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Iron-sulfur clusters are inorganic cofactors of numerous proteins found in all organisms. Fe/S proteins are involved in electron transport, enzyme catalysis and regulation of gene expression. In eukaryotes, the biosynthesis of Fe/S proteins requires three proteinaceous machineries. Mitochondria harbour an iron-sulfur cluster (ISC) assembly machinery which is responsible for the maturation of all cellular Fe/S containing proteins. In addition, maturation of extra-mitochondrial Fe/S containing proteins in Saccharomyces cerevisiae depends on a so far unknown substrate, produced by the ISC assembly machinery and exported via the ABC transporter Atm1 (ISC export machinery) into the cytosol. The substrate is required by the cytosolic ISC assembly (CIA) machinery to assemble Fe/S clusters on extra-mitochondrial target proteins.
In the past four years six CIA proteins were identified and exhibited a crucial function in extra-mitochondrial Fe/S protein biogenesis. The first part of this work presents the identification and characterization of the CIA component Nbp35, a soluble P-loop NTPase. The protein resides in the cytoplasm and a small amount is present in the nucleus. Depletion of Nbp35 strongly impairs the activity of the cytosolic Fe/S protein Leu1 (Isopropylmalate-Isomerase), whereas mitochondrial Fe/S proteins are unaffected. Moreover, defects in the de novo maturation of various cytosolic and nuclear Fe/S proteins were observed in the absence of Nbp35, demonstrating the functional involvement of Nbp35 in the biogenesis of extra-mitochondrial Fe/S proteins. Nbp35 shows sequence similarity to Cfd1, both proteins form in vitro and in vivo a hetero-oligomer. Nbp35 contains a 4Fe/4S cluster at its N-terminus coordinated by four conserved cysteine residues. Maturation of Nbp35 depends on the mitochondrial ISC and CIA machineries. Hence, Nbp35 is involved in its own maturation. To determine the precise molecular function of Nbp35 further biochemical investigations will be necessary.
It has been reported that disruption of the Fe/S cluster biogenesis is associated with mitochondrial iron overload and activation of the iron-sensing transcription factors Aft1/Aft2. These effects are due to defects in the mitochondrial ISC machineries, not the cytosolic CIA machinery. To investigate the cellular consequences of defective Fe/S cluster biogenesis in S. cerevisiae, strains depleted of yeast adrenodoxin homolog Yah1 (ISC assembly), the ABC transporter Atm1 (ISC export) and the soluble P-loop ATPase Nbp35 (CIA) were used. Transcriptional changes were analysed by genome-wide DNA microarray experiments. The analysis revealed a strong and largely similar transcriptional response of yeast cells depleted for the mitochondrial ABC transporter Atm1 or yeast adrenodoxin Yah1, whereas the transcriptional response to Nbp35 depletion was minimal. The Nbp35 mutant showed no effect on iron homeostasis. Therefore, it could conclude that the CIA machinery has no impact on cellular iron homeostasis. The results refute an earlier suggestion that iron regulation in yeast is mediated by a cytosolic Fe/S protein. In addition, this observation indicates that the regulation of cellular iron homeostasis is crucially controlled by mitochondria.
In contrast to Nbp35 depletion the absence of Yah1 and Atm1 depletion induced the expression of Aft1/Aft2 dependent genes and the repression of genes involved in respiration. Moreover, genes involved in the synthesis of ergosterol, biotin, amino acid biosynthesis and the degradation and synthesis of heme display an altered gene expression. Hence, depletion of Atm1p und Yah1 and the disruption of cellular Fe/S protein biogenesis signal an intracellular iron-limitation. Data were confirmed by promoter studies and northern blots using selected iron-dependent genes such as ERG3 (ergosterol-biosynthesis), BIO2 (biotin-biosynthesis), and HEM15 (heme-biosynthesis). Interestingly, heme-biosynthesis does not have a similar impact on iron homeostasis. This means that the ISC assembly and export machinery could be involved in the production of a so far unknown component, necessary for maturation of Fe/S proteins and inhibition of the iron-sensing transcription factor Aft1. In addition, this component could be involved in the regulation of further iron consuming metabolic processes (e.g. respiration, heme-synthesis).
Previous studies already demonstrated that disruption of the ISC machinery is accompanied by a heme-defect, which could explain the loss in activity of heme-containing proteins. Decreased heme-pools in absence of Atm1, Yah1 and under iron-deplete conditions are due to repression of the HEM15 gene encoding a ferrochelatase. Furthermore, previous studies show a biochemical inhibition of the ferrochelatase. The microarray data revealed a general cross-talk between mitochondrial Fe/S cluster assembly and the biosynthesis of heme.