fe-S clusters, a dedicated cytosolic Fe-S protein assembly (CIA) machinery is required. Here, we identify the essential flavoprotein Tah18 as a previously unrecognized CIA component and show by cell biological, biochemical and spectroscopic approaches that the complex of Tah18 and the CIA protein Dre2 is part of an electron transfer chain functioning in an early step of cytosolic Fe-S protein biogenesis. Electrons are transferred from NADPH via the FAD- and FMN-containing Tah18 to the Fe-S clusters of Dre2. This electron transfer chain is required for assembly of target but not scaffold Fe-S proteins, suggesting a need for reduction in the generation of stably inserted Fe-S clusters. The pathway is conserved in eukaryotes, as human Ndor1-Ciapin1 proteins can functionally replace yeast Tah18-Dre2.
- Published
- 2010
- Full Text
- View/download PDF
43. Humans possess two mitochondrial ferredoxins, Fdx1 and Fdx2, with distinct roles in steroidogenesis, heme, and Fe/S cluster biosynthesis.
- Author
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Sheftel AD, Stehling O, Pierik AJ, Elsässer HP, Mühlenhoff U, Webert H, Hobler A, Hannemann F, Bernhardt R, and Lill R
- Subjects
- Adrenodoxin antagonists & inhibitors, Adrenodoxin genetics, Ferredoxins antagonists & inhibitors, Ferredoxins genetics, HeLa Cells, Humans, Iron metabolism, Iron-Sulfur Proteins chemistry, Mitochondrial Proteins antagonists & inhibitors, Mitochondrial Proteins genetics, Models, Biological, Protein Isoforms antagonists & inhibitors, Protein Isoforms chemistry, Protein Isoforms genetics, Protein Isoforms metabolism, RNA Interference, RNA, Small Interfering genetics, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism, Species Specificity, Adrenodoxin chemistry, Adrenodoxin metabolism, Ferredoxins chemistry, Ferredoxins metabolism, Heme biosynthesis, Mitochondrial Proteins chemistry, Mitochondrial Proteins metabolism, Steroids biosynthesis
- Abstract
Mammalian adrenodoxin (ferredoxin 1; Fdx1) is essential for the synthesis of various steroid hormones in adrenal glands. As a member of the [2Fe-2S] cluster-containing ferredoxin family, Fdx1 reduces mitochondrial cytochrome P450 enzymes, which then catalyze; e.g., the conversion of cholesterol to pregnenolone, aldosterone, and cortisol. The high protein sequence similarity between Fdx1 and its yeast adrenodoxin homologue (Yah1) suggested that Fdx1, like Yah1, may be involved in the biosynthesis of heme A and Fe/S clusters, two versatile and essential protein cofactors. Our study, employing RNAi technology to deplete human Fdx1, did not confirm this expectation. Instead, we identified a Fdx1-related mitochondrial protein, designated ferredoxin 2 (Fdx2) and found it to be essential for heme A and Fe/S protein biosynthesis. Unlike Fdx1, Fdx2 was unable to efficiently reduce mitochondrial cytochromes P450 and convert steroids, indicating that the two ferredoxin isoforms are highly specific for their substrates in distinct biochemical pathways. Moreover, Fdx2 deficiency had a severe impact, via impaired Fe/S protein biogenesis, on cellular iron homeostasis, leading to increased cellular iron uptake and iron accumulation in mitochondria. We conclude that mammals depend on two distinct mitochondrial ferredoxins for the specific production of either steroid hormones or heme A and Fe/S proteins.
