Endoplasmic reticulum oxidoreductin provides resilience against reductive stress and hypoxic conditions by mediating luminal redox dynamics.

Oxidative protein folding in the endoplasmic reticulum (ER) depends on the coordinated action of protein disulfide isomerases and ER oxidoreductins (EROs). Strict dependence of ERO activity on molecular oxygen as the final electron acceptor implies that oxidative protein folding and other ER processes are severely compromised under hypoxia. Here, we isolated viable Arabidopsis thaliana ero1 ero2 double mutants that are highly sensitive to reductive stress and hypoxia. To elucidate the specific redox dynamics in the ER in vivo, we expressed the glutathione redox potential (EGSH) sensor Grx1-roGFP2iL-HDEL with a midpoint potential of -240 mV in the ER of Arabidopsis plants. We found EGSH values of -241 mV in wild-type plants, which is less oxidizing than previously estimated. In the ero1 ero2 mutants, luminal EGSH was reduced further to -253 mV. Recovery to reductive ER stress induced by dithiothreitol was delayed in ero1 ero2. The characteristic signature of EGSH dynamics in the ER lumen triggered by hypoxia was affected in ero1 ero2 reflecting a disrupted balance of reductive and oxidizing inputs, including nascent polypeptides and glutathione entry. The ER redox dynamics can now be dissected in vivo, revealing a central role of EROs as major redox integrators to promote luminal redox homeostasis.

The mitochondrial monothiol glutaredoxin S15 is essential for iron-sulfur protein maturation in Arabidopsis thaliana.

The iron-sulfur cluster (ISC) is an ancient and essential cofactor of many proteins involved in electron transfer and metabolic reactions. In Arabidopsis, three pathways exist for the maturation of iron-sulfur proteins in the cytosol, plastids, and mitochondria. We functionally characterized the role of mitochondrial glutaredoxin S15 (GRXS15) in biogenesis of ISC containing aconitase through a combination of genetic, physiological, and biochemical approaches. Two Arabidopsis T-DNA insertion mutants were identified as null mutants with early embryonic lethal phenotypes that could be rescued by GRXS15. Furthermore, we showed that recombinant GRXS15 is able to coordinate and transfer an ISC and that this coordination depends on reduced glutathione (GSH). We found the Arabidopsis GRXS15 able to complement growth defects based on disturbed ISC protein assembly of a yeast Δgrx5 mutant. Modeling of GRXS15 onto the crystal structures of related nonplant proteins highlighted amino acid residues that after mutation diminished GSH and subsequently ISC coordination, as well as the ability to rescue the yeast mutant. When used for plant complementation, one of these mutant variants, GRXS15K83/A, led to severe developmental delay and a pronounced decrease in aconitase activity by approximately 65%. These results indicate that mitochondrial GRXS15 is an essential protein in Arabidopsis, required for full activity of iron-sulfur proteins.

The EXS Domain of PHO1 Participates in the Response of Shoots to Phosphate Deficiency via a Root-to-Shoot Signal.

The response of shoots to phosphate (Pi) deficiency implicates long-distance communication between roots and shoots, but the participating components are poorly understood. We have studied the topology of the Arabidopsis (Arabidopsis thaliana) PHOSPHATE1 (PHO1) Pi exporter and defined the functions of its different domains in Pi homeostasis and signaling. The results indicate that the amino and carboxyl termini of PHO1 are both oriented toward the cytosol and that the protein spans the membrane twice in the EXS domain, resulting in a total of six transmembrane α-helices. Using transient expression in Nicotiana benthamiana leaf, we demonstrated that the EXS domain of PHO1 is essential for Pi export activity and proper localization to the Golgi and trans-Golgi network, although the EXS domain by itself cannot mediate Pi export. In contrast, removal of the amino-terminal hydrophilic SPX domain does not affect the Pi export capacity of the truncated PHO1 in N. benthamiana. While the Arabidopsis pho1 mutant has low shoot Pi and shows all the hallmarks associated with Pi deficiency, including poor shoot growth and overexpression of numerous Pi deficiency-responsive genes, expression of only the EXS domain of PHO1 in the roots of the pho1 mutant results in a remarkable improvement of shoot growth despite low shoot Pi. Transcriptomic analysis of pho1 expressing the EXS domain indicates an attenuation of the Pi signaling cascade and the up-regulation of genes involved in cell wall synthesis and the synthesis or response to several phytohormones in leaves as well as an altered expression of genes responsive to abscisic acid in roots.

Glutathione peroxidase-like enzymes cover five distinct cell compartments and membrane surfaces in Arabidopsis thaliana.

