In the above article, the authors twice made the statement that Cfd1p was the first iron-sulfur cluster assembly factor to be found in the cytoplasm. Other recent publications have suggested that proteins that have been shown to function in Fe-S cluster assembly in mitochondria may exist at low levels in the cytoplasm (Land and Rouault, 1998; Tong and Rouault, 2000; Nakai et al, 2001). Since this article went to press, two additional papers have added to these findings (Tong et al, 2003; Nakai et al, 2004).While some investigators have questioned whether Cfd1p is indeed the first reported cytosolic Fe-S cluster assembly factor, we emphasize that the significance of our finding is in identifying a new class of cluster assembly factor whose localization is exclusively cytoplasmic and whose function is demonstrably cytoplasmic as well.

Iron regulatory protein 1 (IRP1) is regulated through the assembly/disassembly of a [4Fe-4S] cluster, which interconverts IRP1 with cytosolic aconitase. A genetic screen to isolate Saccharomyces cerevisiae strains bearing mutations in genes required for the conversion of IRP1 to c-aconitase led to the identification of a previously uncharacterized, essential gene, which we call CFD1 (cytosolic Fe-S cluster deficient). CFD1 encodes a highly conserved, putative P-loop ATPase. A non-lethal mutation of CFD1 (cfd1-1) reduced c-aconitase specific activity in IRP1-transformed yeast by >90%, although IRP1 in these cells could be readily converted to c-aconitase in vitro upon incubation with iron alone. IRP1-transformed cfd1-1 yeast lacked EPR-detectable Fe-S clusters in c-aconitase, pointing to a defect in Fe-S cluster assembly. The specific activity of another cytosolic Fe-S protein, Leu1p, was also inhibited by >90% in cfd1-1 yeast, whereas activity of mitochondrial Fe-S proteins was not inhibited. Consistent with a cytosolic site of activity, Cfd1p was localized in the cytoplasm. To our knowledge, Cfd1p is the first cytoplasmic Fe-S cluster assembly factor described in eukaryotes.

Background: A Cfd1 and Nbp35 heterocomplex serves as scaffold for cytosolic iron-sulfur cluster assembly. Results: Deficiency in Cfd1-Nbp35 interaction impaired iron turnover on Nbp35. Conclusion: Cfd1 promotes binding and transfer of labile iron-sulfur cluster on the Nbp35 scaffold. Significance: This is the first insight into the unique roles of these P-loop ATPases in cytosolic iron-sulfur cluster assembly.

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Lager beer brewing relies on strains collectively known as Saccharomyces carlsbergensis, which are hybrids between S. cerevisiae and S. eubayanus-like strains. Lager yeasts are particularly adapted to low-temperature fermentations. Selection of new yeast strains for improved traits or fermentation performance is laborious, due to the allotetraploid nature of lager yeasts. Initially, we have generated new F1 hybrids by classical genetics, using spore clones of lager yeast and S. cerevisiae and complementation of auxotrophies of the single strains upon mating. These hybrids were improved on several parameters, including growth at elevated temperature and resistance against high osmolarity or high ethanol concentrations. Due to the uncertainty of chromosomal make-up of lager yeast spore clones, we introduced molecular markers to analyse mating-type composition by PCR. Based on these results, new hybrids between a lager and an ale yeast strain were isolated by micromanipulation. These hybrids were not subject to genetic modification. We generated and verified 13 hybrid strains. All of these hybrid strains showed improved stress resistance as seen in the ale parent, including improved survival at the end of fermentation. Importantly, some of the strains showed improved fermentation rates using 18 Plato at 18-25 C. Uniparental mitochondrial DNA inheritance was observed mostly from the S. cerevisiae parent.