Figure 6.
Ind1 binds an Fe–S cluster in vivo. (A) Wild-type S. cerevisiae cells carrying an empty plasmid (−) or plasmids for intermediate (↑) or high (↑↑) expression of Y. lipolytica IND1–strep were radiolabelled with 55Fe. Cells were harvested after 2 h and, following preparation of a cell extract, Ind1–strep was immunoprecipitated with either Strep-Tactin or Ind1 antibodies coupled to sepharose A. The 55Fe associated with the protein was quantified by scintillation counting. The expression levels of Ind1–strep were confirmed by immunoblotting (lower panels). (B) Iron-55 labelling and immunoprecipitation of Ind1 or endogenous aconitase (Aco1) upon depletion of the cysteine desulphurase Nfs1 (Nfs1↓) or the scaffold protein Isu1 (Isu1↓). Wild-type S. cerevisiae (control), Gal-NFS1 or Gal-ISU1/isu2Δ strains were transformed with a plasmid to overexpress Y. lipolytica IND1–strep, and grown on glucose to downregulate the expression of NFS1 or ISU1 as indicated. Expression of Aco1, Ind1, Nfs1 and Isu1 was visualized by immunoblotting (inset). The scintillation values were corrected for the average background signal obtained from experiments with cells containing an empty plasmid (for Ind1; 1.6 × 103 d.p.m./g cells, see A), or with aco1Δ cells (for aconitase; 1.3 × 103 d.p.m./g cells). Error bars represent the standard deviation, n=3–5.