Kyung-Chang Lee

IscR acts as an activator in response to oxidative stress for the suf operon encoding Fe-S assembly proteins.

In Escherichia coli, Fe‐S clusters are assembled by gene products encoded from the isc and suf operons. Both the iscRSUA and sufABCDSE operons are induced highly by oxidants, reflecting an increased need for providing and maintaining Fe‐S clusters under oxidative stress conditions. Three cis‐acting oxidant‐responsive elements (ORE‐I, II, III) in the upstream of the sufA promoter serve as the binding sites for OxyR, IHF and an uncharacterized factor respectively. Using DNA affinity fractionation, we isolated an ORE‐III‐binding factor that positively regulates the suf operon in response to various oxidants. MALDI‐TOF mass analysis identified it with IscR, known to serve as a repressor of the iscRSUA gene expression under anaerobic condition as a [2Fe‐2S]‐bound form. The iscR null mutation abolished ORE‐III‐binding activity in cell extracts, and caused a significant decrease in the oxidant induction of sufA in vivo. OxyR and IscR contributed almost equally to activate the sufA operon in response to oxidants. Purified IscR that lacked Fe‐S cluster bound to the ORE‐III site and activated transcription from the sufA promoter in vitro. Mutations in Fe‐S‐binding sites of IscR enabled sufA activation in vivo and in vitro. These results support a model that IscR in its demetallated form directly activates sufA transcription, while it de‐represses isc operon, under oxidative stress condition.

Oxidant-Responsive Induction of the suf Operon, Encoding a Fe-S Assembly System, through Fur and IscR in Escherichia coli

The suf operon encoding a Fe-S assembly system is induced by peroxides through activators OxyR and IscR in Escherichia coli. For apo-IscR to bind, oxidation-mediated dissociation of Fur is required. Therefore, a peroxide-responsive signal is transduced through OxyR, IscR, and Fur to achieve oxidation-sensitive and maximal induction of this operon.

Monothiol glutaredoxin Grx5 interacts with Fe-S scaffold proteins Isa1 and Isa2 and supports Fe-S assembly and DNA integrity in mitochondria of fission yeast

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 Deltagrx5 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(+) gene encoding a putative A-type Fe-S scaffold, in addition to mas5(+) and hsc1(+) genes encoding putative chaperones for Fe-S assembly process. Examination of other scaffold and chaperone genes revealed that isa2(+), but not isu1(+) and ssc1(+), complemented the growth phenotype of Deltagrx5 mutant as isa1(+) 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.

Multi-domain CGFS-type glutaredoxin Grx4 regulates iron homeostasis via direct interaction with a repressor Fep1 in fission yeast.

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 of 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.

Study of chromium and chromium nitride coatings deposited by inductively coupled plasma-assisted evaporation

Abstract Inductively coupled plasma (ICP)-assisted reactive evaporation was successfully applied to deposit Cr and CrN x coatings (>4000 HK 0.01 ) at a high deposition rate of 1000 A/min without intentional substrate heating. High plasma density and adequate substrate bias were essential for achieving a stoichiometric and dense microstructure. Films deposited at high ICP power exhibited bulk CrN crystalline properties, and this is thought to result from the extremely enhanced surface processes, even on unheated substrates.

Essential function of Aco2, a fusion protein of aconitase and mitochondrial ribosomal protein bL21, in mitochondrial translation in fission yeast

A possible interaction between aconitase and a mitochondrial ribosomal protein was suggested in a genome‐wide interactome study. In fission yeast Schizosaccharomyces pombe, the aco2+ gene encodes a fusion protein between aconitase and a putative mitochondrial ribosomal protein bL21 (Mrpl49). Two types of aco2 + transcripts are generated via alternative poly (A) site selection, producing both a single aconitase domain protein and the fusion form. The bL21‐fused Aco2 protein resides in mitochondria as well as in the cytosol and the nucleus. The viability defect of aco2 mutation is complemented not by the aconitase domain but by the bL21 domain, which enables mitochondrial translation.