Vacuolar Iron Stores Gated by NRAMP3 and NRAMP4 Are the Primary Source of Iron in Germinating Seeds.

During seed germination, iron (Fe) stored in vacuoles is exported by the redundant NRAMP3 and NRAMP4 transporter proteins. A double nramp3 nramp4 mutant is unable to mobilize Fe stores and does not develop in the absence of external Fe. We used RNA sequencing to compare gene expression in nramp3 nramp4 and wild type during germination and early seedling development. Even though sufficient Fe was supplied, the Fe-responsive transcription factors bHLH38, 39, 100, and 101 and their downstream targets FRO2 and IRT1 mediating Fe uptake were strongly upregulated in the nramp3 nramp4 mutant. Activation of the Fe deficiency response was confirmed by increased ferric chelate reductase activity in the mutant. At early stages, genes important for chloroplast redox control (FSD1 and SAPX), Fe homeostasis (FER1 and SUFB), and chlorophyll metabolism (HEMA1 and NYC1) were downregulated, indicating limited Fe availability in plastids. In contrast, expression of FRO3, encoding a ferric reductase involved in Fe import into the mitochondria, was maintained, and Fe-dependent enzymes in the mitochondria were unaffected in nramp3 nramp4 Together, these data show that a failure to mobilize Fe stores during germination triggered Fe deficiency responses and strongly affected plastids, but not mitochondria.

NBP35 interacts with DRE2 in the maturation of cytosolic iron-sulphur proteins in Arabidopsis thaliana.

Proteins of the cytosolic pathway for iron-sulphur (FeS) cluster assembly are conserved, except that plants lack a gene for CFD1 (Cytosolic FeS cluster Deficient 1). This poses the question of how NBP35 (Nucleotide-Binding Protein 35 kDa), the heteromeric partner of CFD1 in metazoa, functions on its own in plants. Firstly, we created viable mutant alleles of NBP35 in Arabidopsis to overcome embryo lethality of previously reported knockout mutations. RNAi knockdown lines with less than 30% NBP35 protein surprisingly showed no developmental or biochemical differences to wild-type. Substitution of Cys14 to Ala, which destabilized the N-terminal Fe4 S4 cluster in vitro, caused mild growth defects and a significant decrease in the activity of cytosolic FeS enzymes such as aconitase and aldehyde oxidases. The DNA glycosylase ROS1 was only partially decreased in activity and xanthine dehydrogenase not at all. Plants with strongly depleted NBP35 protein in combination with Cys14 to Ala substitution had distorted leaf development and decreased FeS enzyme activities. To find protein interaction partners of NBP35, a yeast-two-hybrid screen was carried out that identified NBP35 and DRE2 (Derepressed for Ribosomal protein S14 Expression). NBP35 is known to form a dimer, and DRE2 acts upstream in the cytosolic FeS protein assembly pathway. The NBP35-DRE2 interaction was not disrupted by Cys14 to Ala substitution. Our results show that NBP35 has a function in the maturation of FeS proteins that is conserved in plants, and is closely allied to the function of DRE2.

New links between SOD1 and metabolic dysfunction from a yeast model of amyotrophic lateral sclerosis.

A number of genes have been linked to familial forms of the fatal motor neuron disease amyotrophic lateral sclerosis (ALS). Over 150 mutations within the gene encoding superoxide dismutase 1 (SOD1) have been implicated in ALS, but why such mutations lead to ALS-associated cellular dysfunction is unclear. In this study, we identify how ALS-linked SOD1 mutations lead to changes in the cellular health of the yeast Saccharomyces cerevisiae We find that it is not the accumulation of aggregates but the loss of Sod1 protein stability that drives cellular dysfunction. The toxic effect of Sod1 instability does not correlate with a loss of mitochondrial function or increased production of reactive oxygen species, but instead prevents acidification of the vacuole, perturbs metabolic regulation and promotes senescence. Central to the toxic gain-of-function seen with the SOD1 mutants examined was an inability to regulate amino acid biosynthesis. We also report that leucine supplementation results in an improvement in motor function in a Caenorhabditis elegans model of ALS. Our data suggest that metabolic dysfunction plays an important role in Sod1-mediated toxicity in both the yeast and worm models of ALS.

Identification of new surfaces of cofilin that link mitochondrial function to the control of multi-drug resistance.

