Mitochondrial [4Fe-4S] protein assembly involves reductive [2Fe-2S] cluster fusion on ISCA1–ISCA2 by electron flow from ferredoxin FDX2

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.

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Iron–sulfur cluster biosynthesis

Biochemical Society Transactions ◽

10.1042/bst0361112 ◽

2008 ◽

Vol 36 (6) ◽

pp. 1112-1119 ◽

Cited By ~ 112

Author(s):

Sibali Bandyopadhyay ◽

Kala Chandramouli ◽

Michael K. Johnson

Keyword(s):

Scaffold Proteins ◽

Cluster Assembly ◽

Cysteine Desulfurase ◽

Iron Sulfur Cluster ◽

Iron Sulfur ◽

Eukaryotic Organisms ◽

Almost All ◽

Cluster Biosynthesis

Iron–sulfur (Fe–S) clusters are present in more than 200 different types of enzymes or proteins and constitute one of the most ancient, ubiquitous and structurally diverse classes of biological prosthetic groups. Hence the process of Fe–S cluster biosynthesis is essential to almost all forms of life and is remarkably conserved in prokaryotic and eukaryotic organisms. Three distinct types of Fe–S cluster assembly machinery have been established in bacteria, termed the NIF, ISC and SUF systems, and, in each case, the overall mechanism involves cysteine desulfurase-mediated assembly of transient clusters on scaffold proteins and subsequent transfer of pre-formed clusters to apo proteins. A molecular level understanding of the complex processes of Fe–S cluster assembly and transfer is now beginning to emerge from the combination of in vivo and in vitro approaches. The present review highlights recent developments in understanding the mechanism of Fe–S cluster assembly and transfer involving the ubiquitous U-type scaffold proteins and the potential roles of accessory proteins such as Nfu proteins and monothiol glutaredoxins in the assembly, storage or transfer of Fe–S clusters.

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Physical and Functional Interactions of a Monothiol Glutaredoxin and an Iron Sulfur Cluster Carrier Protein with the Sulfur-donating Radical S-Adenosyl-l-methionine Enzyme MiaB

Journal of Biological Chemistry ◽

10.1074/jbc.m113.460360 ◽

2013 ◽

Vol 288 (20) ◽

pp. 14200-14211 ◽

Cited By ~ 20

Author(s):

Sylvain Boutigny ◽

Avneesh Saini ◽

Edward E. K. Baidoo ◽

Natasha Yeung ◽

Jay D. Keasling ◽

...

Keyword(s):

Protein Interactions ◽

Carrier Protein ◽

Cluster Assembly ◽

Carrier Proteins ◽

Iron Sulfur Cluster ◽

Iron Sulfur ◽

Initial Cluster ◽

Initial Assembly

The biosynthesis of iron sulfur (FeS) clusters, their trafficking from initial assembly on scaffold proteins via carrier proteins to final incorporation into FeS apoproteins, is a highly coordinated process enabled by multiprotein systems encoded in iscRSUAhscBAfdx and sufABCDSE operons in Escherichia coli. Although these systems are believed to encode all factors required for initial cluster assembly and transfer to FeS carrier proteins, accessory factors such as monothiol glutaredoxin, GrxD, and the FeS carrier protein NfuA are located outside of these defined systems. These factors have been suggested to function both as shuttle proteins acting to transfer clusters between scaffold and carrier proteins and in the final stages of FeS protein assembly by transferring clusters to client FeS apoproteins. Here we implicate both of these factors in client protein interactions. We demonstrate specific interactions between GrxD, NfuA, and the methylthiolase MiaB, a radical S-adenosyl-l-methionine-dependent enzyme involved in the maturation of a subset of tRNAs. We show that GrxD and NfuA physically interact with MiaB with affinities compatible with an in vivo function. We furthermore demonstrate that NfuA is able to transfer its cluster in vitro to MiaB, whereas GrxD is unable to do so. The relevance of these interactions was demonstrated by linking the activity of MiaB with GrxD and NfuA in vivo. We observe a severe defect in in vivo MiaB activity in cells lacking both GrxD and NfuA, suggesting that these proteins could play complementary roles in maturation and repair of MiaB.

