Cysteine Import via the High Affinity Glutathione Transporter Hgt1 Rescues Glutathione Auxotrophy in Yeast.

Glutathione (GSH) is an abundant thiol-containing tripeptide that functions in redox homeostasis, protein folding, and iron metabolism. In Saccharomyces cerevisiae, GSH depletion leads to increased sensitivity to oxidants and other toxic compounds, disruption of Fe-S cluster biogenesis, and eventually cell death. GSH pools are supplied by intracellular biosynthesis and GSH import from the extracellular environment.

Iron homeostasis proteins Grx4 and Fra2 control activity of the Schizosaccharomyces pombe iron repressor Fep1 by facilitating [2Fe-2S] cluster removal.

The Bol2 homolog Fra2 and monothiol glutaredoxin Grx4 together play essential roles in regulating iron homeostasis in Schizosaccharomyces pombe. In vivo studies indicate that Grx4 and Fra2 act as coinhibitory partners that inactivate the transcriptional repressor Fep1 in response to iron deficiency. In Saccharomyces cerevisiae, Bol2 is known to form a [2Fe-2S]-bridged heterodimer with the monothiol Grxs Grx3 and Grx4, with the cluster ligands provided by conserved residues in Grx3/4 and Bol2 as well as GSH.

Iron-sulfur cluster biogenesis, trafficking, and signaling: Roles for CGFS glutaredoxins and BolA proteins.

The synthesis and trafficking of iron-sulfur (Fe-S) clusters in both prokaryotes and eukaryotes requires coordination within an expanding network of proteins that function in the cytosol, nucleus, mitochondria, and chloroplasts in order to assemble and deliver these ancient and essential cofactors to a wide variety of Fe-S-dependent enzymes and proteins. This review focuses on the evolving roles of two ubiquitous classes of proteins that operate in this network: CGFS glutaredoxins and BolA proteins. Monothiol or CGFS glutaredoxins possess a Cys-Gly-Phe-Ser active site that coordinates an Fe-S cluster in a homodimeric complex.

Iron-sulfur cluster signaling: The common thread in fungal iron regulation.

Iron homeostasis in fungi involves balancing iron uptake and storage with iron utilization to achieve adequate, nontoxic levels of this essential nutrient. Extensive work in the nonpathogenic yeast Saccharomyces cerevisiae and Schizosaccharomyces pombe has uncovered unique iron regulation networks for each organism that control iron metabolism via distinct molecular mechanisms. However, common themes have emerged from these studies.

The conserved CDC motif in the yeast iron regulator Aft2 mediates iron-sulfur cluster exchange and protein-protein interactions with Grx3 and Bol2.

The Saccharomyces cerevisiae transcriptional activator Aft1 and its paralog Aft2 respond to iron deficiency by upregulating expression of proteins required for iron uptake at the plasma membrane, vacuolar iron transport, and mitochondrial iron metabolism, with the net result of mobilizing iron from extracellular sources and intracellular stores. Conversely, when iron levels are sufficient, Aft1 and Aft2 interact with the cytosolic glutaredoxins Grx3 and Grx4 and the BolA protein Bol2, which promote Aft1/2 dissociation from DNA and subsequent export from the nucleus. Previous studies unveiled the molecular mechanism for iron-dependent inhibition of Aft1/2 activity, demonstrating that the [2Fe-2S]-bridged Grx3-Bol2 heterodimer transfers a cluster to Aft2, driving Aft2 dimerization and dissociation from DNA.

Characterization of Glutaredoxin Fe-S Cluster-Binding Interactions Using Circular Dichroism Spectroscopy.

Monothiol glutaredoxins (Grxs) with a conserved Cys-Gly-Phe-Ser (CGFS) active site are iron-sulfur (Fe-S) cluster-binding proteins that interact with a variety of partner proteins and perform crucial roles in iron metabolism including Fe-S cluster transfer, Fe-S cluster repair, and iron signaling. Various analytical and spectroscopic methods are currently being used to monitor and characterize glutaredoxin Fe-S cluster-dependent interactions at the molecular level. The electronic, magnetic, and vibrational properties of the protein-bound Fe-S cluster provide a convenient handle to probe the structure, function, and coordination chemistry of Grx complexes.

Checks and balances for the iron bank.

The RNA-binding iron regulatory proteins IRP1 and IRP2 are inactivated by either Fe-S cluster insertion or protein degradation mediated by the E3 ligase component FBXL5. However, the mechanisms for coordination between Fe-S cluster assembly, FBXL5, and IRP1/IRP2 activity are poorly defined. A new study reveals that FBXL5 plays a critical role in limiting IRP1 and IRP2 overaccumulation when cytosolic Fe-S cluster assembly is impaired in order to maintain optimal iron levels for cell viability.

Endoplasmic Reticulum Transport of Glutathione by Sec61 Is Regulated by Ero1 and Bip.

In the endoplasmic reticulum (ER), Ero1 catalyzes disulfide bond formation and promotes glutathione (GSH) oxidation to GSSG. Since GSSG cannot be reduced in the ER, maintenance of the ER glutathione redox state and levels likely depends on ER glutathione import and GSSG export. We used quantitative GSH and GSSG biosensors to monitor glutathione import into the ER of yeast cells.

