RESUMO
Poly(A) (pA) tail binding proteins (PABPs) control mRNA polyadenylation, stability, and translation. In a purified system, S. cerevisiae PABPs, Pab1p and Nab2p, are individually sufficient to provide normal pA tail length. However, it is unknown how this occurs in more complex environments. Here we find that the nuclear exosome subunit Rrp6p counteracts the in vitro and in vivo extension of mature pA tails by the noncanonical pA polymerase Trf4p. Moreover, PABP loading onto nascent pA tails is controlled by Rrp6p; while Pab1p is the major PABP, Nab2p only associates in the absence of Rrp6p. This is because Rrp6p can interact with Nab2p and displace it from pA tails, potentially leading to RNA turnover, as evidenced for certain pre-mRNAs. We suggest that a nuclear mRNP surveillance step involves targeting of Rrp6p by Nab2p-bound pA-tailed RNPs and that pre-mRNA abundance is regulated at this level.
Assuntos
Exorribonucleases/metabolismo , Poli A/química , Proteínas de Ligação a Poli(A)/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Bases , Núcleo Celular/metabolismo , Citoplasma/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo , Exossomos/metabolismo , Regulação Fúngica da Expressão Gênica , Modelos Biológicos , Dados de Sequência Molecular , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Poli A/genética , Ligação Proteica , RNA/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
In this issue of Molecular Cell, Johnson et al. (2009) reveal a physical link between the yeast Pcf11p and Yra1p proteins, providing insights into the coupling of mRNA 3' end formation and export.
Assuntos
Ribonucleoproteínas/metabolismo , Transporte Ativo do Núcleo Celular , Proteínas Fúngicas/metabolismo , Modelos Biológicos , RNA Mensageiro/metabolismoRESUMO
The yeast THO complex and the associated RNA helicase Sub2p are important mRNP maturation factors. Transcripts produced in THO/sub2 mutants are subject to degradation by a surveillance mechanism that involves the nuclear RNA exosome. Here we show that inefficient polyadenylation forms the basis of this accelerated mRNA decay. A genetic screen reveals extensive interactions between deletions of THO subunits and mRNA 3' end processing mutants. Nuclear run-ons strengthen this link by showing premature transcription termination close to polyadenylation sites in THO/sub2 mutants in vivo. Moreover, in vitro, pre-mRNA substrates are poorly polyadenylated and consequently unstable in extracts from THO/sub2 mutant strains. Decreased polyadenylation correlates with a specific downregulation of the poly(A)-polymerase cofactor Fip1p by the ubiquitin/proteasome pathway. Both polyadenylation defects and Fip1p instability depend on the nuclear exosome component Rrp6p and its activator Trf4p. We suggest that removal of aberrant mRNA is facilitated by direct regulation of polyadenylation activity.
Assuntos
Adenosina Trifosfatases/metabolismo , Núcleo Celular/metabolismo , Mutação/genética , Poliadenilação , Saccharomyces cerevisiae/enzimologia , Sistemas de Transporte de Aminoácidos/metabolismo , Códon sem Sentido , Regulação para Baixo , Complexos Multiproteicos/metabolismo , Poli A/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Precursores de RNA/metabolismo , Estabilidade de RNA , RNA Fúngico/metabolismo , RNA Mensageiro , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcrição Gênica , Ubiquitina/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
BACKGROUND: The opportunistic pathogen Candida glabrata is a member of the Saccharomycetaceae yeasts. Like its close relative Saccharomyces cerevisiae, it underwent a whole-genome duplication followed by an extensive loss of genes. Its genome contains a large number of very long tandem repeats, called megasatellites. In order to determine the whole replication program of the C. glabrata genome and its general chromosomal organization, we used deep-sequencing and chromosome conformation capture experiments. RESULTS: We identified 253 replication fork origins, genome wide. Centromeres, HML and HMR loci, and most histone genes are replicated early, whereas natural chromosomal breakpoints are located in late-replicating regions. In addition, 275 autonomously replicating sequences (ARS) were identified during ARS-capture experiments, and their relative fitness was determined during growth competition. Analysis of ARSs allowed us to identify a 17-bp consensus, similar to the S. cerevisiae ARS consensus sequence but slightly more constrained. Megasatellites are not in close proximity to replication origins or termini. Using chromosome conformation capture, we also show that early origins tend to cluster whereas non-subtelomeric megasatellites do not cluster in the yeast nucleus. CONCLUSIONS: Despite a shorter cell cycle, the C. glabrata replication program shares unexpected striking similarities to S. cerevisiae, in spite of their large evolutionary distance and the presence of highly repetitive large tandem repeats in C. glabrata. No correlation could be found between the replication program and megasatellites, suggesting that their formation and propagation might not be directly caused by replication fork initiation or termination.
