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Title: The iron uptake repressor Fep1 in the fission yeast binds Fe-S cluster through conserved cysteines

Abstract

Iron homeostasis is tightly regulated since iron is an essential but toxic element in the cell. The GATA-type transcription factor Fep1 and its orthologs contribute to iron homeostasis in many fungi by repressing genes for iron uptake when intracellular iron is high. Even though the function and interaction partners of Fep1 have been elucidated extensively In Schizosaccharomyces pombe, the mechanism behind iron-sensing by Fep1 remains elusive. It has been reported that Fep1 interacts with Fe-S-containing monothiol glutaredoxin Grx4 and Grx4-Fra2 complex. In this study, we demonstrate that Fep1 also binds iron, in the form of Fe-S cluster. Spectroscopic and biochemical analyses of as isolated and reconstituted Fep1 suggest that the dimeric Fep1 binds Fe-S clusters. The mutation study revealed that the cluster-binding depended on the conserved cysteines located between the two zinc fingers in the DNA binding domain. EPR analyses revealed [Fe-S]-specific peaks indicative of mixed presence of [2Fe-2S], [3Fe-4S], or [4Fe-4S]. The finding that Fep1 is an Fe-S protein fits nicely with the model that the Fe-S-trafficking Grx4 senses intracellular iron environment and modulates the activity of Fep1. - Highlights: • Fep1, a prototype fungal iron uptake regulator, was isolated stably from Schizosaccharomyces pombe. • Fep1 exhibits UV–visible absorptionmore » spectrum, characteristic of [Fe-S] proteins. • The iron and sulfide contents in purified or reconstituted Fep1 also support [Fe-S]. • The conserved cysteines are critical for [Fe-S]-binding. • EPR spectra at 5 K and 123 K suggest a mixed population of [Fe-S].« less

Authors:
; ; ;
Publication Date:
OSTI Identifier:
22606209
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 478; Journal Issue: 1; Other Information: Copyright (c) 2016 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ABSORPTION; ABSORPTION SPECTRA; CYSTEINE; DNA; ELECTRON SPIN RESONANCE; HOMEOSTASIS; IRON; MUTATIONS; SULFIDES; TOXICITY; TRANSCRIPTION; TRANSCRIPTION FACTORS; UPTAKE; YEASTS

