Components involved in assembly and dislocation of iron-sulfur clusters on the scaffold protein Isu1p

Mühlenhoff, Ulrich; Gerber, Jana; Richhardt, Nadine; Lill, Roland

doi:10.1093/emboj/cdg446

Cited by 356 publications

(360 citation statements)

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“…Both results are in accord with studies of in vivo and in vitro studies of the ISC assembly machinery in yeast mitochondria. In vivo evidence that Ssq1 and Jac1 are involved in a step after cluster assembly on the Isu1 scaffold protein was provided by 55 Feimmunoprecipation studies which revealed that Fe accumulation on Isu1 accompanies depletion of Ssq1 and Jac1 (26). Furthermore, recent in vitro studies indicated that Ssq1 and Jac1 are not important for Fe-S cluster synthesis on Isu1 (35).…”

Section: Discussionmentioning

confidence: 99%

“…Indeed there are conflicting reports as to whether the role of the chaperone system is to facilitate the assembly of clusters on IscU (25) or the transfer of clusters preassembled on IscU to acceptor proteins (26). Immunoprecipitation studies in yeast indicated that functional depletion of the homologous Fe-S cluster assembly-associated chaperones resulted in increased accumulation of Fe on Isu (eukaryotic IscU), which suggested a role in mediating cluster transfer to acceptor proteins (26). In contrast, cluster transfer from Thermotoga maritima [2Fe-2S]IscU and human [2Fe-2S]Isu to apo human Fd was found to be inhibited by the nonspecific T. maritima DnaK/DnaJ chaperone system, irrespective of the presence or absence of nucleotides (25).…”

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HscA and HscB Stimulate [2Fe-2S] Cluster Transfer from IscU to Apoferredoxin in an ATP-Dependent Reaction

2006

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The role of the Azotobacter vinelandii HscA/HscB co-chaperone system in ISC-mediated iron-sulfur cluster biogenesis has been investigated in vitro by using CD and EPR spectrometry to monitor the effect of HscA, HscB, MgATP, and MgADP on the time course of cluster transfer from [2Fe-2S] IscU to apo-Isc ferredoxin. CD spectra indicate that both HscB and HscA interact with [2Fe-2S] IscU and the rate of cluster transfer was stimulated more than 20-fold in the presence stoichiometric HscA and HscB and excess MgATP. No stimulation was observed in the absence of either HscB or MgATP and cluster transfer was found to be an ATP-dependent reaction based on concomitant phosphate production and the enhanced rates of cluster transfer in the presence of KCl which is known to stimulated HscA ATPase activity. The results demonstrate a role of the ISC HscA/HscB cochaperone system in facilitating efficient [2Fe-2S] cluster transfer from the IscU scaffold protein to acceptor proteins and that [2Fe-2S] cluster transfer from IscU is an ATP-dependent process. The data are consistent with the proposed regulation of the HscA ATPase cycle by HscB and IscU (Silberg, J. J., Tapley, T. L., Hoff, K. G., and Vickery, L. E. (2004) J. Biol. Chem. 279,[53924][53925][53926][53927][53928][53929][53930][53931], and mechanistic proposals for coupling of the HscA ATPase cycle with cluster transfer from [2Fe-2S] IscU to apo-IscFdx are discussed.Iron-sulfur cluster biogenesis proteins in bacteria are commonly encoded by a highly conserved isc (iron-sulfur cluster) gene cluster, iscRSUA-hscBA-fdx (1). Moreover, with the exception of the regulatory protein IscR, homologs of each of these proteins have been shown to be involved in Fe-S cluster biogenesis in eukaryotic organisms (2). Extensive biochemical and genetic studies have established well-defined roles for IscS and IscU, but specific roles for the IscA, HscA, HscB and Fdx proteins remain elusive. IscS is a cysteine desulfurase that catalyzes the conversion of cysteine to alanine (3;4) and thereby provides the inorganic sulfur for assembly or [4Fe-4S] clusters on the IscU scaffold protein (5). In vitro studies have shown thatIscA has the ability to function as either an alternative scaffold for IscS-mediated cluster assembly of or [4Fe-4S] clusters (6;7) or as a specific Fe donor for cluster assembly on IscU (8), but the physiologically relevant role has yet to be determined. Likewise, the specific redox role of the Isc [2Fe-2S] 2+,+ Fdx (ferredoxin) in IscS-mediated cluster assembly has yet to be identified, with reduction of S 0 to S 2− , oxidation of Fe 2+ to Fe 3+ in the assembly of [2Fe-2S] 2+ clusters, and reductive coupling of two [2Fe-2S] 2+ clusters on IscU to yield one [4Fe-4S] 2+ cluster as the most likely candidates (5;9).In vivo gene knockout studies have demonstrated that both HscA (heat shock cognate 66-kDa; Hsc66) and HscB (heat shock cognate 20-kDa; Hsc20), and their eukaryotic homologs, Ssq1 and Jac1, have crucial roles in the maturation of Fe-S proteins in both prokary...