Assuntos
Candida glabrata/genética , Cromossomos Fúngicos , Replicação do DNA , Genoma Fúngico , Ciclo Celular/genética , Genes FúngicosRESUMO
Sub2p/UAP56 is a highly conserved DEAD-box RNA helicase involved in the packaging and nuclear export of mRNA/protein particles (mRNPs). In Saccharomyces cerevisiae, Sub2p is recruited to active chromatin by the pentameric THO complex and incorporated into the larger transcription-export (TREX) complex. Sub2p also plays a role in the maintenance of genome integrity as its inactivation causes severe transcription-dependent recombination of DNA. Despite the central role of Sub2p in early mRNP biology, little is known about its function. Here, we report the presence of an N-terminal motif (NTM) conserved specifically in the Sub2p branch of RNA helicases. Mutation of the NTM causes nuclear accumulation of poly(A)(+) RNA and impaired growth without affecting core helicase functions. Thus, the NTM functions as an autonomous unit. Moreover, two sub2 mutants, that are deficient in ATP binding, act in a trans-dominant negative fashion for growth and induce high recombination rates in vivo. Although wild-type Sub2p is prevented access to transcribed loci in such a background, this does not mechanistically explain the phenotype.
Assuntos
Adenosina Trifosfatases/genética , Instabilidade Genômica , Mutação/genética , Transporte de RNA/genética , RNA Fúngico/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Western Blotting , Núcleo Celular/genética , Cromatina/genética , Imunoprecipitação da Cromatina , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Transcrição GênicaRESUMO
Production of messenger ribonucleoprotein particles (mRNPs) is subjected to quality control (QC). In Saccharomyces cerevisiae, the RNA exosome and its cofactors are part of the nuclear QC machinery that removes, or stalls, aberrant molecules, thereby ensuring that only correctly formed mRNPs are exported to the cytoplasm. The Ccr4-Not complex, which constitutes the major S. cerevisiae cytoplasmic deadenylase, has recently been implied in nuclear exosome-related processes. Consistent with a possible nuclear function of the complex, the deletion or mutation of Ccr4-Not factors also elicits transcription phenotypes. Here we use genetic depletion of the Mft1p protein of the THO transcription/mRNP packaging complex as a model system to link the Ccr4-Not complex to nuclear mRNP QC. We reveal strong genetic interactions between alleles of the Ccr4-Not complex with both the exosomal RRP6 and MFT1 genes. Moreover, Rrp6p-dependent in vivo QC phenotypes of Δmft1 cells can be rescued by codeletion of several Ccr4-Not components. We discuss how the Ccr4-Not complex may connect with the mRNP QC pathway.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Genes Fúngicos/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/metabolismo , Ribonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Ciclo Celular/genética , Exorribonucleases/genética , Exorribonucleases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Ligação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Repressoras/genética , Ribonucleases/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transcrição Gênica , Ubiquitina-Proteína Ligases/genéticaRESUMO
In eukaryotic cells, primary transcripts are processed and bound by proteins before export to the cytoplasm. Nuclear production of export-competent messenger ribonucleoprotein particles (mRNPs) is a complicated process, and mRNP biogenic events that function sub-optimally are rapidly attacked by surveillance leading to degradation of the mRNA. Export of nuclear mRNAs is therefore constantly challenged by the opposing force of mRNA retention and decay. This balance ensures that only 'perfect' transcripts persist, and that non-functional and potentially deleterious transcripts are removed early in their biogenesis. Thus, eukaryotic systems of mRNP quality control can be viewed as simple Darwinian principles operating at the molecular level.
