A Novel Role for Human Nfs1 in the Cytoplasm

Marelja, Zvonimir; Stöcklein, Walter; Nimtz, Manfred; Leimkühler, Silke

doi:10.1074/jbc.m804064200

Cited by 108 publications

(77 citation statements)

References 48 publications

(73 reference statements)

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“…S4). The NFS1⅐ISD11 complex was purified as an apparent octamer ([NFS1 2 -ISD11 2 ] 2 ) and ISCU as an apparent dimer (data not shown) consistent with previous reports (36,41). These proteins were tested for their ability to bind to rec-FXN 81-210 and rec-FXN 42-210 by use of pulldown assays using histidine-tagged versions of NFS1 or ISCU, a method used in previous studies of Yfh1/Isu1 and Yfh1/Nfs1 interactions (32,42).…”

Section: The Length Of the Fxn N Terminus Determines The Stability Ofsupporting

confidence: 86%

“…The bulk of native NFS1 was eluted in one high molecular weight pool as expected (36), together with the high molecular weight pool of native FXN (Fig. 9, A and B).…”

Section: B and D The Length Of The Fxn N Terminus Determines Formasupporting

confidence: 64%

“…Two DNA fragments corresponding to residues 56 -458 of NFS1 and 6 -92 of ISD11 (32,36) were cloned into the XhoI and BamHI sites of vector pET15b (Novagen), and the NcoI and EcoRI sites of vector pCDFDuet (Novagen), respectively. This resulted in the addition of an N-terminal His 6 tag followed by a thrombin cleavage site on NFS1.…”

Section: Expression and Purification Of Recombinant Proteins Used In mentioning

confidence: 99%

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Normal and Friedreich Ataxia Cells Express Different Isoforms of Frataxin with Complementary Roles in Iron-Sulfur Cluster Assembly

Gakh

Bedekovics

Duncan

et al. 2010

Journal of Biological Chemistry

123

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Friedreich ataxia (FRDA) is an autosomal recessive degenerative disease caused by insufficient expression of frataxin (FXN), a mitochondrial iron-binding protein required for Fe-S cluster assembly. The development of treatments to increase FXN levels in FRDA requires elucidation of the steps involved in the biogenesis of functional FXN. The FXN mRNA is translated to a precursor polypeptide that is transported to the mitochondrial matrix and processed to at least two forms, FXN Friedreich ataxia (FRDA) 2 (OMIM number 229300) is an autosomal recessive disease with an estimated incidence of 1:40,000. Most FRDA patients are apparently healthy at birth and during the first 5-10 years of life; then their gait becomes increasingly unsteady and wide-based and their voluntary movements uncoordinated. Many patients develop hypertrophic cardiomyopathy as well as diabetes, muscle weakness, and skeletal deformities. Although cognitive functions remain largely intact during disease progression, patients develop significant communication difficulties due to dysarthria, which is often compounded by vision and hearing loss. The majority of patients eventually become wheelchair-bound and dependent on others for most daily activities. Cardiac failure is a frequent cause of death at a young age (1).The FRDA locus encodes a mitochondrial protein designated frataxin (FXN), which is expressed at much lower levels in FRDA patients compared with normal individuals (2). In most patients, FXN deficiency results from the presence of an expanded GAA repeat in the first intron of the FRDA gene (2) that causes transcriptional silencing (reviewed in Ref.3). Although FXN is ubiquitously expressed, certain cells (dorsal root ganglia neurons, cardiomyocytes, and pancreatic beta cells) are exquisitely sensitive to frataxin depletion, and the degenerative loss of these particular cells accounts for the major clinical aspects of FRDA (1).Extensive biochemical studies have shown that frataxins across species are conserved iron-binding proteins that can either provide iron for Fe-S cluster assembly and heme synthesis or store iron as a stable mineral (reviewed in Ref. 4). The loss of these properties accounts for impaired iron utilization and increased iron toxicity linked to frataxin deficiency in the mitochondria of such diverse organisms as Saccharomyces cerevisiae, Drosophila, mouse, and humans (5-8). In humans, the mitochondrial alterations caused by FXN deficiency lead to tissue-specific changes in various cellular pathways involved in antioxidant, metabolic, and inflammatory responses, thereby amplifying the pathophysiology of FRDA and promoting disease progression (9 -13).

show abstract

Section: The Length Of the Fxn N Terminus Determines The Stability Ofsupporting

confidence: 86%

“…The bulk of native NFS1 was eluted in one high molecular weight pool as expected (36), together with the high molecular weight pool of native FXN (Fig. 9, A and B).…”

Section: B and D The Length Of The Fxn N Terminus Determines Formasupporting

confidence: 64%

Section: Expression and Purification Of Recombinant Proteins Used In mentioning

confidence: 99%

See 1 more Smart Citation

Normal and Friedreich Ataxia Cells Express Different Isoforms of Frataxin with Complementary Roles in Iron-Sulfur Cluster Assembly