Schizosaccharomyces pombe Grx4 regulates the transcriptional repressor Php4 via [2Fe-2S] cluster binding.

The fission yeast Schizosaccharomyces pombe expresses the CCAAT-binding factor Php4 in response to iron deprivation. Php4 forms a transcription complex with Php2, Php3, and Php5 to repress the expression of iron proteins as a means to economize iron usage. Previous in vivo results demonstrate that the function and location of Php4 are regulated in an iron-dependent manner by the cytosolic CGFS type glutaredoxin Grx4.

The Escherichia coli BolA Protein IbaG Forms a Histidine-Ligated [2Fe-2S]-Bridged Complex with Grx4.

Two ubiquitous protein families have emerged as key players in iron metabolism, the CGFS-type monothiol glutaredoxins (Grxs) and the BolA proteins. Monothiol Grxs and BolA proteins form heterocomplexes that have been implicated in Fe-S cluster assembly and trafficking. The Escherichia coli genome encodes members of both of these proteins families, namely, the monothiol glutaredoxin Grx4 and two BolA family proteins, BolA and IbaG.

The Myeloablative Drug Busulfan Converts Cysteine to Dehydroalanine and Lanthionine in Redoxins.

The myeloablative agent busulfan (1,4-butanediol dimethanesulfonate) is an old drug that is used routinely to eliminate cancerous bone marrow prior to hematopoietic stem cell transplant. The myeloablative activity and systemic toxicity of busulfan have been ascribed to its ability to cross-link DNA. In contrast, here we demonstrate that incubation of busulfan with the thiol redox proteins glutaredoxin or thioredoxin at pH 7.

Cytosolic Fe-S Cluster Protein Maturation and Iron Regulation Are Independent of the Mitochondrial Erv1/Mia40 Import System.

The sulfhydryl oxidase Erv1 partners with the oxidoreductase Mia40 to import cysteine-rich proteins in the mitochondrial intermembrane space. In Saccharomyces cerevisiae, Erv1 has also been implicated in cytosolic Fe-S protein maturation and iron regulation. To investigate the connection between Erv1/Mia40-dependent mitochondrial protein import and cytosolic Fe-S cluster assembly, we measured Mia40 oxidation and Fe-S enzyme activities in several erv1 and mia40 mutants.

Molecular mechanism and structure of the Saccharomyces cerevisiae iron regulator Aft2.

The paralogous iron-responsive transcription factors Aft1 and Aft2 (activators of ferrous transport) regulate iron homeostasis in Saccharomyces cerevisiae by activating expression of iron-uptake and -transport genes when intracellular iron is low. We present the previously unidentified crystal structure of Aft2 bound to DNA that reveals the mechanism of DNA recognition via specific interactions of the iron-responsive element with a Zn(2+)-containing WRKY-GCM1 domain in Aft2. We also show that two Aft2 monomers bind a [2Fe-2S] cluster (or Fe(2+)) through a Cys-Asp-Cys motif, leading to dimerization of Aft2 and decreased DNA-binding affinity.

Iron sensing and regulation in Saccharomyces cerevisiae: Ironing out the mechanistic details.

Regulation of iron metabolism in Saccharomyces cerevisiae is achieved at the transcriptional level by low (Aft1 and Aft2) and high iron-sensing (Yap5) transcription factors, and at the post-transcriptional level by mRNA-binding proteins (Cth1 and Cth2). In this review we highlight recent studies unveiling the critical role that iron-sulfur clusters play in control of Aft1/2 and Yap5 activity, as well as the complex relationship between iron homeostasis and thiol redox metabolism. In addition, new insights into the localization and regulation of Cth1/Cth2 have added another layer of complexity to the cell's adaptation to iron deficiency.

The iron metallome in eukaryotic organisms.

This chapter is focused on the iron metallome in eukaryotes at the cellular and subcellular level, including properties, utilization in metalloproteins, trafficking, storage, and regulation of these processes. Studies in the model eukaryote Saccharomyces cerevisiae and mammalian cells will be highlighted. The discussion of iron properties will center on the speciation and localization of intracellular iron as well as the cellular and molecular mechanisms for coping with both low iron bioavailability and iron toxicity.

Monothiol glutaredoxins and A-type proteins: partners in Fe-S cluster trafficking.

Monothiol glutaredoxins (Grxs) are proposed to function in Fe-S cluster storage and delivery, based on their ability to exist as apo monomeric forms and dimeric forms containing a subunit-bridging [Fe(2)S(2)](2+) cluster, and to accept [Fe(2)S(2)](2+) clusters from primary scaffold proteins. In addition yeast cytosolic monothiol Grxs interact with Fra2 (Fe repressor of activation-2), to form a heterodimeric complex with a bound [Fe(2)S(2)](2+) cluster that plays a key role in iron sensing and regulation of iron homeostasis. In this work, we report on in vitro UV-visible CD studies of cluster transfer between homodimeric monothiol Grxs and members of the ubiquitous A-type class of Fe-S cluster carrier proteins ((Nif)IscA and SufA).

