Sequence-specific Interaction between Mitochondrial Fe-S Scaffold Protein Isu and Hsp70 Ssq1 Is Essential for Their in Vivo Function

Dutkiewicz, Rafał; Schilke, Brenda; Cheng, Sara Sara; Knieszner, Helena; Craig, Elizabeth A.; Marszałek, Jarosław

doi:10.1074/jbc.m402947200

Cited by 93 publications

(87 citation statements)

References 36 publications

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“…Ssq1p binds to Isu1p/Isu2p with high specificity by interacting with a highly conserved PVK tripeptide motif on the Isu proteins (25,26). Alterations in this motif disrupt the interaction with Ssq1p and result in deleterious in vivo phenotypes (27). Conversely, SSQ1 mutant proteins with severely reduced affinities for the Isu proteins display a similar phenotype as SSQ1 deletion cells, underscoring the importance of the Ssq1p-Isu protein interaction in vivo (28).…”

mentioning

confidence: 83%

“…Cells were grown in rich yeast extract/peptone, synthetic complete minimal or "iron-poor" minimal (lacking added iron chloride) medium, containing the required carbon sources (35). For overproduction of Isu1p, wild-type ISU1 and the mutant alleles with the P134S, V135E, and K136A amino acid exchanges (27) were inserted into yeast vector p426-GPD under the control of the strong TDH3 promoter (36).…”

Section: Methodsmentioning

confidence: 99%

“…To further investigate this issue, we took advantage of Isu1p mutants that carry single amino acid substitutions in the highly conserved Ssq1p binding motif comprised by residues PVK (27). These Isu1p mutant proteins have previously been , and Mge1p (ϩCME), under experimental conditions as described in the legend to Fig.…”

Section: Spectroscopic Detection Of Fe/s Cluster Formation On Isu1p Imentioning

confidence: 99%

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The Hsp70 Chaperone Ssq1p Is Dispensable for Iron-Sulfur Cluster Formation on the Scaffold Protein Isu1p

Dutkiewicz

Marszałek

Schilke

et al. 2006

Journal of Biological Chemistry

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The specialized yeast mitochondrial chaperone system, composed of the Hsp70 Ssq1p, its co-chaperone J-protein Jac1p, and the nucleotide release factor Mge1p, perform a critical function in the biogenesis of iron-sulfur (Fe/S) proteins. Using a spectroscopic assay, we have analyzed the potential role of the chaperones in Fe/S cluster assembly on the scaffold protein Isu1p in vitro in the presence of the cysteine desulfurase Nfs1p. In the absence of chaperones, the kinetics of Fe/S cluster formation on Isu1p were compatible with a chemical reconstitution pathway with Nfs1p functioning as a sulfide donor. Addition of Ssq1p improved the rates of Fe/S cluster assembly 3-fold. However, this stimulatory effect of Ssq1p required neither ATP nor Jac1p and could be fully attributed to the activation of the Nfs1p desulfurase activity by Ssq1p. Furthermore, chaperone-stimulated Fe/S cluster assembly did not involve the specific interaction between Isu1p and Ssq1p, since the effect was observed with Isu1p mutant proteins defective in this interaction, suggesting that nonspecific binding of Ssq1p to Nfs1p helped to prevent its unfolding. Consistent with this idea, these Isu1p mutants were capable of binding an Fe/S cluster in vivo but failed to restore the growth and Fe/S cluster assembly defects of a Isu1p/ Isu2p-deficient yeast strain. Taken together, these data suggest that Ssq1p/Jac1p/Mge1p are not important for Fe/S cluster synthesis on Isu1p. Hence, consistent with previous in vivo data, these chaperones likely function in steps subsequent to the de novo synthesis of the Fe/S cluster on Isu1p.