Assuntos
Núcleo Celular/metabolismo , Ribonucleoproteínas/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Regulação Fúngica da Expressão Gênica , Modelos Biológicos , Poliadenilação/fisiologia , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , Processamento Pós-Transcricional do RNA/fisiologia , Splicing de RNA/fisiologia , Transporte de RNA/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleoproteínas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcrição GênicaRESUMO
Megasatellites are large tandem repeats found in all fungal genomes but especially abundant in the opportunistic pathogen Candida glabrata. They are encoded in genes involved in cell-cell interactions, either between yeasts or between yeast and human cells. In the present work, we have been using an iterative genetic system to delete several Candida glabrata megasatellite-containing genes and found that 2 of them were positively involved in adhesion to epithelial cells, whereas 3 genes negatively controlled adhesion. Two of the latter, CAGL0B05061g or CAGL0A04851g, were also negative regulators of yeast-to-yeast adhesion, making them central players in controlling Candida glabrata adherence properties. Using a series of synthetic Saccharomyces cerevisiae strains in which the FLO1 megasatellite was replaced by other tandem repeats of similar length but different sequences, we showed that the capacity of a strain to flocculate in liquid culture was unrelated to its capacity to adhere to epithelial cells or to invade agar. Finally, to understand how megasatellites were initially created and subsequently expanded, an experimental evolution system was set up, in which modified yeast strains containing different megasatellite seeds were grown in bioreactors for more than 200 generations and selected for their ability to sediment at the bottom of the culture tube. Several flocculation-positive mutants were isolated. Functionally relevant mutations included general transcription factors as well as a 230-kbp segmental duplication.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Candida glabrata/genética , Floculação , Genoma Fúngico , Humanos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
We pursued the characterization of the divisome of the spherical-celled cyanobacterium Synechocystis PCC6803, through deletion, site-directed mutagenesis, GFP tagging, two-hybrid and co-immunoprecipitation assays. We presently report that the DivIVA-like protein Cdv3 is essential to both cell growth and division, whereas the AmiC, AmpH, FtsE, FtsN, SpoIID, YlmD, YlmE and YlmG proteins are dispensable. With the exception of the self-interacting protein YlmD, none of these dispensable factors appeared to interact with ZipN, the crucial cytokinetic factor we previously characterized. By contrast, we found that ZipN interacts with itself and the self-interacting protein Cdv3, as well as with all other crucial cytokinetic factors we previously characterized, namely: FtsZ, FtsI, FtsQ, SepF and ZipS. We also identified ZipN amino acids selectively involved in ZipN interaction with one of its following partners, Cdv3, FtsQ or SepF. Finally, we found no direct interaction between Cdv3, SepF and ZipS. Collectively, these results indicate that ZipN is a central player of divisome assembly in cyanobacteria, similarly to the FtsA protein of E. coli that is absent in cyanobacteria and chloroplast.
Assuntos
Proteínas de Bactérias/metabolismo , Citocinese , Synechocystis/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Clonagem Molecular , Imunoprecipitação , Viabilidade Microbiana , Mutagênese Sítio-Dirigida , Mapeamento de Interação de Proteínas , Synechocystis/genética , Synechocystis/metabolismoRESUMO
In the yeast Saccharomyces cerevisiae, mutation of some effectors of mRNA nuclear export leads to the rapid accumulation of HSP104 RNA in transcription site-associated foci. We have screened the S. cerevisiae complement of viable gene deletion mutants for their inability to export HSP104 RNA. The 15 strains identified comprise deletions of components of the THO, Thp1p/Sac3p, and nuclear pore complexes. In all three mutant classes, retained RNA overlaps the HSP104 transcription site. Thus, an early block to HSP104 RNA export is general. Incubation of the identified deletion strains, as well as seven additional mutants, under conditions where mRNA export is blocked results in rapid dissipation of nucleolar protein and RNA constituents. Time course experiments show that dissipation of nucleolar antigens succeeds mRNA retention and is reversed when the load of nuclear mRNA ceases. Consistent with a causal role of excess nuclear mRNA, nucleolar morphology in an mRNA export mutant environment remains intact when transcription by RNA polymerase II is inhibited.
