Molybdenum cofactor-biosynthesis and crosstalk to FeS metabolism in Neurospora crassa after ectopic expression of Moco biosynthesis step 1 proteins

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Molybdenum cofactor (Moco) is the catalytically active prosthetic group in Mo-containing enyzmes. In all eukaryotes studied so far, the first step of Moco biosynthesis resides in the mitochondria while all subsequent steps proceed in the cytosol. This step is catalyzed by two proteins where the first one (Nit-7A) harbors two FeS clusters of the [4Fe-4S] type. But GTP as starting compound is also available in the cytosol. And the cytosol harbors also a FeS cluster synthesis machinery. So the question arises why Moco biosynthesis step 1 is located in the mitochondria. Using the filamentous fungus Neurospora crassa as yeast-like model system we wish to analyse the crosstalk between Moco-biosynthesis and FeS metabolism. In yeast (S. cerevisiae) all principles of eukaryotic FeS cluster biogenesis have been successfully worked out, but yeast has no Mo metabolism. In a first project line, we plan to express the N. crassa nit-7 gene in yeast thus establishing Moco biosynthesis step 1 in yeast. A successful expression of nit-7 in yeast would open up a whole new perspective because the complete arsenal of yeast FeS biogenesis research tools would be at our hands. Clearly, this attempt would also allow to uncover whether the yeast mitochondrial exporter Atm1p which facilitates the export of FeS cluster equivalents, would be able to export also the product of Moco biosynthesis step 1, which we have postulated for its plant homolog ATM3. For a second project line, in preliminary experiments we have already expressed Nit-7A ectopically and stably in the cytosol of N. crassa in a nit-7 knock out-background. We plan to characterize this strain (i) phenotypically (growth on nitrate-media creates a strong demand for Moco and thus also for FeS), (ii) biochemically (re-isolation of ectopically expressed Nit-7A and Nit-7AB and analysis of FeS-clusters), and (iii) via transcriptome analysis (nitrate-induced transcriptional changes in FeS cluster assembly and Moco biosynthesis) in order to address the following questions: What is the donor of FeS clusters for ectopically expressed Nit-7A in the cytosol? Is this process co-regulated with Moco- and FeS-biosynthesis? Are there differences between fungal and human step 1 Moco biosynthsis? To meet the objectives of this proposal, a tight collaboration with four groups within the Priority Program has been prepared.