scholarly journals 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

2012 ◽  
Vol 287 (15) ◽  
pp. 12365-12378 ◽  
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.

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

2016 ◽  
Vol 473 (14) ◽  
pp. 2073-2085 ◽  
Author(s):  
Daili J.A. Netz ◽  
Heide M. Genau ◽  
Benjamin D. Weiler ◽  
Eckhard Bill ◽  
Antonio J. Pierik ◽  
...  
Keyword(s):  
Protein Assembly ◽  
Cysteine Residues ◽  
S Protein ◽  
Essential Protein ◽  
Iron Sulfur ◽  
Protein Biogenesis ◽  
Sulfur Protein ◽  
Iron Sulfur Protein

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.

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

2005 ◽  
Vol 25 (24) ◽  
pp. 10833-10841 ◽  
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.

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

2008 ◽  
Vol 28 (17) ◽  
pp. 5517-5528 ◽  
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.

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

2020 ◽  
Vol 401 (6-7) ◽  
pp. 855-876 ◽  
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.

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

2019 ◽  
Vol 1866 (2) ◽  
pp. 240-251 ◽  
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

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

2017 ◽  
Vol 3 (5) ◽  
pp. e184 ◽  
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.

scholarly journals Disulfide bond formation during the folding of influenza virus hemagglutinin.

1992 ◽  
Vol 118 (2) ◽  
pp. 227-244 ◽  
Author(s):  
M S Segal ◽  
J M Bye ◽  
J F Sambrook ◽  
M J Gething
Keyword(s):  
Influenza Virus ◽  
Disulfide Bonds ◽  
Intracellular Transport ◽  
Cellular Protein ◽  
Cysteine Residues ◽  
Mutant Proteins ◽  
Influenza Virus Hemagglutinin ◽  
Stalk Domain ◽  
Do So

To study the importance of individual sulfhydryl residues during the folding and assembly in vivo of influenza virus hemagglutinin (HA), we have constructed and expressed a series of mutant HA proteins in which cysteines involved in three disulfide bonds have been substituted by serine residues. Investigations of the structure and intracellular transport of the mutant proteins indicate that (a) cysteine residues in the ectodomain are essential both for efficient folding of HA and for stabilization of the folded molecule; (b) cysteine residues in the globular portion of the ectodomain are likely to form native disulfide bonds rapidly and directly, without involvement of intermediate, nonnative linkages; and (c) cysteine residues in the stalk portion of the ectodomain also appear not to form intermediate disulfide bonds, even though they have the opportunity to do so, being separated from their correct partners by hundreds of amino acids including two or more other sulfhydryl residues. We propose a role for the cellular protein BiP in shielding the cysteine residues of the stalk domain during the folding process, thus preventing them from forming intermediate, nonnative disulfide bonds.

scholarly journals Charcot-Marie-Tooth 2B mutations in rab7 cause dosage-dependent neurodegeneration due to partial loss of function

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Smita Cherry ◽  
Eugene Jennifer Jin ◽  
Mehmet Neset Özel ◽  
Zhiyuan Lu ◽  
Egemen Agi ◽  
...  
Keyword(s):  
Motor Neurons ◽  
Protein Function ◽  
Quantitative Imaging ◽  
Small Gtpase ◽  
Loss Of Function ◽  
Gain Of Function ◽  
Mutant Proteins ◽  
Charcot Marie Tooth ◽  
Function Mechanism

The small GTPase Rab7 is a key regulator of endosomal maturation in eukaryotic cells. Mutations in rab7 are thought to cause the dominant neuropathy Charcot-Marie-Tooth 2B (CMT2B) by a gain-of-function mechanism. Here we show that loss of rab7, but not overexpression of rab7 CMT2B mutants, causes adult-onset neurodegeneration in a Drosophila model. All CMT2B mutant proteins retain 10–50% function based on quantitative imaging, electrophysiology, and rescue experiments in sensory and motor neurons in vivo. Consequently, expression of CMT2B mutants at levels between 0.5 and 10-fold their endogenous levels fully rescues the neuropathy-like phenotypes of the rab7 mutant. Live imaging reveals that CMT2B proteins are inefficiently recruited to endosomes, but do not impair endosomal maturation. These findings are not consistent with a gain-of-function mechanism. Instead, they indicate a dosage-dependent sensitivity of neurons to rab7-dependent degradation. Our results suggest a therapeutic approach opposite to the currently proposed reduction of mutant protein function.

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