Assuntos
Proteínas Nucleares/metabolismo , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Antígenos de Fungos/genética , Antígenos de Fungos/metabolismo , Sequência de Bases , Nucléolo Celular/metabolismo , Deleção de Genes , Genes Fúngicos , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Sondas de Oligonucleotídeos/genética , RNA Polimerase II/metabolismo , RNA Fúngico/genética , RNA Mensageiro/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcrição GênicaRESUMO
Assembly of the tubulin-like cytoskeletal protein FtsZ into a ring structure at midcell establishes the location of the nascent division sites in prokaryotes. However, it is not yet known how the assembly and contraction of the Z ring are regulated, especially in cyanobacteria, the environmentally crucial organisms for which only one FtsZ partner protein, ZipN, has been described so far. Here, we characterized SepF and Ftn6, two novel septal proteins, in the spherical-celled strain Synechocystis PCC 6803. Both proteins were found to be indispensable to Synechocystis sp. strain PCC 6803. The depletion of both SepF and Ftn6 resulted in delayed cytokinesis and the generation of giant cells but did not prevent FtsZ polymerization, as shown by the visualization of green fluorescent protein (GFP)-tagged FtsZ polymers. These GFP-tagged Z-ring-like structures often appeared to be abnormal, because these reporter cells respond to the depletion of either SepF or Ftn6 with an increased abundance of total, natural, and GFP-tagged FtsZ proteins. In agreement with their septal localization, we found that both SepF and Ftn6 interact physically with FtsZ. Finally, we showed that SepF, but not Ftn6, stimulates the formation and/or stability of FtsZ polymers in vitro.
Assuntos
Proteínas de Bactérias/fisiologia , Synechocystis/citologia , Synechocystis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Microscopia de Fluorescência , Ligação Proteica/genética , Ligação Proteica/fisiologia , Multimerização Proteica/genética , Multimerização Proteica/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Synechocystis/genéticaRESUMO
Because very little is known about cell division in noncylindrical bacteria and cyanobacteria, we investigated 10 putative cytokinetic proteins in the unicellular spherical cyanobacterium Synechocystis strain PCC 6803. Concerning the eight penicillin-binding proteins (PBPs), which define three classes, we found that Synechocystis can survive in the absence of one but not two PBPs of either class A or class C, whereas the unique class B PBP (also termed FtsI) is indispensable. Furthermore, we showed that all three classes of PBPs are required for normal cell size. Similarly, the putative FtsQ and FtsW proteins appeared to be required for viability and normal cell size. We also used a suitable bacterial two-hybrid system to characterize the interaction web among the eight PBPs, FtsQ, and FtsW, as well as ZipN, the crucial FtsZ partner that occurs only in cyanobacteria and plant chloroplasts. We showed that FtsI, FtsQ, and ZipN are self-interacting proteins and that both FtsI and FtsQ interact with class A PBPs, as well as with ZipN. Collectively, these findings indicate that ZipN, in interacting with FtsZ and both FtsI and FtQ, plays a similar role to the Escherichia coli FtsA protein, which is missing in cyanobacteria and chloroplasts.
Assuntos
Proteínas de Bactérias/fisiologia , Proteínas de Membrana/fisiologia , Proteínas de Ligação às Penicilinas/fisiologia , Synechocystis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Citometria de Fluxo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Biológicos , Mutação , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/metabolismo , Ligação Proteica/genética , Ligação Proteica/fisiologia , Synechocystis/genética , Synechocystis/crescimento & desenvolvimento , Técnicas do Sistema de Duplo-HíbridoRESUMO
BACKGROUND: DNA replication and cell cycle as well as their relationship have been extensively studied in the two model organisms E. coli and B. subtilis. By contrast, little is known about these processes in cyanobacteria, even though they are crucial to the biosphere, in utilizing solar energy to renew the oxygenic atmosphere and in producing the biomass for the food chain. Recent studies have allowed the identification of several cell division factors that are specifics to cyanobacteria. Among them, Ftn6 has been proposed to function in the recruitment of the crucial FtsZ proteins to the septum or the subsequent Z-ring assembly and possibly in chromosome segregation. RESULTS: In this study, we identified an as yet undescribed domain located in the conserved N-terminal region of Ftn6. This 77 amino-acids-long domain, designated here as FND (Ftn6 N-Terminal Domain), exhibits striking sequence and structural similarities with the DNA-interacting module, listed in the PFAM database as the DnaD-like domain (pfam04271). We took advantage of the sequence similarities between FND and the DnaD-like domains to construct a homology 3D-model of the Ftn6 FND domain from the model cyanobacterium Synechocystis PCC6803. Mapping of the conserved residues exposed onto the FND surface allowed us to identify a highly conserved area that could be engaged in Ftn6-specific interactions. CONCLUSION: Overall, similarities between FND and DnaD-like domains as well as previously reported observations on Ftn6 suggest that FND may function as a DNA-interacting module thereby providing an as yet missing link between DNA replication and cell division in cyanobacteria. Consistently, we also showed that Ftn6 is involved in tolerance to DNA damages generated by UV rays.
