The Fe/S Assembly Protein IscU Behaves as a Substrate for the Molecular Chaperone Hsc66 from Escherichia coli

Silberg, Jonathan J.; Hoff, Kevin G.; Tapley, Timothy L.; Vickery, Larry E.

doi:10.1074/jbc.m009542200

Cited by 104 publications

(124 citation statements)

References 28 publications

(40 reference statements)

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“…ATPase Assays-Steady-state ATPase rates were determined at 23°C in HKM buffer (50 mM Hepes, pH 7.3, 150 mM KCl, 10 mM MgCl 2 ) containing 1 mM DTT and 0.5 mM ATP by measuring phosphate released using the EnzCheck coupled enzyme phosphate assay kit (20) (Molecular Probes) as previously reported (2,3,10,13,14,21). Under these conditions Hsc66 has a basal turnover number of Х0.10 min Ϫ1 .…”

Section: Methodsmentioning

confidence: 99%

“…Surface Plasmon Resonance Analysis-Surface plasmon resonance studies were carried out at 25°C using a Biacore 2000 instrument as previously described (10,13,14). Hsc66 in the presence of 1 mM ATP and 10 mM magnesium chloride was randomly cross-linked to the surface of the sensor chip by amine coupling.…”

Section: Methodsmentioning

confidence: 99%

“…IscU is a small, dimeric protein that is capable of forming labile iron-sulfur clusters in vitro and is proposed to serve as a template for iron-sulfur cluster formation in vivo (10 -12). Recent studies have provided evidence that IscU behaves as a substrate for Hsc66 (13), and Hsc66 binding of IscU may serve to regulate the type or stability of iron-sulfur clusters formed on IscU.…”

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confidence: 99%

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Contributions of the LPPVK Motif of the Iron-Sulfur Template Protein IscU to Interactions with the Hsc66-Hsc20 Chaperone System

Hoff¹,

Cupp‐Vickery

Vickery

2003

Journal of Biological Chemistry

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Contributions of the LPPVK Motif of the Iron-Sulfur Template Protein IscU to Interactions with the Hsc66-Hsc20 Chaperone System

Hoff¹,

Cupp‐Vickery

Vickery

2003

Journal of Biological Chemistry

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“…HscA and HscB function together as a nucleotide-dependent molecular chaperone system that is specific for IscU (15)(16)(17). HscA is a specialized member of the ubiquitous 70-kDa heat shock protein (Hsp70 or DnaK) family of molecular chaperones that facilitate protein folding, (dis)assembly and transport via nucleotide-dependent binding to unfolded, misfolded or unstable polypeptides in order to prevent non-specific aggregation processes (18).…”

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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 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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“…The hscB gene in bacteria encodes a 20-kDa J-type co-chaperone, designated Hsc20, that functions with the hsp70-class molecular chaperone HscA/Hsc66 (Seaton and Vickery 1994;Kawula and Lelivelt 1994;Vickery et al 1997). In vitro studies have shown that HscB/Hsc20 regulates interactions between the Fe-S cluster assembly protein IscU and the HscA/ Hsc66 chaperone (Hoff et al 2000;Silberg et al 2001;Hoff et al 2002). Homologues of the bacterial hscB gene are found in eukaryotes, and the homologue in Saccharomyces cerevisiae, JAC1, is a nuclear gene whose protein product is targeted to mitochondria (Strain et al 1998;Kim et al 2001;Lutz et al 2001;Voisine et al 2001).…”

Section: Introductionmentioning

confidence: 99%

Identification of a novel candidate gene in the iron-sulfur pathway implicated in ataxia-susceptibility: human gene encoding HscB, a J-type co-chaperone

Sun

Gargus

et al. 2003

J Hum Genet

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Iron-sulfur proteins participate in a wide range of biochemical processes, including many that are central to mitochondrial electron transfer and energy metabolism. Mutations in two such proteins, frataxin and ABCB7, cause Friedreich ataxia and X-linked sideroblastic anemia with ataxia, respectively, rendering other participants in this pathway functional candidates for hereditary ataxia syndromes. Recently frataxin was shown to have an identical phylogenetic distribution with two genes and was most likely specifically involved in the same sub-process in iron-sulfur cluster assembly as one gene, designated hscB, in bacteria. To set the stage for an analysis of the potential role of this candidate gene in human disease, we defined the human HscB cDNA, its genomic locus, and its pattern of expression in normal human tissues. The isolated human HscB cDNA spans 785 bp and encodes a conserved 235-amino-acid protein, including a putative mitochondrial import leader. The HscB gene is found at chromosome 22q11-12 and is composed of six exons and five introns. Northern blot analyses of RNA from adult and fetal tissues defined a pattern of expression in mitochondria-rich tissues similar to that of frataxin, an expression pattern compatible with its implied role in mitochondrial energetics and related disease phenotypes.

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The Fe/S Assembly Protein IscU Behaves as a Substrate for the Molecular Chaperone Hsc66 from Escherichia coli

Cited by 104 publications

References 28 publications

Contributions of the LPPVK Motif of the Iron-Sulfur Template Protein IscU to Interactions with the Hsc66-Hsc20 Chaperone System

Contributions of the LPPVK Motif of the Iron-Sulfur Template Protein IscU to Interactions with the Hsc66-Hsc20 Chaperone System

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

Identification of a novel candidate gene in the iron-sulfur pathway implicated in ataxia-susceptibility: human gene encoding HscB, a J-type co-chaperone

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