The conserved protein Dre2 uses essential [2Fe–2S] and [4Fe–4S] clusters for its function in cytosolic iron–sulfur protein assembly

The essential protein Dre2 uses iron–sulfur (Fe–S) clusters to transfer electrons for cytosolic Fe–S protein biogenesis. Biochemical, cell biological and spectroscopic approaches demonstrate that recombinant Dre2 binds oxygen-labile [2Fe–2S] and [4Fe–4S] clusters at two conserved C-terminal motifs with four cysteine residues each.

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A Bridging [4Fe-4S] Cluster and Nucleotide Binding Are Essential for Function of the Cfd1-Nbp35 Complex as a Scaffold in Iron-Sulfur Protein Maturation

Journal of Biological Chemistry ◽

10.1074/jbc.m111.328914 ◽

2012 ◽

Vol 287 (15) ◽

pp. 12365-12378 ◽

Cited By ~ 66

Author(s):

Daili J. A. Netz ◽

Antonio J. Pierik ◽

Martin Stümpfig ◽

Eckhard Bill ◽

Anil K. Sharma ◽

...

Keyword(s):

Protein Function ◽

Nucleotide Binding ◽

Protein Assembly ◽

Cysteine Residues ◽

Binding Motif ◽

S Protein ◽

Mutant Proteins ◽

Iron Sulfur ◽

Sulfur Protein

The essential P-loop NTPases Cfd1 and Nbp35 of the cytosolic iron-sulfur (Fe-S) protein assembly machinery perform a scaffold function for Fe-S cluster synthesis. Both proteins contain a nucleotide binding motif of unknown function and a C-terminal motif with four conserved cysteine residues. The latter motif defines the Mrp/Nbp35 subclass of P-loop NTPases and is suspected to be involved in transient Fe-S cluster binding. To elucidate the function of these two motifs, we first created cysteine mutant proteins of Cfd1 and Nbp35 and investigated the consequences of these mutations by genetic, cell biological, biochemical, and spectroscopic approaches. The two central cysteine residues (CPXC) of the C-terminal motif were found to be crucial for cell viability, protein function, coordination of a labile [4Fe-4S] cluster, and Cfd1-Nbp35 hetero-tetramer formation. Surprisingly, the two proximal cysteine residues were dispensable for all these functions, despite their strict evolutionary conservation. Several lines of evidence suggest that the C-terminal CPXC motifs of Cfd1-Nbp35 coordinate a bridging [4Fe-4S] cluster. Upon mutation of the nucleotide binding motifs Fe-S clusters could no longer be assembled on these proteins unless wild-type copies of Cfd1 and Nbp35 were present in trans. This result indicated that Fe-S cluster loading on these scaffold proteins is a nucleotide-dependent step. We propose that the bridging coordination of the C-terminal Fe-S cluster may be ideal for its facile assembly, labile binding, and efficient transfer to target Fe-S apoproteins, a step facilitated by the cytosolic iron-sulfur (Fe-S) protein assembly proteins Nar1 and Cia1 in vivo.

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Faculty Opinions recommendation of The essential WD40 protein Cia1 is involved in a late step of cytosolic and nuclear iron-sulfur protein assembly.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1029526.345457 ◽

2005 ◽

Author(s):

Joan Broderick

Keyword(s):

Protein Assembly ◽

Iron Sulfur ◽

Late Step ◽

Sulfur Protein ◽

Iron Sulfur Protein

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From the discovery to molecular understanding of cellular iron-sulfur protein biogenesis

Biological Chemistry ◽

10.1515/hsz-2020-0117 ◽

2020 ◽

Vol 401 (6-7) ◽

pp. 855-876 ◽

Cited By ~ 13

Author(s):

Roland Lill

Keyword(s):