Glutathione peroxidase-like enzymes (GPXLs) constitute a family of eight peroxidases in Arabidopsis thaliana. In contrast to the eponymous selenocysteine glutathione peroxidases in mammalian cells that use glutathione as electron donor, GPXLs rely on cysteine instead of selenocysteine for activity and depend on the thioredoxin system for reduction. Although plant GPXLs have been implicated in important agronomic traits such as drought tolerance, photooxidative tolerance and immune responses, there remain major ambiguities regarding their subcellular localization. Because their site of action is a prerequisite for an understanding of their function, we investigated the localization of all eight GPXLs in stable Arabidopsis lines expressing N-terminal and C-terminal fusions with redox-sensitive green fluorescent protein 2 (roGFP2) using confocal microscopy. GPXL1 and GPXL7 were found in plastids, while GPXL2 and GPXL8 are cytosolic nuclear. The N-terminal target peptide of GPXL6 is sufficient to direct roGFP2 into mitochondria. Interestingly, GPXL3, GPXL4 and GPXL5 all appear to be membrane bound. GPXL3 was found exclusively in the secretory pathway where it is anchored by a single N-terminal transmembrane domain. GPXL4 and GPXL5 are anchored to the plasma membrane. Presence of an N-terminal myristoylation motif and genetic disruption of membrane association through targeted mutagenesis point to myristoylation as essential for membrane localization.

Endoplasmic reticulum: reduced and oxidized glutathione revisited.

The reducing power of glutathione, expressed by its reduction potential EGSH, is an accepted measure for redox conditions in a given cell compartment. In the endoplasmic reticulum (ER), EGSH is less reducing than elsewhere in the cell. However, attempts to determine EGSH(ER) have been inconsistent and based on ineligible assumptions. Using a codon-optimized and evidently glutathione-specific glutaredoxin-coupled redox-sensitive green fluorescent protein (roGFP) variant, we determined EGSH(ER) in HeLa cells as -208±4 mV (at pH 7.0). At variance with existing models, this is not oxidizing enough to maintain the known redox state of protein disulfide isomerase family enzymes. Live-cell microscopy confirmed ER hypo-oxidation upon inhibition of ER Ca(2+) import. Conversely, stressing the ER with a glycosylation inhibitor did not lead to more reducing conditions, as reported for yeast. These results, which for the first time establish the oxidative capacity of glutathione in the ER, illustrate a context-dependent interplay between ER stress and EGSH(ER). The reported development of ER-localized EGSH sensors will enable more targeted in vivo redox analyses in ER-related disorders.

Five Arabidopsis reticulon isoforms share endoplasmic reticulum location, topology, and membrane-shaping properties.

The cortical endoplasmic reticulum (ER) in tobacco (Nicotiana tabacum) epidermal cells is a network of tubules and cisternae undergoing dramatic rearrangements. Reticulons are integral membrane proteins involved in shaping ER tubules. Here, we characterized the localization, topology, effect, and interactions of five Arabidopsis thaliana reticulons (RTNs), isoforms 1-4 and 13, in the cortical ER. Our results indicate that RTNLB13 and RTNLB1-4 colocate to and constrict the tubular ER membrane. All five RTNs preferentially accumulate on ER tubules and are excluded from ER cisternae. All isoforms share the same transmembrane topology, with N and C termini facing the cytosol and four transmembrane domains. We show by Förster resonance energy transfer and fluorescence lifetime imaging microscopy that several RTNs have the capacity to interact with themselves and each other, and we suggest that oligomerization is responsible for their residence in the ER membrane. We also show that a complete reticulon homology domain is required for both RTN residence in high-curvature ER membranes and ER tubule constriction, yet it is not necessary for homotypic interactions.

Analysis of plant mitochondrial function using fluorescent protein sensors.

Mitochondrial physiology sets the basis for function of the organelle and vice versa. While a limited range of in vivo parameters, such as oxygen consumption, has been classically accessible for measurement, a growing collection of fluorescent protein sensors can now give insights into the physiology of plant mitochondria. Nevertheless, the meaningful application of these sensors in mitochondria is technically challenging and requires rigorous experimental standards. Here we exemplify the application of three genetically encoded sensors to monitor glutathione redox potential, pH, and calcium in the matrix of mitochondria in intact plants. We describe current methods for quantitative imaging and analysis in living root tips by confocal microscopy and discuss methodological limitations.

Redesign of genetically encoded biosensors for monitoring mitochondrial redox status in a broad range of model eukaryotes.

