Figure 4.

Measurement of enzyme activities and iron content in yeast. (A) Aconitase activity was measured in whole-cell extracts from cells grown exponentially at 28°C in yeast nitrogen base (YNB) medium plus 0.6% glucose. (B and C) Succinate dehydrogenase activity and cytocrome c oxidase activities were measured in a mitochondria-enriched fraction obtained from cells grown as described before. The values for isu1Δisu2Δ/isu1 ^G97V and isu1Δisu2Δ/ISU1/isu1 ^G97V strains are expressed as percentage of the activities obtained in the strains isu1Δisu2Δ/ISU1 and isu1Δisu2Δ/ISU1/ISU1. (D) Cellular iron content was quantified in cells grown up to early stationary phase in YNB 0.2% glucose and 2% galactose medium. *<0.05 (unpaired two-tailed t-test), **<0.01 (unpaired two-tailed t-test). (E) Gal-ISU1/isu2Δ cells and isu1Δ cells expressing Myc-tagged Isu1 were radiolabelled with ⁵⁵Fe and ⁵⁵Fe incorporation into Isu1-Myc was determined by immunoprecipitation with α-Myc antibodies followed by scintillation counting. Wild-type cells harbouring the empty vector (e.v.) served as control. Isu1-myc protein levels in isu1Δ cells were determined by immunostaining with α-Myc antibodies. Porin (Por1) served as a loading control. (F) Gal-ISU1/isu2Δ cells expressing Isu1 from vector pFL39 and the reporter plasmid pFET3-GFP were cultivated in SD or SGal medium supplemented with 50 µM ferric ammonium citrate. At an optical density=0.5, the GFP-specific fluorescence emission of whole cells was determined. Error bars indicate the SDs (n=3).