Assuntos
Proteínas de Bactérias/química , DNA Bacteriano/genética , Proteínas de Ligação a DNA/química , Synechocystis/química , Synechocystis/genética , Sequência de Aminoácidos , Dano ao DNA , Deleção de Genes , Viabilidade Microbiana/efeitos da radiação , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Synechocystis/crescimento & desenvolvimento , Synechocystis/efeitos da radiaçãoRESUMO
To identify the proteins associated with the RNA polymerase III (Pol III) machinery in exponentially growing yeast cells, we developed our own tandem chromatin affinity purification procedure (TChAP) after in vivo cross-link, allowing a reproducible and good recovery of the protein bait and its associated partners. In contrast to TFIIIA that could only be purified as a free protein, this protocol allows us to capture free Pol III together with Pol III bound on its target genes. Transcription factors, elongation factors, RNA-associated proteins and proteins involved in Pol III biogenesis were identified by mass spectrometry. Interestingly, the presence of all the TFIIIB subunits found associated with Pol III together with the absence of TFIIIC and chromatin factors including histones suggest that DNA-bound Pol III purified using TChAP is mainly engaged in transcription reinitiation.
Assuntos
Cromatografia de Afinidade/métodos , RNA Polimerase III/metabolismo , Fatores de Transcrição/isolamento & purificação , Fatores de Transcrição/metabolismo , Cromatina/química , Cromatina/metabolismo , Reagentes de Ligações Cruzadas/farmacologia , Redes Reguladoras de Genes , Espectrometria de Massas , Ligação Proteica , RNA Polimerase III/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/química , Iniciação da Transcrição GenéticaRESUMO
Before polyadenylated mRNA is exported from the nucleus, the 3'-end processing complex is removed by a poorly described mechanism. In this study, we asked whether factors involved in mRNP maturation and export are also required for disassembly of the cleavage and polyadenylation complex. An RNA immunoprecipitation assay monitoring the amount of the cleavage factor (CF) IA component Rna15p associated with poly(A)(+) RNA reveals defective removal of Rna15p in mutants of the nuclear export receptor Mex67p as well as other factors important for assembly of an export-competent mRNP. In contrast, Rna15p is not retained in mutants of export factors that function primarily on the cytoplasmic side of the nuclear pore. Consistent with a functional interaction between Mex67p and the 3'-end processing complex, a mex67 mutant accumulates unprocessed SSA4 transcripts and exhibits a severe growth defect when this mutation is combined with mutation of Rna15p or another CF IA subunit, Rna14p. RNAs that become processed in a mex67 mutant have longer poly(A) tails both in vivo and in vitro. This influence of Mex67p on 3'-end processing is conserved, as depletion of its human homolog, TAP/NXF1, triggers mRNA hyperadenylation. Our results indicate a function for nuclear mRNP assembly factors in releasing the 3'-end processing complex once polyadenylation is complete.
Assuntos
Núcleo Celular/metabolismo , Poliadenilação , Ribonucleoproteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Alelos , Linhagem Celular , Humanos , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcrição Gênica , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
When produced in Escherichia coli, the CGFS-type monothiol Grxs from this organism (EcGrx4p) and the model cyanobacterium Synechocystis (SyGrx3p) exist as a dimeric iron-sulfur containing holoprotein or as a monomeric apoprotein in solution. Spectroscopic and site-directed mutagenesis analyses show that the SyGrx3 holoprotein contains a subunit-bridging [2Fe-2S] cluster that is ligated by the catalytic cysteine located in the CGFS motif of each monomer and the cysteines of two molecules of glutathione. The biochemical characterization of several monothiol Grxs from the cyanobacteria Gloeobacter violaceus (GvGrx3p) and Thermosynechococcus elongatus (TeGrx3p), the yeast Saccharomyces cerevisiae (ScGrx3p, ScGrx4p, and ScGrx5p), the plant Arabidopsis thaliana (AtGrx5p), and human (HsGrx5p) indicate that the incorporation of a GSH-ligated [2Fe-2S] center is a common feature of prokaryotic and eukaryotic CGFS-active site monothiol Grxs. In light of these results, the involvement of these enzymes in the sensing of iron and/or the biogenesis and transfer of Fe-S cluster is discussed.