- Published
- 2010
- Full Text
- View/download PDF
44. Involvement of mitochondrial ferredoxin and para-aminobenzoic acid in yeast coenzyme Q biosynthesis.
- Author
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Pierrel F, Hamelin O, Douki T, Kieffer-Jaquinod S, Mühlenhoff U, Ozeir M, Lill R, and Fontecave M
- Subjects
- Aminophenols metabolism, Mutation, Oxidation-Reduction, 4-Aminobenzoic Acid metabolism, Adrenodoxin metabolism, Ferredoxin-NADP Reductase metabolism, Membrane Proteins metabolism, Mitochondria metabolism, Parabens metabolism, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism, Ubiquinone biosynthesis
- Abstract
Yeast ubiquinone or coenzyme Q(6) (Q(6)) is a redox active lipid that plays a crucial role in the mitochondrial electron transport chain. At least nine proteins (Coq1p-9p) participate in Q(6) biosynthesis from 4-hydroxybenzoate (4-HB). We now show that the mitochondrial ferredoxin Yah1p and the ferredoxin reductase Arh1p are required for Q(6) biosynthesis, probably for the first hydroxylation of the pathway. Conditional Gal-YAH1 and Gal-ARH1 mutants accumulate 3-hexaprenyl-4-hydroxyphenol and 3-hexaprenyl-4-aminophenol. Para-aminobenzoic acid (pABA) is shown to be the precursor of 3-hexaprenyl-4-aminophenol and to compete with 4-HB for the prenylation reaction catalyzed by Coq2p. Yeast cells convert U-((13)C)-pABA into (13)C ring-labeled Q(6), a result that identifies pABA as a new precursor of Q(6) and implies an additional NH(2)-to-OH conversion in Q(6) biosynthesis. Our study identifies pABA, Yah1p, and Arh1p as three actors in Q(6) biosynthesis., (2010 Elsevier Ltd. All rights reserved.)
- Published
- 2010
- Full Text
- View/download PDF
45. Iron regulation through the back door: iron-dependent metabolite levels contribute to transcriptional adaptation to iron deprivation in Saccharomyces cerevisiae.
- Author
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Ihrig J, Hausmann A, Hain A, Richter N, Hamza I, Lill R, and Mühlenhoff U
- Subjects
- 3-Isopropylmalate Dehydrogenase genetics, CCAAT-Binding Factor genetics, Ceruloplasmin genetics, Cytochromes c genetics, Cytochromes c metabolism, DNA-Binding Proteins genetics, Down-Regulation genetics, Ferrochelatase metabolism, Gene Expression drug effects, Gene Expression genetics, Hydro-Lyases genetics, Hydro-Lyases metabolism, Iron Chelating Agents pharmacology, Iron Deficiencies, Isomerases genetics, Isomerases metabolism, Malates pharmacology, Peroxidases genetics, Phenanthrolines pharmacology, Promoter Regions, Genetic genetics, Saccharomyces cerevisiae drug effects, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism, Terminator Regions, Genetic genetics, Trans-Activators genetics, Transcription Factors genetics, Tristetraprolin genetics, Up-Regulation genetics, Gene Expression Regulation, Fungal physiology, Heme metabolism, Homeostasis physiology, Iron metabolism, Malates metabolism, Saccharomyces cerevisiae metabolism
- Abstract
Budding yeast (Saccharomyces cerevisiae) responds to iron deprivation both by Aft1-Aft2-dependent transcriptional activation of genes involved in cellular iron uptake and by Cth1-Cth2-specific degradation of certain mRNAs coding for iron-dependent biosynthetic components. Here, we provide evidence for a novel principle of iron-responsive gene expression. This regulatory mechanism is based on the modulation of transcription through the iron-dependent variation of levels of regulatory metabolites. As an example, the LEU1 gene of branched-chain amino acid biosynthesis is downregulated under iron-limiting conditions through depletion of the metabolic intermediate alpha-isopropylmalate, which functions as a key transcriptional coactivator of the Leu3 transcription factor. Synthesis of alpha-isopropylmalate involves the iron-sulfur protein Ilv3, which is inactivated under iron deficiency. As another example, decreased mRNA levels of the cytochrome c-encoding CYC1 gene under iron-limiting conditions involve heme-dependent transcriptional regulation via the Hap1 transcription factor. Synthesis of the iron-containing heme is directly correlated with iron availability. Thus, the iron-responsive expression of genes that are downregulated under iron-limiting conditions is conferred by two independent regulatory mechanisms: transcriptional regulation through iron-responsive metabolites and posttranscriptional mRNA degradation. Only the combination of the two processes provides a quantitative description of the response to iron deprivation in yeast.
