Urzica E, et al. (2009) Crucial role of conserved cysteine residues in the assembly of two iron-sulfur clusters on the CIA protein Nar1. Biochemistry 48(22):4946-58
Abstract: Iron-sulfur (Fe/S) protein maturation in the eukaryotic cytosol and nucleus requires conserved components of the essential CIA machinery. The CIA protein Nar1 performs a specific function in transferring an Fe/S cluster that is assembled de novo on the Cfd1-Nbp35 scaffold to apoproteins. Here, we used systematic site-directed mutagenesis and a combination of in vitro and in vivo studies to show that Nar1 holds two Fe/S clusters at conserved N- and C-terminal cysteine motifs. A wealth of biochemical studies suggests that the assembly of these Fe/S clusters on Nar1 cannot be studied in E. coli, as the recombinant protein does not contain the native Fe/S clusters. We therefore followed Fe/S cluster incorporation directly in yeast by a 55Fe radiolabelling method in vivo, and we measured the functional consequences of Nar1 mutations in the assembly of cytosolic Fe/S proteins. We find that both Fe/S clusters are essential for Nar1 function and cell viability. Molecular modelling using a structurally, but not functionally related bacterial iron-only hydrogenase as a template provided compelling structural explanations for our mutational data. The C-terminal Fe/S cluster is stably buried within Nar1, whereas the N-terminal one is exposed at the protein surface and hence may be more easily lost. Fe/S cluster insertion into the C-terminal location depends on the N-terminal motif, suggesting a participation of the latter motif in the assembly process of the C-terminal cluster. The vicinity of the two Fe/S centres suggests a close functional cooperation during cytosolic Fe/S protein maturation.
|Status: Published||Type: Journal Article||PubMed ID: 19385603|
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