The development of genetically encoded redox biosensors has paved the way toward chemically specific, quantitative, dynamic, and compartment-specific redox measurements in cells and organisms. In particular, redox-sensitive green fluorescent proteins (roGFPs) have attracted major interest as tools to monitor biological redox changes in real time and in vivo. Most recently, the engineering of a redox relay that combines glutaredoxin (Grx) with roGFP2 as a translational fusion (Grx1-roGFP2) led to a biosensor for the glutathione redox potential (EGSH ). The expression of this probe in mitochondria is of particular interest as mitochondria are the major source of oxidants, and their redox status is closely connected to cell fate decisions. While Grx1-roGFP2 can be expressed in mammalian mitochondria, it fails to enter mitochondria in various nonmammalian model organisms. Here we report that inversion of domain order from Grx1-roGFP2 to roGFP2-Grx1 yields a biosensor with perfect mitochondrial targeting while fully maintaining its biosensor capabilities. The redesigned probe thus allows extending in vivo observations of mitochondrial redox homeostasis to important nonmammalian model organisms, particularly plants and insects.

The oxidative protein folding machinery in plant cells.

Formation of intra-molecular disulfides and concomitant oxidative protein folding is essential for stability and catalytic function of many soluble and membrane-bound proteins in the endomembrane system, the mitochondrial inter-membrane space and the thylakoid lumen. Disulfide generation from free cysteines in nascent polypeptide chains is generally a catalysed process for which distinct pathways exist in all compartments. A high degree of similarities between highly diverse eukaryotic and bacterial systems for generation of protein disulfides indicates functional conservation of key processes throughout evolution. However, while many aspects about molecular function of enzymatic systems promoting disulfide formation have been demonstrated for bacterial and non-plant eukaryotic organisms, it is now clear that the plant machinery for oxidative protein folding displays distinct details, suggesting that the different pathways have been adapted to plant-specific requirements in terms of compartmentation, molecular function and regulation. Here, we aim to evaluate biological diversity by comparing the plant systems for oxidative protein folding to the respective systems from non-plant eukaryotes.

Development of roGFP2-derived redox probes for measurement of the glutathione redox potential in the cytosol of severely glutathione-deficient rml1 seedlings.

Glutathione is important for detoxification, as a cofactor in biochemical reactions and as a thiol-redox buffer. The cytosolic glutathione buffer is normally highly reduced with glutathione redox potentials (E GSH ) of more negative than -310 mV. Maintenance of such negative redox potential is achieved through continuous reduction of glutathione disulfide by glutathione reductase (GR). Deviations from steady state glutathione redox homeostasis have been discussed as a possible mean to alter the activity of redox-sensitive proteins through switching of critical thiol residues. To better understand such signaling mechanisms it is essential to be able to measure E GSH over a wide range from highly negative redox potentials down to potentials found in mutants that show already severe phenotypes. With the advent of redox-sensitive GFPs (roGFPs), understanding the in vivo dynamics of the thiol-based redox buffer system became within reach. The original roGFP versions, roGFP1 and roGFP2, however, have midpoint potentials between -280 and -290 mV rendering them fully oxidized in the ER and almost fully reduced in the cytosol, plastids, mitochondria, and peroxisomes. To extend the range of suitable probes we have engineered a roGFP2 derivative, roGFP2-iL, with a midpoint potential of about -238 mV. This value is within the range of redox potentials reported for homologous roGFP1-iX probes, albeit with different excitation properties. To allow rapid and specific equilibration with the glutathione pool, fusion constructs with human glutaredoxin 1 (GRX1) were generated and characterized in vitro. GRX1-roGFP2-iL proved to be suitable for in vivo redox potential measurements and extends the range of E GSH values that can be measured in vivo with roGFP2-based probes from about -320 mV for GRX1-roGFP2 down to about -210 mV for GRX1-roGFP2-iL. Using both probes in the cytosol of severely glutathione-deficient rml1 seedlings revealed an E GSH of about -260 mV in this mutant.

A role for SNAP25 in internalization of kainate receptors and synaptic plasticity.

Regulation of surface insertion and internalization of AMPA and NMDA receptors has emerged as a key mechanism for the control of synaptic strength. Regulatory elements for synaptic kainate receptors (KARs) are, however, largely undetermined. We have found that SNAP25 is critical for the synaptic removal of KARs, acting via GluK5 (i.e., KA2) subunits. SNAP25 coimmunoprecipitates with protein complexes containing PICK1, GRIP1, and GluK5 and colocalizes with GluK5 in both hippocampal neurons and transfected HEK293 cells. In hippocampal slices, purified SNAP25 antibodies and blocking peptides caused a GluK5-dependent run-up of KARs-mediated EPSC (EPSC(KAR)) recorded from CA3 pyramidal neurons when included in the patch pipette and prevented activity-dependent long-term depression of EPSC(KAR). As EPSC(KAR) LTD, SNAP25/PICK1/GluK5 interactions are dynamically regulated by PKC.