- Published
- 2010
- Full Text
- View/download PDF
46. Crucial role of conserved cysteine residues in the assembly of two iron-sulfur clusters on the CIA protein Nar1.
- Author
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Urzica E, Pierik AJ, Mühlenhoff U, and Lill R
- Subjects
- Amino Acid Motifs genetics, Cell Survival genetics, Cell Survival physiology, Cysteine genetics, Cysteine physiology, Cytosol metabolism, Electron Spin Resonance Spectroscopy, Hydrogenase genetics, Iron-Sulfur Proteins genetics, Models, Molecular, Mutagenesis, Site-Directed, Protein Structure, Tertiary genetics, Recombinant Proteins genetics, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Saccharomyces cerevisiae chemistry, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae growth & development, Saccharomyces cerevisiae Proteins genetics, Conserved Sequence genetics, Cysteine chemistry, Cytosol chemistry, Hydrogenase chemistry, Hydrogenase physiology, Iron-Sulfur Proteins chemistry, Iron-Sulfur Proteins physiology, Saccharomyces cerevisiae Proteins chemistry, Saccharomyces cerevisiae Proteins physiology
- Abstract
Iron-sulfur (Fe/S) protein maturation in the eukaryotic cytosol and nucleus requires conserved components of the essential CIA machinery. The CIA protein Nar1 performs a specific function in transferring an Fe/S cluster that is assembled de novo on the Cfd1-Nbp35 scaffold to apoproteins. Here, we used systematic site-directed mutagenesis and a combination of in vitro and in vivo studies to show that Nar1 holds two Fe/S clusters at conserved N- and C-terminal cysteine motifs. A wealth of biochemical studies suggests that the assembly of these Fe/S clusters on Nar1 cannot be studied in Escherichia coli, as the recombinant protein does not contain the native Fe/S clusters. We therefore followed Fe/S cluster incorporation directly in yeast by a (55)Fe radiolabeling method in vivo, and we measured the functional consequences of Nar1 mutations in the assembly of cytosolic Fe/S proteins. We find that both Fe/S clusters are essential for Nar1 function and cell viability. Molecular modeling using a structurally but not functionally related bacterial iron-only hydrogenase as a template provided compelling structural explanations for our mutational data. The C-terminal Fe/S cluster is stably buried within Nar1, whereas the N-terminal one is exposed at the protein surface and hence may be more easily lost. Insertion of an Fe/S cluster into the C-terminal location depends on the N-terminal motif, suggesting the participation of the latter motif in the assembly process of the C-terminal cluster. The vicinity of the two Fe/S centers suggests a close functional cooperation during cytosolic Fe/S protein maturation.
