Crucial role of conserved cysteine residues in the assembly of two iron-sulfur clusters on the CIA protein Nar1.
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 Escherichia 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 (55)Fe radiolabeling 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 modeling 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. Insertion of an Fe/S cluster into the C-terminal location depends on the N-terminal motif, suggesting the participation of the latter motif in the assembly process of the C-terminal cluster. The vicinity of the two Fe/S centers suggests a close functional cooperation during cytosolic Fe/S protein maturation.
Urzica
Eugen
E
Institut für Zytobiologie, Philipps Universität Marburg, Robert-Koch-Strasse 6, D-35033 Marburg, Germany.
Pierik
Antonio J
AJ
Mühlenhoff
Ulrich
U
Lill
Roland
R
D020816:Amino Acid Motifs / Q000235:genetics; D002470:Cell Survival / Q000235:genetics / Q000502:physiology; D017124:Conserved Sequence / Q000235:genetics; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D003600:Cytosol / Q000737:chemistry* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D006864:Hydrogenase / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*
D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D006864:Hydrogenase; C501975:Nar1 protein, S cerevisiae; D003545:Cysteine