Citation Formats

Kim, Hyo-Jin, Lee, Kang-Lok, Kim, Kyoung-Dong, and Roe, Jung-Hye, E-mail: jhroe@snu.ac.kr. The iron uptake repressor Fep1 in the fission yeast binds Fe-S cluster through conserved cysteines. United States: N. p., 2016. Web. doi:10.1016/J.BBRC.2016.07.070.
Kim, Hyo-Jin, Lee, Kang-Lok, Kim, Kyoung-Dong, & Roe, Jung-Hye, E-mail: jhroe@snu.ac.kr. The iron uptake repressor Fep1 in the fission yeast binds Fe-S cluster through conserved cysteines. United States. doi:10.1016/J.BBRC.2016.07.070.
Kim, Hyo-Jin, Lee, Kang-Lok, Kim, Kyoung-Dong, and Roe, Jung-Hye, E-mail: jhroe@snu.ac.kr. Fri . "The iron uptake repressor Fep1 in the fission yeast binds Fe-S cluster through conserved cysteines". United States. doi:10.1016/J.BBRC.2016.07.070.
@article{osti_22606209,
title = {The iron uptake repressor Fep1 in the fission yeast binds Fe-S cluster through conserved cysteines},
author = {Kim, Hyo-Jin and Lee, Kang-Lok and Kim, Kyoung-Dong and Roe, Jung-Hye, E-mail: jhroe@snu.ac.kr},
abstractNote = {Iron homeostasis is tightly regulated since iron is an essential but toxic element in the cell. The GATA-type transcription factor Fep1 and its orthologs contribute to iron homeostasis in many fungi by repressing genes for iron uptake when intracellular iron is high. Even though the function and interaction partners of Fep1 have been elucidated extensively In Schizosaccharomyces pombe, the mechanism behind iron-sensing by Fep1 remains elusive. It has been reported that Fep1 interacts with Fe-S-containing monothiol glutaredoxin Grx4 and Grx4-Fra2 complex. In this study, we demonstrate that Fep1 also binds iron, in the form of Fe-S cluster. Spectroscopic and biochemical analyses of as isolated and reconstituted Fep1 suggest that the dimeric Fep1 binds Fe-S clusters. The mutation study revealed that the cluster-binding depended on the conserved cysteines located between the two zinc fingers in the DNA binding domain. EPR analyses revealed [Fe-S]-specific peaks indicative of mixed presence of [2Fe-2S], [3Fe-4S], or [4Fe-4S]. The finding that Fep1 is an Fe-S protein fits nicely with the model that the Fe-S-trafficking Grx4 senses intracellular iron environment and modulates the activity of Fep1. - Highlights: • Fep1, a prototype fungal iron uptake regulator, was isolated stably from Schizosaccharomyces pombe. • Fep1 exhibits UV–visible absorption spectrum, characteristic of [Fe-S] proteins. • The iron and sulfide contents in purified or reconstituted Fep1 also support [Fe-S]. • The conserved cysteines are critical for [Fe-S]-binding. • EPR spectra at 5 K and 123 K suggest a mixed population of [Fe-S].},
doi = {10.1016/J.BBRC.2016.07.070},
journal = {Biochemical and Biophysical Research Communications},
number = 1,
volume = 478,
place = {United States},
year = {Fri Sep 09 00:00:00 EDT 2016},
month = {Fri Sep 09 00:00:00 EDT 2016}
}
  • Research highlights: {yields} Monothiol glutaredoxin Grx4 allows Fep1-mediated de-repression of iron uptake genes at low iron. {yields} Grx4 directly interacts with Fep1 in vivo and in vitro. {yields} The Cys172 in the CGFS motif of Grx4 is necessary for cell proliferation and iron regulation. {yields} The Cys172 of Grx4 is required for normal interaction with Fep1. -- Abstract: The fission yeast Schizosaccharomyces pombe contains two CGFS-type monothiol glutaredoxins, Grx4 and Grx5, which are localized primarily in the nucleus and mitochondria, respectively. We observed involvement of Grx4 in regulating iron-responsive gene expression, which is modulated by a repressor Fep1. Lack ofmore » Grx4 caused defects not only in growth but also in the expression of both iron-uptake and iron-utilizing genes regardless of iron availability. In order to unravel how Grx4 is involved in Fep1-mediated regulation, interaction between them was investigated. Co-immunoprecipitation and bimolecular fluorescence complementation (BiFC) revealed that Grx4 physically interacts with Fep1 in vivo. BiFC revealed localized nuclear dots produced by interaction of Grx4 with Fep1. Mutation of cysteine-172 in the CGFS motif to serine (C172S) produced effects similarly observed under Grx4 depletion, such as the loss of iron-dependent gene regulation and the absence of nuclear dots in BiFC analysis. These results suggest that the ability of Grx4 to bind iron, most likely Fe-S cofactor, could be critical in interacting with and modulating the activity of Fep1.« less