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The Essential WD40 Protein Cia1 Is Involved in a Late Step of Cytosolic and Nuclear Iron-Sulfur Protein Assembly

Molecular and Cellular Biology ◽

10.1128/mcb.25.24.10833-10841.2005 ◽

2005 ◽

Vol 25 (24) ◽

pp. 10833-10841 ◽

Cited By ~ 97

Author(s):

Janneke Balk ◽

Daili J. Aguilar Netz ◽

Katharina Tepper ◽

Antonio J. Pierik ◽

Roland Lill

Keyword(s):

Specific Interaction ◽

Protein Assembly ◽

Cluster Assembly ◽

Iron Sulfur Cluster ◽

Additional Function ◽

S Protein ◽

Iron Sulfur ◽

Late Step ◽

S Proteins

ABSTRACT The assembly of cytosolic and nuclear iron-sulfur (Fe/S) proteins in yeast is dependent on the iron-sulfur cluster assembly and export machineries in mitochondria and three recently identified extramitochondrial proteins, the P-loop NTPases Cfd1 and Nbp35 and the hydrogenase-like Nar1. However, the molecular mechanism of Fe/S protein assembly in the cytosol is far from being understood, and more components are anticipated to take part in this process. Here, we have identified and functionally characterized a novel WD40 repeat protein, designated Cia1, as an essential component required for Fe/S cluster assembly in vivo on cytosolic and nuclear, but not mitochondrial, Fe/S proteins. Surprisingly, Nbp35 and Nar1, themselves Fe/S proteins, could assemble their Fe/S clusters in the absence of Cia1, demonstrating that these components act before Cia1. Consequently, Cia1 is involved in a late step of Fe/S cluster incorporation into target proteins. Coimmunoprecipitation assays demonstrated a specific interaction between Cia1 and Nar1. In contrast to the mostly cytosolic Nar1, Cia1 is preferentially localized to the nucleus, suggesting an additional function of Cia1. Taken together, our results indicate that Cia1 is a new member of the cytosolic Fe/S protein assembly (CIA) machinery participating in a step after Nbp35 and Nar1.

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The mitochondrial Hsp70 chaperone Ssq1 facilitates Fe/S cluster transfer from Isu1 to Grx5 by complex formation

Molecular Biology of the Cell ◽

10.1091/mbc.e12-09-0644 ◽

2013 ◽

Vol 24 (12) ◽

pp. 1830-1841 ◽

Cited By ~ 83

Author(s):

Marta A. Uzarska ◽

Rafal Dutkiewicz ◽

Sven-Andreas Freibert ◽

Roland Lill ◽

Ulrich Mühlenhoff

Keyword(s):

Complex Formation ◽

Synthesis Reaction ◽

The Core ◽

Iron Sulfur ◽

Cluster Transfer ◽

Hsp70 Chaperone ◽

Mitochondrial Hsp70 ◽

S Proteins

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.

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IOP1 Protein Is an External Component of the Human Cytosolic Iron-Sulfur Cluster Assembly (CIA) Machinery and Functions in the MMS19 Protein-dependent CIA Pathway

Journal of Biological Chemistry ◽

10.1074/jbc.m112.416602 ◽

2013 ◽

Vol 288 (23) ◽

pp. 16680-16689 ◽

Cited By ~ 29

Author(s):

Mineaki Seki ◽

Yukiko Takeda ◽

Kazuhiro Iwai ◽

Kiyoji Tanaka

Keyword(s):

Cluster Assembly ◽

Core Complex ◽

Core Component ◽

Iron Sulfur Cluster ◽

Sulfur Cluster ◽

The Core ◽

Iron Sulfur ◽

External Component

The emerging link between iron metabolism and genome integrity is increasingly clear. Recent studies have revealed that MMS19 and cytosolic iron-sulfur cluster assembly (CIA) factors form a complex and have central roles in CIA pathway. However, the composition of the CIA complex, particularly the involvement of the Fe-S protein IOP1, is still unclear. The roles of each component are also largely unknown. Here, we show that MMS19, MIP18, and CIAO1 form a tight “core” complex and that IOP1 is an “external” component of this complex. Although IOP1 and the core complex form a complex both in vivo and in vitro, IOP1 behaves differently in vivo. A deficiency in any core component leads to down-regulation of all of the components. In contrast, IOP1 knockdown does not affect the level of any core component. In MMS19-overproducing cells, other core components are also up-regulated, but the protein level of IOP1 remains unchanged. IOP1 behaves like a target protein in the CIA reaction, like other Fe-S helicases, and the core complex may participate in the maturation process of IOP1. Alternatively, the core complex may catch and hold IOP1 when it becomes mature to prevent its degradation. In any case, IOP1 functions in the MMS19-dependent CIA pathway. We also reveal that MMS19 interacts with target proteins. MIP18 has a role to bridge MMS19 and CIAO1. CIAO1 also binds IOP1. Based on our in vivo and in vitro data, new models of the CIA machinery are proposed.