- Published
- 2009
- Full Text
- View/download PDF
47. Saccharomyces cerevisiae Grx6 and Grx7 are monothiol glutaredoxins associated with the early secretory pathway.
- Author
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Izquierdo A, Casas C, Mühlenhoff U, Lillig CH, and Herrero E
- Subjects
- DNA-Binding Proteins metabolism, Endoplasmic Reticulum metabolism, Endoplasmic Reticulum ultrastructure, Enzyme Inhibitors pharmacology, Gene Expression Regulation, Fungal drug effects, Gene Expression Regulation, Fungal physiology, Glutaredoxins genetics, Glycosylation drug effects, Golgi Apparatus metabolism, Golgi Apparatus ultrastructure, Oxidoreductases Acting on Sulfur Group Donors metabolism, Saccharomyces cerevisiae Proteins genetics, Secretory Vesicles genetics, Secretory Vesicles metabolism, Secretory Vesicles ultrastructure, Transcription Factors metabolism, Glutaredoxins metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism, Signal Transduction genetics
- Abstract
Saccharomyces cerevisiae Grx6 and Grx7 are two monothiol glutaredoxins whose active-site sequences (CSYS and CPYS, respectively) are reminiscent of the CPYC active-site sequence of classical dithiol glutaredoxins. Both proteins contain an N-terminal transmembrane domain which is responsible for their association to membranes of the early secretory pathway vesicles, facing the luminal side. Thus, Grx6 localizes at the endoplasmic reticulum and Golgi compartments, while Grx7 is mostly at the Golgi. Expression of GRX6 is modestly upregulated by several stresses (calcium, sodium, and peroxides) in a manner dependent on the Crz1-calcineurin pathway. Some of these stresses also upregulate GRX7 expression under the control of the Msn2/4 transcription factor. The N glycosylation inhibitor tunicamycin induces the expression of both genes along with protein accumulation. Mutants lacking both glutaredoxins display reduced sensitivity to tunicamycin, although the drug is still able to manifest its inhibitory effect on a reporter glycoprotein. Grx6 and Grx7 have measurable oxidoreductase activity in vivo, which is increased in the presence of tunicamycin. Both glutaredoxins could be responsible for the regulation of the sulfhydryl oxidative state at the oxidant conditions of the early secretory pathway vesicles. However, the differences in location and expression responses against stresses suggest that their functions are not totally overlapping.
- Published
- 2008
- Full Text
- View/download PDF
48. Cellular and mitochondrial remodeling upon defects in iron-sulfur protein biogenesis.
- Author
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Hausmann A, Samans B, Lill R, and Mühlenhoff U
- Subjects
- ATP-Binding Cassette Transporters genetics, ATP-Binding Cassette Transporters metabolism, Adenosine Triphosphatases genetics, Adenosine Triphosphatases metabolism, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Fungal Proteins genetics, Fungal Proteins metabolism, GTP-Binding Proteins genetics, GTP-Binding Proteins metabolism, Gene Deletion, Gene Expression Regulation, Fungal, Heme metabolism, Homeostasis, Iron metabolism, Iron-Sulfur Proteins genetics, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism, Transcription Factors genetics, Transcription Factors metabolism, Transcription, Genetic genetics, Iron-Sulfur Proteins biosynthesis, Mitochondria metabolism
- Abstract
Biogenesis of iron-sulfur (Fe/S) proteins in eukaryotes is an essential process involving the mitochondrial iron-sulfur cluster (ISC) assembly and export machineries and the cytosolic iron/sulfur protein assembly (CIA) apparatus. To define the integration of Fe/S protein biogenesis into cellular homeostasis, we compared the global transcriptional responses to defects in the three biogenesis systems in Saccharomyces cerevisiae using DNA microarrays. Depletion of a member of the CIA machinery elicited only weak (up to 2-fold) alterations in gene expression with no clear preference for any specific cellular process. In contrast, depletion of components of the mitochondrial ISC assembly and export systems induced strong and largely overlapping transcriptional responses of more than 200 genes (2-100-fold changes). These alterations were strikingly similar, yet not identical, to the transcriptional profiles developed upon iron starvation. Hence, mitochondria and their ISC systems serve as primary physiological regulators exerting a global control of numerous iron-dependent processes. First, ISC depletion activates the iron-responsive transcription factors Aft1/2p leading to increased cellular iron acquisition. Second, respiration and heme metabolism are repressed ensuring the balanced utilization of iron by the two major iron-consuming processes, iron-sulfur protein and heme biosynthesis. Third, the decreased respiratory activity is compensated by induction of genes involved in glucose acquisition. Finally, transcriptional remodeling of the citric acid cycle and the biosyntheses of ergosterol and biotin reflect the iron dependence of these pathways. Together, our data suggest a model in which mitochondria perform a global regulatory role in numerous cellular processes linked to iron homeostasis.