  • The binding affinity of (S)-2-amino-6-nitrohexanoic acid to human arginase I was studied using surface plasmon resonance (K{sub d} = 60 {mu}M), and the X-ray crystal structure of the enzyme-inhibitor complex was determined at 1.6 {angstrom} resolution to reveal multiple nitro-metal coordination interactions.
  • Mitochondrial monothiol glutaredoxins that bind Fe-S cluster are known to participate in Fe-S cluster assembly. However, their precise role has not been well understood. Among three monothiol glutaredoxins (Grx3, 4, and 5) in Schizosaccharomyces pombe only Grx5 resides in mitochondria. The {Delta}grx5 mutant requires cysteine on minimal media, and does not grow on non-fermentable carbon source such as glycerol. We found that the mutant is low in the activity of Fe-S enzymes in mitochondria as well as in the cytoplasm. Screening of multi-copy suppressor of growth defects of the mutant identified isa1{sup +} gene encoding a putative A-type Fe-S scaffold,more » in addition to mas5{sup +} and hsc1{sup +} genes encoding putative chaperones for Fe-S assembly process. Examination of other scaffold and chaperone genes revealed that isa2{sup +}, but not isu1{sup +} and ssc1{sup +}, complemented the growth phenotype of {Delta}grx5 mutant as isa1{sup +} did, partly through restoration of Fe-S enzyme activities. The mutant also showed a significant decrease in the amount of mitochondrial DNA. We demonstrated that Grx5 interacts in vivo with Isa1 and Isa2 proteins in mitochondria by observing bimolecular fluorescence complementation. These results indicate that Grx5 plays a central role in Fe-S assembly process through interaction with A-type Fe-S scaffold proteins Isa1 and Isa2, each of which is an essential protein in S. pombe, and supports mitochondrial genome integrity as well as Fe-S assembly.« less
  • Sulfur dioxide reacts with [PPN]{sub 2}[MFe{sub 3}(CO){sub 14}] (M = Cr, Mo, W) (PPN = bistriphenylphosphonium iminium) to produce [PPN]{sub 2}[Fe{sub 3}(CO){sub 9}({mu}{sub 3},{eta}{sup 2}-SO{sub 2})] (1) and [PPN]{sub 2}[Fe{sub 3}(CO){sub 8}({mu}-SO{sub 2}){mu}{sub 3}-S] (2), which were characterized by infrared spectroscopy, {sup 13}C NMR, and X-ray crystallography. Further reaction of 1 with sulfur dioxide results in the formation of 2 in 48% yield. Reaction of SO{sub 2} with [PPN]{sub 2}[Fe{sub 4}(CO){sub 13}] yields [PPN]{sub 2}[Fe{sub 2}(CO){sub 6}({mu}-SO{sub 2}){sub 2}] (3) which was characterized by infrared spectroscopy, {sup 13}C NMR, mass spectrometry, and X-ray crystallography. One equivalent of sulfur dioxide withmore » [PPN]{sub 2}[MFe{sub 3}(CO){sub 14}C] (M = Cr, W) produces [PPN]{sub 2}[Fe{sub 3}(CO){sub 8}({mu}-SO{sub 2})-({mu}{sub 3}-CCO)] (4), which on further reaction with SO{sub 2} gives the known cluster [PPN]{sub 2}[Fe{sub 3}(CO){sub 7}({mu}-SO{sub 2}){sub 2}({mu}{sub 3}-CCO)] (5). An excess of sulfur dioxide with [MFe{sub 3}(CO){sub n}C]{sup x{minus}} (M = Cr, W: n = 13, x = 2; M = Rh: n = 12, x = 1; M = Mn: n = 13, x = 1) produced 5 as the only identified product.« less
  • Frataxin, a conserved nuclear-encoded mitochondrial protein, plays a direct role in iron-sulfur cluster biosynthesis within the ISC assembly pathway. Humans with frataxin deficiency have Friedreich's ataxia, a neurodegenerative disorder characterized by mitochondrial iron overload and disruption in Fe-S cluster synthesis. Biochemical and genetic studies have shown frataxin interacts with the iron-sulfur cluster assembly scaffold protein (in yeast, there are two, Isu1 and Isu2), indicating frataxin plays a direct role in cluster assembly, possibly by serving as an iron chaperone in the assembly pathway. Here we provide molecular details of how yeast frataxin (Yfh1) interacts with Isu1 as a structural modulemore » to improve our understanding of the multiprotein complex assembly that completes Fe-S cluster assembly; this complex also includes the cysteine desulfurase (Nfs1 in yeast) and the accessory protein (Isd11), together in the mitochondria. Thermodynamic binding parameters for protein partner and iron binding were measured for the yeast orthologs using isothermal titration calorimetry. Nuclear magnetic resonance spectroscopy was used to provide the molecular details to understand how Yfh1 interacts with Isu1. X-ray absorption studies were used to electronically and structurally characterize how iron is transferred to Isu1 and then incorporated into an Fe-S cluster. These results were combined with previously published data to generate a structural model for how the Fe-S cluster protein assembly complex can come together to accomplish Fe-S cluster assembly.« less