Gakh

Bedekovics

Duncan

et al. 2010

Journal of Biological Chemistry

123

View full text Add to dashboard Cite

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“…A thrombin cleavage site was also introduced immediately after the 6-histidine tag on Isd11. Nfs1 and Isd11 were co-expressed in E. coli essentially as described for the human orthologs (15). Briefly, BL21 cells were grown at 25°C in 1 liter of Luria broth containing 30 g/ml streptomycin.…”

Section: Methodsmentioning

confidence: 99%

Oligomeric Yeast Frataxin Drives Assembly of Core Machinery for Mitochondrial Iron-Sulfur Cluster Synthesis

Li¹,

Gakh²,

Smith

et al. 2009

Journal of Biological Chemistry

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Mitochondrial biosynthesis of iron-sulfur clusters (ISCs) is a vital process involving the delivery of elemental iron and sulfur to a scaffold protein via molecular interactions that are still poorly defined. Analysis of highly conserved components of the yeast ISC assembly machinery shows that the iron-chaperone, Yfh1, and the sulfur-donor complex, Nfs1-Isd11, directly bind to each other. This interaction is mediated by direct Yfh1-Isd11 contacts. Moreover, both Yfh1 and Nfs1-Isd11 can directly bind to the scaffold, Isu1. Binding of Yfh1 to Nfs1-Isd11 or Isu1 requires oligomerization of Yfh1 and can occur in an iron-independent manner. However, more stable contacts are formed when Yfh1 oligomerization is normally coupled with the binding and oxidation of Fe 2؉ . Our observations challenge the view that iron delivery for ISC synthesis is mediated by Fe 2؉ -loaded monomeric Yfh1. Rather, we find that the iron oxidation-driven oligomerization of Yfh1 promotes the assembly of stable multicomponent complexes in which the iron donor and the sulfur donor simultaneously interact with each other as well as with the scaffold. Moreover, the ability to store ferric iron enables oligomeric Yfh1 to adjust iron release depending on the presence of Isu1 and the availability of elemental sulfur and reducing equivalents. In contrast, the use of anaerobic conditions that prevent Yfh1 oligomerization results in inhibition of ISC assembly on Isu1. These findings suggest that iron-dependent oligomerization is a mechanism by which the iron donor promotes assembly of the core machinery for mitochondrial ISC synthesis.

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“…However, this possibility was excluded by showing that Nfs lacking the mitochondrial targeting sequence cannot complement thiolation of either cytosolic or mitochondrial tRNAs (52). In mammalian cells, Nfs was also detected in the cytosol (49) and was shown to be involved in the biosynthesis of the molybdenum cofactor (53). We can speculate that such a role may be also attributed to the mitochondrial Nfs in the protist studied, since the molybdenum cofactor synthetase of T. brucei (Tb927.11.11920) has a 0.99 probability of mitochondrial localization, according to Mitoprot (54).…”

Section: Discussionmentioning

confidence: 99%

Mitochondrial and Nucleolar Localization of Cysteine Desulfurase Nfs and the Scaffold Protein Isu in Trypanosoma brucei

et al. 2014

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Trypanosoma brucei has a complex life cycle during which its single mitochondrion is subjected to major metabolic and morphological changes. While the procyclic stage (PS) of the insect vector contains a large and reticulated mitochondrion, its counterpart in the bloodstream stage (BS) parasitizing mammals is highly reduced and seems to be devoid of most functions. We show here that key Fe-S cluster assembly proteins are still present and active in this organelle and that produced clusters are incorporated into overexpressed enzymes. Importantly, the cysteine desulfurase Nfs, equipped with the nuclear localization signal, was detected in the nucleolus of both T. brucei life stages. The scaffold protein Isu, an interacting partner of Nfs, was also found to have a dual localization in the mitochondrion and the nucleolus, while frataxin and both ferredoxins are confined to the mitochondrion. Moreover, upon depletion of Isu, cytosolic tRNA thiolation dropped in the PS but not BS parasites.

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A Novel Role for Human Nfs1 in the Cytoplasm

Cited by 108 publications

References 48 publications

Normal and Friedreich Ataxia Cells Express Different Isoforms of Frataxin with Complementary Roles in Iron-Sulfur Cluster Assembly

Normal and Friedreich Ataxia Cells Express Different Isoforms of Frataxin with Complementary Roles in Iron-Sulfur Cluster Assembly

Oligomeric Yeast Frataxin Drives Assembly of Core Machinery for Mitochondrial Iron-Sulfur Cluster Synthesis

Mitochondrial and Nucleolar Localization of Cysteine Desulfurase Nfs and the Scaffold Protein Isu in Trypanosoma brucei

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