Abstract
Defects in frataxin result in Friedreich ataxia, a genetic disease characterized by early onset of neurodegeneration, cardiomyopathy, and diabetes. Frataxin is a conserved mitochondrial protein that controls iron needed for iron-sulfur cluster assembly and heme synthesis and also detoxifies excess iron. Studies in vitro have shown that either monomeric or oligomeric frataxin delivers iron to other proteins, whereas ferritin-like frataxin particles convert redox-active iron to an inert mineral. We have investigated how these different forms of frataxin are regulated in vivo. In Saccharomyces cerevisiae, only monomeric yeast frataxin (Yfh1) was detected in unstressed cells when mitochondrial iron uptake was maintained at a steady, low nanomolar level. Increments in mitochondrial iron uptake induced stepwise assembly of Yfh1 species ranging from trimer to > or = 24-mer, independent of interactions between Yfh1 and its major iron-binding partners, Isu1/Nfs1 or aconitase. The rate-limiting step in Yfh1 assembly was a structural transition that preceded conversion of monomer to trimer. This step was induced, independently or synergistically, by mitochondrial iron increments, overexpression of wild type Yfh1 monomer, mutations that stabilize Yfh1 trimer, or heat stress. Faster assembly kinetics correlated with reduced oxidative damage and higher levels of aconitase activity, respiratory capacity, and cell survival. However, deregulation of Yfh1 assembly resulted in Yfh1 aggregation, aconitase sequestration, and mitochondrial DNA depletion. The data suggest that Yfh1 assembly responds to dynamic changes in mitochondrial iron uptake or stress exposure in a highly controlled fashion and that this may enable frataxin to simultaneously promote respiratory function and stress tolerance.
Highlights
Frataxin binds iron and can promote iron delivery to different iron-binding proteins as well as detoxify iron
In an attempt to genetically manipulate the oligomeric state of frataxin in yeast, we expressed two Yfh1 proteins with stabilizing mutations, Yfh1-Y73A and Yfh1-T118A/ V120A, in a yeast frataxin knock out strain
Frataxin is synthesized in the cytoplasm as a precursor polypeptide that is imported by mitochondria and processed to mature form by mitochondrial processing peptidase [46]
Summary
S. cerevisiae Expression Constructs and Strains—The mutant yfh1-Y73A and yfh1-T118A/V120A alleles were created by PCR, cloned into low copy plasmid YCplac22-YFH1 [34] under the control of the natural YFH1 promoter, and sequenced completely. Aliquots of these samples were frozen immediately, whereas additional aliquots were first subject to a 15-min aerobic incubation or a 5-min treatment with 100 M H2O2 (from a 8.8 M solution) (Sigma) at NOVEMBER 14, 2008 VOLUME 283 NUMBER 46. These samples were subsequently used for (i) detection of protein carbonyls (100 g and total protein) using an OxiBlot kit (Chemicon) per manufacturer’s protocol; (ii) in-gel aconitase activity assays (150 g of total protein) [44]; and (iii) measurement of mitochondrial iron content (10 – 600 g of total protein, depending on growth conditions) [45]. Glycerol instead of dextrose), the cells were scored for viability or respiratory capacity [33]
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