ADF/cofilin family proteins are essential regulators of actin cytoskeletal dynamics. Recent evidence also implicates cofilin in the regulation of mitochondrial function. Here, we identify new functional surfaces of cofilin that are linked with mitochondrial function and stress responses in the budding yeast Saccharomyces cerevisiae. Our data link surfaces of cofilin that are involved in separable activities of actin filament disassembly or stabilisation, to the regulation of mitochondrial morphology and the activation status of Ras, respectively. Importantly, charge alterations to conserved surfaces of cofilin that do not interfere with its actin regulatory activity lead to a dramatic increase in respiratory function that triggers a retrograde signal to upregulate a battery of ABC transporters and concurrent metabolic changes that support multi-drug resistance. We hypothesise that cofilin functions within a bio-sensing system that connects the cytoskeleton and mitochondrial function to environmental challenge.

Using yeast models to probe the molecular basis of amyotrophic lateral sclerosis.

ALS (amyotrophic lateral sclerosis) is a fatal neurodegenerative disease attributable to the death of motor neurons. Associated with ALS are mutations in the genes encoding SOD1 (superoxide dismutase 1), FUS (fused in Sarcoma) protein and TDP-43 (TAR DNA-binding protein-43) each of which leads to aggregation of the respective protein. For example, the ALS-associated mutations in the hSOD1 (human SOD1) gene typically destabilize the native SOD homodimer, leading to misfolding, aggregation and degradation of SOD1. The ALS-associated pathology is not a consequence of the functional inactivation of SOD1 itself, but is rather due to a toxic gain-of-function triggered by mutant SOD1. Recently, the molecular basis of a number of human neurodegenerative diseases resulting from protein misfolding and aggregation, including fALS (familial ALS), was probed by using the baker's yeast, Saccharomyces cerevisiae, as a highly tractable model. Such studies have, for example, identified novel mutant SOD1-specific interactions and demonstrated that mutant SOD1 disrupts mitochondrial homoeostasis. Features of ALS associated with TDP-43 aggregation have also been recapitulated in S. cerevisiae including the identification of modulators of the toxicity of TDP-43. In this paper, we review recent studies of ALS pathogenesis using S. cerevisiae as a model organism and summarize the potential mechanisms involved in ALS progression.

Quantitative Analyses of the Yeast Oxidative Protein Folding Pathway In Vitro and In Vivo.

Aims: Efficient oxidative protein folding (OPF) in the endoplasmic reticulum (ER) is a key requirement of the eukaryotic secretory pathway. In particular, protein folding linked to the formation of disulfide bonds, an activity dependent on the enzyme protein disulfide isomerase (PDI), is crucial. For the de novo formation of disulfide bonds, reduced PDI must be reoxidized by an ER-located oxidase (ERO1). Despite some knowledge of this pathway, the kinetic parameters with which these components act and the importance of specific parameters, such as PDI reoxidation by Ero1, for the overall performance of OPF in vivo remain poorly understood. Results: We established an in vitro system using purified yeast (Saccharomyces cerevisiae) PDI (Pdi1p) and ERO1 (Ero1p) to investigate OPF. This necessitated the development of a novel reduction/oxidation processing strategy to generate homogenously oxidized recombinant yeast Ero1p. This new methodology enabled the quantitative assessment of the interaction of Pdi1p and Ero1p in vitro by measuring oxygen consumption and reoxidation of reduced RNase A. The resulting quantitative data were then used to generate a simple model that can describe the oxidizing capacity of Pdi1p and Ero1p in vitro and predict the in vivo effect of modulation of the levels of these proteins. Innovation: We describe a model that can be used to explore the OPF pathway and its control in a quantitative way. Conclusion: Our study informs and provides new insights into how OPF works at a molecular level and provides a platform for the design of more efficient heterologous protein expression systems in yeast.

Loss of cytochrome c oxidase promotes RAS-dependent ROS production from the ER resident NADPH oxidase, Yno1p, in yeast.

Many disease states, including the aging process, are associated with the accumulation of mitochondria harboring respiratory dysfunction. Mitochondrial dysfunction is often accompanied by increased ROS levels that can contribute to cellular dysfunction and disease etiology. Here we use the model eukaryote S. cerevisiae to investigate whether reduced cytochrome c oxidase (COX) activity, commonly reported in aging organisms and associated with neurodegenerative disorders, leads to ROS production from mitochondria. We provide evidence that although reduced COX complex activity correlates with ROS accumulation, mitochondria are not the major production center. Instead we show that COX-deficient mitochondria activate Ras upon their outer membrane that establishes a pro-ROS accumulation environment by suppressing antioxidant defenses and the ERAD-mediated turnover of the ER-localized NADPH oxidase Yno1p. Our data suggest that dysfunctional mitochondria can serve as a signaling platform to promote the loss of redox homeostasis, ROS accumulation, and accelerate aging in yeast.