De Novo ◽

Current Knowledge ◽

Protein Stabilization ◽

S Protein ◽

Cellular Iron ◽

Iron Sulfur ◽

Protein Biogenesis ◽

Sulfur Protein ◽

Initial Discovery ◽

S Proteins

AbstractProtein cofactors often are the business ends of proteins, and are either synthesized inside cells or are taken up from the nutrition. A cofactor that strictly needs to be synthesized by cells is the iron-sulfur (Fe/S) cluster. This evolutionary ancient compound performs numerous biochemical functions including electron transfer, catalysis, sulfur mobilization, regulation and protein stabilization. Since the discovery of eukaryotic Fe/S protein biogenesis two decades ago, more than 30 biogenesis factors have been identified in mitochondria and cytosol. They support the synthesis, trafficking and target-specific insertion of Fe/S clusters. In this review, I first summarize what led to the initial discovery of Fe/S protein biogenesis in yeast. I then discuss the function and localization of Fe/S proteins in (non-green) eukaryotes. The major part of the review provides a detailed synopsis of the three major steps of mitochondrial Fe/S protein biogenesis, i.e. the de novo synthesis of a [2Fe-2S] cluster on a scaffold protein, the Hsp70 chaperone-mediated transfer of the cluster and integration into [2Fe-2S] recipient apoproteins, and the reductive fusion of [2Fe-2S] to [4Fe-4S] clusters and their subsequent assembly into target apoproteins. Finally, I summarize the current knowledge of the mechanisms underlying the maturation of cytosolic and nuclear Fe/S proteins.

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Depletion of thiol reducing capacity impairs cytosolic but not mitochondrial iron-sulfur protein assembly machineries

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research ◽

10.1016/j.bbamcr.2018.11.003 ◽

2019 ◽

Vol 1866 (2) ◽

pp. 240-251 ◽

Cited By ~ 7

Author(s):

Joseph J. Braymer ◽

Martin Stümpfig ◽

Stefanie Thelen ◽

Ulrich Mühlenhoff ◽

Roland Lill

Keyword(s):

Protein Assembly ◽

Iron Sulfur ◽

Mitochondrial Iron ◽

Sulfur Protein ◽

Iron Sulfur Protein ◽

Reducing Capacity

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IBA57 mutations abrogate iron-sulfur cluster assembly leading to cavitating leukoencephalopathy

Neurology Genetics ◽

10.1212/nxg.0000000000000184 ◽

2017 ◽

Vol 3 (5) ◽

pp. e184 ◽

Cited By ~ 12

Author(s):

Akihiko Ishiyama ◽

Chika Sakai ◽

Yuichi Matsushima ◽

Satoru Noguchi ◽

Satomi Mitsuhashi ◽

...

Keyword(s):

Protein Expression ◽

Lipoic Acid ◽

Protein Assembly ◽

Assembly System ◽

Iron Sulfur Cluster ◽

Sulfur Cluster ◽

Iron Sulfur ◽

Mitochondrial Iron ◽

Sulfur Protein ◽

Iron Sulfur Protein

Objective:To determine the molecular factors contributing to progressive cavitating leukoencephalopathy (PCL) to help resolve the underlying genotype-phenotype associations in the mitochondrial iron-sulfur cluster (ISC) assembly system.Methods:The subjects were 3 patients from 2 families who showed no inconsistencies in either clinical or brain MRI findings as PCL. We used exome sequencing, immunoblotting, and enzyme activity assays to establish a molecular diagnosis and determine the roles of ISC-associated factors in PCL.Results:We performed genetic analyses on these 3 patients and identified compound heterozygosity for the IBA57 gene, which encodes the mitochondrial iron-sulfur protein assembly factor. Protein expression analysis revealed substantial decreases in IBA57 protein expression in myoblasts and fibroblasts. Immunoblotting revealed substantially reduced expression of SDHB, a subunit of complex II, and lipoic acid synthetase (LIAS). Levels of pyruvate dehydrogenase complex-E2 and α-ketoglutarate dehydrogenase-E2, which use lipoic acid as a cofactor, were also reduced. In activity staining, SDH activity was clearly reduced, but it was ameliorated in mitochondrial fractions from rescued myoblasts. In addition, NFU1 protein expression was also decreased, which is required for the assembly of a subset of iron-sulfur proteins to SDH and LIAS in the mitochondrial ISC assembly system.Conclusions:Defects in IBA57 essentially regulate NFU1 expression, and aberrant NFU1 ultimately affects SDH activity and LIAS expression in the ISC biogenesis pathway. This study provides new insights into the role of the iron-sulfur protein assembly system in disorders related to mitochondrial energy metabolism associated with leukoencephalopathy with cavities.

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