Abstract
Biogenesis of iron-sulfur ([Fe-S]) proteins in eukaryotes requires the function of complex proteinaceous machineries in both mitochondria and cytosol. In contrast to the mitochondrial pathway, little is known about [Fe-S] protein assembly in the cytosol. So far, four highly conserved proteins (Cfd1, Nbp35, Nar1 and Cia1) have been identified as members of the cytosolic [Fe-S] protein assembly machinery, but their molecular function is unresolved. Using in vivo and in vitro approaches, we found that the soluble P-loop NTPases Cfd1 and Nbp35 form a complex and bind up to three [4Fe-4S] clusters, one at the N terminus of Nbp35 and one each at a new C-terminal cysteine-rich motif present in both proteins. These labile [Fe-S] clusters can be rapidly transferred and incorporated into target [Fe-S] apoproteins in a Nar1- and Cia1-dependent fashion. Our data suggest that the Cfd1–Nbp35 complex functions as a novel scaffold for [Fe-S] cluster assembly in the eukaryotic cytosol.
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Acknowledgements
We thank R.K. Thauer (Max-Planck-Institute, Marburg) for access to the EPR spectrometer and providing purified ferredoxin, and J. Balk and W. Walden for discussion. This work was supported by grants from Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 593, Graduiertenkolleg 1216, Gottfried-Wilhelm Leibniz program), the European Commission (MitEURO) and Fonds der chemischen Industrie.
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Supplementary information
Supplementary Fig. 1
Stable complex formation between Cfd1 and Nbp35. (PDF 123 kb)
Supplementary Fig. 2
Spectroscopic properties of Nbp35 and its C253A mutant. (PDF 73 kb)
Supplementary Fig. 3
The [Fe-S] clusters bound to Cfd1, Nbp35 or the Cfd1–Nbp35 complex are labile in the presence of oxygen. (PDF 103 kb)
Supplementary Table 1
Constructs used in this work. (PDF 21 kb)
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Netz, D., Pierik, A., Stümpfig, M. et al. The Cfd1–Nbp35 complex acts as a scaffold for iron-sulfur protein assembly in the yeast cytosol. Nat Chem Biol 3, 278–286 (2007). https://doi.org/10.1038/nchembio872
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DOI: https://doi.org/10.1038/nchembio872
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