Real-time monitoring of subcellular H2O2 distribution in Chlamydomonas reinhardtii.

Hydrogen peroxide (H2O2) is recognized as an important signaling molecule in plants. We sought to establish a genetically encoded, fluorescent H2O2 sensor that allows H2O2 monitoring in all major subcompartments of a Chlamydomonas cell. To this end, we used the Chlamydomonas Modular Cloning toolbox to target the hypersensitive H2O2 sensor reduction-oxidation sensitive green fluorescent protein2-Tsa2ΔCR to the cytosol, nucleus, mitochondrial matrix, chloroplast stroma, thylakoid lumen, and endoplasmic reticulum (ER). The sensor was functional in all compartments, except for the ER where it was fully oxidized. Employing our novel sensors, we show that H2O2 produced by photosynthetic linear electron transport (PET) in the stroma leaks into the cytosol but only reaches other subcellular compartments if produced under nonphysiological conditions. Furthermore, in heat-stressed cells, we show that cytosolic H2O2 levels closely mirror temperature up- and downshifts and are independent from PET. Heat stress led to similar up- and downshifts of H2O2 levels in the nucleus and, more mildly, in mitochondria but not in the chloroplast. Our results thus suggest the establishment of steep intracellular H2O2 gradients under normal physiological conditions with limited diffusion into other compartments. We anticipate that these sensors will greatly facilitate future investigations of H2O2 biology in plant cells.

An intracellular assay for activity screening and characterization of glutathione-dependent oxidoreductases.

The thioredoxin fold superfamily is highly diverse and contains many enzymatically active glutathione-dependent thiol-disulfide oxidoreductases, for example glutaredoxins and protein disulfide isomerases. However, many thioredoxin fold proteins remain completely uncharacterized, their cellular function is unknown, and it is unclear if they have a redox-dependent enzymatic activity with glutathione or not. Investigation of enzymatic activity traditionally involved time-consuming in vitro characterization of recombinant proteins, limiting the capacity to study novel mechanisms and structure-function relationships. To accelerate our investigation of glutathione-dependent oxidoreductases, we have developed a high-throughput and semi-quantitative assay in yeast. We combined overexpression of the glutathione transporter OPT1 with genetic fusion constructs between glutathione-dependent oxidoreductases and redox-sensitive green fluorescent protein 2 (roGFP2) to allow the rapid characterization of enzymatic activity with physiological substrates. We show that the kinetics of roGFP2 oxidation by glutathione disulfide correlate well with the in vitro-determined activity of the genetically fused glutaredoxins or mutants thereof. Our assay thus allows direct screening of glutaredoxin activity and rapid investigation of structure-function relationships. We also demonstrate that our assay can be used to monitor roGFP2 oxidation by S-nitrosoglutathione (GSNO). We show that glutaredoxins efficiently catalyze oxidation of roGFP2 by GSNO in both live yeast cells and in vitro. In summary, we have established a novel assay for activity screening and characterization of glutathione-dependent oxidoreductases.

One cysteine is enough: A monothiol Grx can functionally replace all cytosolic Trx and dithiol Grx.

Glutaredoxins are small proteins of the thioredoxin superfamily that are present throughout life. Most glutaredoxins fall into two major subfamilies. Class I glutaredoxins are glutathione-dependent thiol-disulfide oxidoreductases whilst class II glutaredoxins coordinate Fe-S clusters. Class I glutaredoxins are typically dithiol enzymes with two active-site cysteine residues, however, some enzymatically active monothiol glutaredoxins are also known. Whilst both monothiol and dithiol class I glutaredoxins mediate protein deglutathionylation, it is widely claimed that only dithiol glutaredoxins are competent to reduce protein disulfide bonds. In this study, using a combination of yeast 'viability rescue', growth, and redox-sensitive GFP-based assays, we show that two different monothiol class I glutaredoxins can each facilitate the reduction of protein disulfide bonds in ribonucleotide reductase, methionine sulfoxide reductase and roGFP2. Our observations thus challenge the generalization of the dithiol mechanism for glutaredoxin catalysis and raise the question of why most class I glutaredoxins have two active-site cysteine residues.

Glutathione: subcellular distribution and membrane transport 1.

Glutathione (γ-l-glutamyl-l-cysteinylglycine) is a small tripeptide found at millimolar concentrations in nearly all eukaryotes as well as many prokaryotic cells. Glutathione synthesis is restricted to the cytosol in animals and fungi and to the cytosol and plastids in plants. Nonetheless, glutathione is found in virtually all subcellular compartments. This implies that transporters must exist that facilitate glutathione transport into and out of the various subcellular compartments. Glutathione may also be exported and imported across the plasma membrane in many cells. However, in most cases, the molecular identity of these transporters remains unclear. Whilst glutathione transport is essential for the supply and replenishment of subcellular glutathione pools, recent evidence supports a more active role for glutathione transport in the regulation of subcellular glutathione redox homeostasis. However, our knowledge of glutathione redox homeostasis at the level of specific subcellular compartments remains remarkably limited and the role of glutathione transport remains largely unclear. In this review, we discuss how new tools and techniques have begun to yield insights into subcellular glutathione distribution and glutathione redox homeostasis. In particular, we discuss the known and putative glutathione transporters and examine their contribution to the regulation of subcellular glutathione redox homeostasis.