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

HscA and HscB Stimulate [2Fe-2S] Cluster Transfer from IscU to Apoferredoxin in an ATP-Dependent Reaction

2006

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“…The use of genetic depletion methods to diminish the cellular amounts of these usually essential proteins allows the investigation of their role in vivo. For instance, the stages at which these factors perform their function in the biosynthetic pathway can be determined 12,37 . In turn, this experimental approach opens the way to address the phenotypes associated with the depletion of the ISC and CIA components.…”

Section: Introductionmentioning

confidence: 99%

Analysis of iron–sulfur protein maturation in eukaryotes

2009

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“…The ISC machinery comprises ISCU, a scaffold protein on which Fe-S clusters are assembled, NFS1, a pyridoxal phosphatedependent cysteine desulfurase, ISD11 and FXN [20][21][22][23] . The eukaryotic ISC machinery has evolved from bacteria and thus some of the primary steps of Fe-S cluster assembly have been conserved 16,24,25 .…”

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Mammalian frataxin directly enhances sulfur transfer of NFS1 persulfide to both ISCU and free thiols

et al. 2015

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Friedreich's ataxia is a severe neurodegenerative disease caused by the decreased expression of frataxin, a mitochondrial protein that stimulates iron-sulfur (Fe-S) cluster biogenesis. In mammals, the primary steps of Fe-S cluster assembly are performed by the NFS1-ISD11-ISCU complex via the formation of a persulfide intermediate on NFS1. Here we show that frataxin modulates the reactivity of NFS1 persulfide with thiols. We use maleimide-peptide compounds along with mass spectrometry to probe cysteine-persulfide in NFS1 and ISCU. Our data reveal that in the presence of ISCU, frataxin enhances the rate of two similar reactions on NFS1 persulfide: sulfur transfer to ISCU leading to the accumulation of a persulfide on the cysteine C104 of ISCU, and sulfur transfer to small thiols such as DTT, L-cysteine and GSH leading to persulfuration of these thiols and ultimately sulfide release. These data raise important questions on the physiological mechanism of Fe-S cluster assembly and point to a unique function of frataxin as an enhancer of sulfur transfer within the NFS1-ISD11-ISCU complex.

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Components involved in assembly and dislocation of iron-sulfur clusters on the scaffold protein Isu1p

Cited by 356 publications

References 54 publications

HscA and HscB Stimulate [2Fe-2S] Cluster Transfer from IscU to Apoferredoxin in an ATP-Dependent Reaction

HscA and HscB Stimulate [2Fe-2S] Cluster Transfer from IscU to Apoferredoxin in an ATP-Dependent Reaction

Analysis of iron–sulfur protein maturation in eukaryotes

Mammalian frataxin directly enhances sulfur transfer of NFS1 persulfide to both ISCU and free thiols

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