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Human Nbp35 Is Essential for both Cytosolic Iron-Sulfur Protein Assembly and Iron Homeostasis

Molecular and Cellular Biology ◽

10.1128/mcb.00545-08 ◽

2008 ◽

Vol 28 (17) ◽

pp. 5517-5528 ◽

Cited By ~ 77

Author(s):

Oliver Stehling ◽

Daili J. A. Netz ◽

Brigitte Niggemeyer ◽

Ralf Rösser ◽

Richard S. Eisenstein ◽

...

Keyword(s):

Mammalian Cells ◽

Iron Homeostasis ◽

Regulatory Protein ◽

Protein Assembly ◽

Cluster Assembly ◽

S Protein ◽

Cellular Iron ◽

Iron Sulfur ◽

S Proteins

ABSTRACT The maturation of cytosolic iron-sulfur (Fe/S) proteins in mammalian cells requires components of the mitochondrial iron-sulfur cluster assembly and export machineries. Little is known about the cytosolic components that may facilitate the assembly process. Here, we identified the cytosolic soluble P-loop NTPase termed huNbp35 (also known as Nubp1) as an Fe/S protein, and we defined its role in the maturation of Fe/S proteins in HeLa cells. Depletion of huNbp35 by RNA interference decreased cell growth considerably, indicating its essential function. The deficiency in huNbp35 was associated with an impaired maturation of the cytosolic Fe/S proteins glutamine phosphoribosylpyrophosphate amidotransferase and iron regulatory protein 1 (IRP1), while mitochondrial Fe/S proteins remained intact. Consequently, huNbp35 is specifically involved in the formation of extramitochondrial Fe/S proteins. The impaired maturation of IRP1 upon huNbp35 depletion had profound consequences for cellular iron metabolism, leading to decreased cellular H-ferritin, increased transferrin receptor levels, and higher transferrin uptake. These properties clearly distinguished huNbp35 from its yeast counterpart Nbp35, which is essential for cytosolic-nuclear Fe/S protein assembly but plays no role in iron regulation. huNbp35 formed a complex with its close homologue huCfd1 (also known as Nubp2) in vivo, suggesting the existence of a heteromeric P-loop NTPase complex that is required for both cytosolic Fe/S protein assembly and cellular iron homeostasis.

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Structures and functions of mitochondrial ABC transporters

Biochemical Society Transactions ◽

10.1042/bst20150118 ◽

2015 ◽

Vol 43 (5) ◽

pp. 943-951 ◽

Cited By ~ 27

Author(s):

Theresia A. Schaedler ◽

Belinda Faust ◽

Chitra A. Shintre ◽

Elisabeth P. Carpenter ◽

Vasundara Srinivasan ◽

...

Keyword(s):

Abc Transporters ◽

In Vivo Studies ◽

Iron Sulfur ◽

Haem Biosynthesis ◽

Cofactor Biosynthesis ◽

Substrate Binding Sites ◽

Cluster Biosynthesis ◽

Physiological Substrates

A small number of physiologically important ATP-binding cassette (ABC) transporters are found in mitochondria. Most are half transporters of the B group forming homodimers and their topology suggests they function as exporters. The results of mutant studies point towards involvement in iron cofactor biosynthesis. In particular, ABC subfamily B member 7 (ABCB7) and its homologues in yeast and plants are required for iron-sulfur (Fe-S) cluster biosynthesis outside of the mitochondria, whereas ABCB10 is involved in haem biosynthesis. They also play a role in preventing oxidative stress. Mutations in ABCB6 and ABCB7 have been linked to human disease. Recent crystal structures of yeast Atm1 and human ABCB10 have been key to identifying substrate-binding sites and transport mechanisms. Combined with in vitro and in vivo studies, progress is being made to find the physiological substrates of the different mitochondrial ABC transporters.

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