- Published
- 2008
- Full Text
- View/download PDF
49. Mitochondrial Iba57p is required for Fe/S cluster formation on aconitase and activation of radical SAM enzymes.
- Author
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Gelling C, Dawes IW, Richhardt N, Lill R, and Mühlenhoff U
- Subjects
- Carrier Proteins genetics, Enzyme Activation, Iron-Sulfur Proteins genetics, Mitochondria metabolism, Mutation, Plasmids, Saccharomyces cerevisiae enzymology, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Aconitate Hydratase metabolism, Carrier Proteins biosynthesis, Iron-Sulfur Proteins biosynthesis, Mitochondrial Proteins metabolism, S-Adenosylmethionine chemistry, Saccharomyces cerevisiae Proteins metabolism, Sulfurtransferases metabolism
- Abstract
A genome-wide screen for Saccharomyces cerevisiae iron-sulfur (Fe/S) cluster assembly mutants identified the gene IBA57. The encoded protein Iba57p is located in the mitochondrial matrix and is essential for mitochondrial DNA maintenance. The growth phenotypes of an iba57Delta mutant and extensive functional studies in vivo and in vitro indicate a specific role for Iba57p in the maturation of mitochondrial aconitase-type and radical SAM Fe/S proteins (biotin and lipoic acid synthases). Maturation of other Fe/S proteins occurred normally in the absence of Iba57p. These observations identify Iba57p as a novel dedicated maturation factor with specificity for a subset of Fe/S proteins. The Iba57p primary sequence is distinct from any known Fe/S assembly factor but is similar to certain tetrahydrofolate-binding enzymes, adding a surprising new function to this protein family. Iba57p physically interacts with the mitochondrial ISC assembly components Isa1p and Isa2p. Since all three proteins are conserved in eukaryotes and bacteria, the specificity of the Iba57/Isa complex may represent a biosynthetic concept that is universally used in nature. In keeping with this idea, the human IBA57 homolog C1orf69 complements the iba57Delta growth defects, demonstrating its conserved function throughout the eukaryotic kingdom.
- Published
- 2008
- Full Text
- View/download PDF
50. Maturation of iron-sulfur proteins in eukaryotes: mechanisms, connected processes, and diseases.
- Author
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Lill R and Mühlenhoff U
- Subjects
- Animals, Apoproteins chemistry, Cytosol metabolism, Enzyme Activation, Gene Expression Regulation, Fungal, Humans, Iron chemistry, Mitochondria metabolism, Models, Biological, RNA, Transfer chemistry, Sulfur chemistry, Carbon-Sulfur Lyases chemistry, Ferredoxins chemistry, Iron-Sulfur Proteins chemistry
- Abstract
Iron-sulfur (Fe/S) proteins are involved in a wide variety of cellular processes such as enzymatic reactions, respiration, cofactor biosynthesis, ribosome biogenesis, regulation of gene expression, and DNA-RNA metabolism. Assembly of Fe/S clusters, small inorganic cofactors, is assisted by complex proteinaceous machineries, which use cysteine as a source of sulfur, combine it with iron to synthesize an Fe/S cluster on scaffold proteins, and finally incorporate the cluster into recipient apoproteins. In eukaryotes, such as yeast and human cells, more than 20 components are known that facilitate the maturation of Fe/S proteins in mitochondria, cytosol, and nucleus. These biogenesis components also perform crucial roles in other cellular pathways, e.g., in the regulation of iron homeostasis or the modification of tRNA. Numerous diseases including several neurodegenerative and hematological disorders have been associated with defects in Fe/S protein biogenesis, underlining the central importance of this process for life.
- Published
- 2008
- Full Text
- View/download PDF
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