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Iron is an essential trace element for life and has a fundamental role in many metabolic processes including mitochondrial respiration, amino acid and nucleotide synthesis, ribosome assembly, DNA replication and DNA repair. However, elevated levels of cellular iron are highly toxic, necessitating sophisticated strategies for intracellular transport and sensing of iron to avoid both iron deprivation and iron overload. The current work was concentrated on the mechanism of mitochondrial iron supply and the mode of sensing high iron concentrations within the cytoplasm.
Mitochondria utilize the majority of the cellular iron for the biogenesis of iron-sulfur (Fe/S) proteins and heme. Previous studies have identified a critical role of the mitochondrial carrier proteins Mrs3 and Mrs4 in iron uptake, yet deletion of their genes in yeast is not lethal. Since the process of Fe/S protein biogenesis is essential for cell viability, alternative pathways for iron uptake into mitochondria must exist. Recently, overproduction of the mitochondrial carrier Rim2 was shown to suppress the phenotype of mrs3/4Δ cells in iron transport suggesting that this carrier protein may serve as another iron transporter in S. cerevisiae. Surprisingly, Rim2 was also shown to function as a pyrimidine nucleotide transporter raising the question of how the latter well-defined role may be compatible with an iron transport function. The first part of this work provides evidence that the deletion of RIM2 in mrs3/4Δ cells exacerbated their poor growth, and had a further negative impact on the biogenesis of mitochondrial Fe/S proteins. Conversely, overexpression of RIM2 was able to restore Fe/S protein maturation and heme synthesis of mrs3/4Δ cells to wild-type levels suggesting that high concentrations of Rim2 facilitate mitochondrial uptake of reasonable amounts of iron. Direct biochemical evidence for this idea was derived from in vitro transport experiments in collaboration with Dr. Wiesenberger and colleagues using sub-mitochondrial particles and a trapped fluorescent dye which is quenched by iron. Collectively, the combination of in vivo, in organello, and in vitro studies suggest that the mitochondrial carrier Rim2 obligatorily co-translocates pyrimidine nucleotides and divalent metals including iron. Under physiological conditions, however, the majority of iron is transported via Mrs3 and Mrs4.
The second part of this work was focused on the iron-sensing transcription factor Yap5 from S. cerevisiae that plays a central role in the adaptation of budding yeast to toxic iron levels. The divalent metal transporter Ccc1, the only known vacuolar iron importer in fungi and plants is crucial for this detoxification. Recently, the transcription factor Yap5 was shown to activate the expression of CCC1. The biochemical mechanism underlying iron sensing by Yap5 is so far unknown. Here, I show that the activator domain of Yap5 (tYap5) binds radioactive iron (55Fe) when tYap5 is overproduced in yeast. Circular dichroism, electron paramagnetic resonance, and Mössbauer spectroscopy of recombinant tYap5 showed that the protein binds a [2Fe-2S] cluster after chemical reconstitution. This [2Fe-2S] cluster is coordinated by the N-terminal cysteine-rich domain (n-CRD) of the activator domain and is essential for transcriptional activity. Fe/S cluster binding to tYap5 induces a conformational change that likely modulates the transcriptional activity of Yap5. Surprisingly, in vitro Yap5 binds a second Fe/S cofactor at the C-terminal CRD, yet this metal binding motif is not involved in iron sensing in vivo. The Fe/S cluster binding motif within the n-CRD regulatory domain of Yap5 is conserved in the basic leucine zipper transcription factor HapX that is wide-spread in fungi and that is crucial for virulence of fungal pathogens. Hence, the Fe/S cluster of Yap5 represents a novel sensor module that is frequently utilized by eukaryotic stress response regulators. Taken together, the work on Yap5 has provided fundamental new insights into the mechanism of high iron sensing and regulation by iron-responsive transcription factors in fungi.