Functions and cellular compartmentation of the thioredoxin and glutathione pathways in yeast.

Significance: The thioredoxin (TRX) and glutathione (GSH) pathways are universally conserved thiol-reductase systems that drive an array of cellular functions involving reversible disulfide formation. Here we consider these pathways in Saccharomyces cerevisiae, focusing on their cell compartment-specific functions, as well as the mechanisms that explain extreme differences of redox states between compartments.

Recent Advances: Recent work leads to a model in which the yeast TRX and GSH pathways are not redundant, in contrast to Escherichia coli.

Redox properties of the disulfide bond of human Cu,Zn superoxide dismutase and the effects of human glutaredoxin 1.

The intramolecular disulfide bond in hSOD1 [human SOD1 (Cu,Zn superoxide dismutase 1)] plays a key role in maintaining the protein's stability and quaternary structure. In mutant forms of SOD1 that cause familial ALS (amyotrophic lateral sclerosis), this disulfide bond is more susceptible to chemical reduction, which may lead to destabilization of the dimer and aggregation. During hSOD1 maturation, disulfide formation is catalysed by CCS1 (copper chaperone for SOD1).

Monothiol CGFS glutaredoxins and BolA-like proteins: [2Fe-2S] binding partners in iron homeostasis.

Monothiol glutaredoxins (Grxs) with a signature CGFS active site and BolA-like proteins have recently emerged as novel players in iron homeostasis. Elegant genetic and biochemical studies examining the functional and physical interactions of CGFS Grxs in the fungi Saccharomyces cerevisiae and Schizosaccharomyces pombe have unveiled their essential roles in intracellular iron signaling, iron trafficking, and the maturation of Fe-S cluster proteins. Biophysical and biochemical analyses of the [2Fe-2S] bridging interaction between CGFS Grxs and a BolA-like protein in S.

Redox-sensitive YFP sensors monitor dynamic nuclear and cytosolic glutathione redox changes.

Intracellular redox homeostasis is crucial for many cellular functions but accurate measurements of cellular compartment-specific redox states remain technically challenging. To better characterize redox control in the nucleus, we targeted a yellow fluorescent protein-based redox sensor (rxYFP) to the nucleus of the yeast Saccharomyces cerevisiae. Parallel analyses of the redox state of nucleus-rxYFP and cytosol-rxYFP allowed us to monitor distinctively dynamic glutathione (GSH) redox changes within these two compartments under a given condition.

Human glutaredoxin 3 forms [2Fe-2S]-bridged complexes with human BolA2.

Human glutaredoxin 3 (Glrx3) is an essential [2Fe-2S]-binding protein with ill-defined roles in immune cell response, embryogenesis, cancer cell growth, and regulation of cardiac hypertrophy. Similar to other members of the CGFS monothiol glutaredoxin (Grx) family, human Glrx3 forms homodimers bridged by two [2Fe-2S] clusters that are ligated by the conserved CGFS motifs and glutathione (GSH). We recently demonstrated that the yeast homologues of human Glrx3 and the yeast BolA-like protein Fra2 form [2Fe-2S]-bridged heterodimers that play a key role in signaling intracellular iron availability.

Histidine 103 in Fra2 is an iron-sulfur cluster ligand in the [2Fe-2S] Fra2-Grx3 complex and is required for in vivo iron signaling in yeast.

The BolA homologue Fra2 and the cytosolic monothiol glutaredoxins Grx3 and Grx4 together play a key role in regulating iron homeostasis in Saccharomyces cerevisiae. Genetic studies indicate that Grx3/4 and Fra2 regulate activity of the iron-responsive transcription factors Aft1 and Aft2 in response to mitochondrial Fe-S cluster biosynthesis. We have previously shown that Fra2 and Grx3/4 form a [2Fe-2S](2+)-bridged heterodimeric complex with iron ligands provided by the active site cysteine of Grx3/4, glutathione, and a histidine residue.

The yeast iron regulatory proteins Grx3/4 and Fra2 form heterodimeric complexes containing a [2Fe-2S] cluster with cysteinyl and histidyl ligation.

The transcription of iron uptake and storage genes in Saccharomyces cerevisiae is primarily regulated by the transcription factor Aft1. Nucleocytoplasmic shuttling of Aft1 is dependent upon mitochondrial Fe-S cluster biosynthesis via a signaling pathway that includes the cytosolic monothiol glutaredoxins (Grx3 and Grx4) and the BolA homologue Fra2. However, the interactions between these proteins and the iron-dependent mechanism by which they control Aft1 localization are unclear.

Activation of Cu,Zn-superoxide dismutase in the absence of oxygen and the copper chaperone CCS.

Eukaryotic Cu,Zn-superoxide dismutases (SOD1s) are generally thought to acquire the essential copper cofactor and intramolecular disulfide bond through the action of the CCS copper chaperone. However, several metazoan SOD1s have been shown to acquire activity in vivo in the absence of CCS, and the Cu,Zn-SOD from Caenorhabditis elegans has evolved complete independence from CCS. To investigate SOD1 activation in the absence of CCS, we compared and contrasted the CCS-independent activation of C.