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mentioning

confidence: 83%

Section: Methodsmentioning

confidence: 99%

Section: Spectroscopic Detection Of Fe/s Cluster Formation On Isu1p Imentioning

confidence: 99%

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The Hsp70 Chaperone Ssq1p Is Dispensable for Iron-Sulfur Cluster Formation on the Scaffold Protein Isu1p

Dutkiewicz

Marszałek

Schilke

et al. 2006

Journal of Biological Chemistry

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“…On the other hand, the molecular chaperone J-protein HSCB (Jac1 in yeast) and its partner mtHsp70 (Ssq1 in yeast) play a critical role in the transfer of Fe-S clusters from the ISCU/Isu1 scaffold to recipient proteins (63). Both are indispensable processes for the homeostasis of iron metabolism in the mitochondrial compartment.…”

Section: Discussionmentioning

confidence: 99%

The Presence of Multiple Cellular Defects Associated with a Novel G50E Iron-Sulfur Cluster Scaffold Protein (ISCU) Mutation Leads to Development of Mitochondrial Myopathy

Saha¹,

Kumar²,

Srivastava³

et al. 2014

Journal of Biological Chemistry

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Background: Muscle-specific deficiency of iron-sulfur (Fe-S) cluster scaffold protein (ISCU) leads to myopathy. Results: Cells carrying the myopathy-associated G50E ISCU mutation demonstrate impaired Fe-S cluster biogenesis and mitochondrial dysfunction. Conclusion: Reduced mitochondrial respiration as a result of diminished Fe-S cluster synthesis results in muscle weakness in myopathy patients. Significance: The molecular mechanism behind disease progression should provide invaluable information to combat ISCU myopathy.

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“…The conserved tripeptide PVK (residues 134-136 in Isu) is known to be critical for interaction with Ssq1. Alterations of single residues in this motif disrupt the interaction and result in cell inviability (16). The interface between Isu and Nfs1 is complex.…”

mentioning

confidence: 99%

Cysteine desulfurase Nfs1 and Pim1 protease control levels of Isu, the Fe-S cluster biogenesis scaffold

Song

Marszałek

Craig

2012

Proc. Natl. Acad. Sci. U.S.A.

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Fe-S clusters are critical prosthetic groups for proteins involved in various critical biological processes. Before being transferred to recipient apo-proteins, Fe-S clusters are assembled on the highly conserved scaffold protein Isu, the abundance of which is regulated posttranslationally on disruption of the cluster biogenesis system. Here we report that Isu is degraded by the Lon-type AAA+ ATPase protease of the mitochondrial matrix, Pim1. Nfs1, the cysteine desulfurase responsible for providing sulfur for cluster formation, is required for the increased Isu stability occurring after disruption of cluster formation on or transfer from Isu. Physical interaction between the Isu and Nfs1 proteins, not the enzymatic activity of Nfs1, is the important factor in increased stability. Analysis of several conditions revealed that high Isu levels can be advantageous or disadvantageous, depending on the physiological condition. During the stationary phase, elevated Isu levels were advantageous, resulting in prolonged chronological lifespan. On the other hand, under iron-limiting conditions, high Isu levels were deleterious. Compared with cells expressing normal levels of Isu, such cells grew poorly and exhibited reduced activity of the heme-containing enzyme ferric reductase. Our results suggest that modulation of the degradation of Isu by the Pim1 protease is a regulatory mechanism serving to rapidly help balance the cell's need for critical ironrequiring processes under changing environmental conditions. proteolysis | Hsp70 | aging | Saccharomyces cerevisiae

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Sequence-specific Interaction between Mitochondrial Fe-S Scaffold Protein Isu and Hsp70 Ssq1 Is Essential for Their in Vivo Function

Cited by 93 publications

References 36 publications

The Hsp70 Chaperone Ssq1p Is Dispensable for Iron-Sulfur Cluster Formation on the Scaffold Protein Isu1p

The Hsp70 Chaperone Ssq1p Is Dispensable for Iron-Sulfur Cluster Formation on the Scaffold Protein Isu1p

The Presence of Multiple Cellular Defects Associated with a Novel G50E Iron-Sulfur Cluster Scaffold Protein (ISCU) Mutation Leads to Development of Mitochondrial Myopathy

Cysteine desulfurase Nfs1 and Pim1 protease control levels of Isu, the Fe-S cluster biogenesis scaffold

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