PMC

Fig. 3. Wild-type CFD1 restores glutamate-dependent growth to AR27/IRP1. Mutant strain AR27 or the cfd1-disrupted strain NS1 transformed with the indicated CFD1 allele carried on pRS313 was transformed with IRP1. Approximately 10⁴ transformed cells were spotted in 10 µl of water onto selective SD medium, with (+) or without (–) glutamate as indicated, and allowed to grow for 4 days at 30°C.

Fig. 6. IRP1-transformed cfd1-1 yeast lack [3Fe–4S] c-aconitase. Strains 0615d and AR27 were transformed with IRP1 and transformants were grown to mid-log phase in selective SD medium, harvested, and prepared for whole-cell EPR as described in Materials and methods. Non-transformed yeast were grown in SD-complete medium. Where indicated, cells were exposed to 0.6 mM H₂O₂ for 15 min prior to harvest. Spectra shown are the difference spectra resulting after subtracting non-transformed cell spectra from IRP1-transformed cell spectra.

Fig. 1. IRP1-transformed AR27 mutant strain displays a severe growth defect on glutamate-free medium. Fresh overnight cultures of 0615d or AR27, each transformed with IRP1, were harvested, washed twice with sterile water, and resuspended into sterile water at an OD₆₀₀ of 0.5. Ten microliters of this suspension and 10-fold serial dilutions were spotted onto SD medium lacking uracil, with (+) or without (–) glutamate. Cells were allowed to grow at 30°C for 4 days before photographing.

Fig. 7. Inhibition caused by the cfd1-1 mutation is specific to cytosolic Fe–S proteins. (A) Strains 0615d and AR27 were transformed with ACO1 and transformants were grown to mid-log phase in selective SD medium, harvested, and extracts were prepared for assay of aconitase (ACO1), succinate dehydrogenase (SDH), Leu1p, malate dehydrogenase (MDH) and NAD⁺-dependent isocitrate dehydrogenase (IDH) as described in Materials and methods. (B) Protein immunoblotting performed with 50 µg of total extract protein using antiserum specific for mitochondrial aconitase (Aco1p) or Leu1p.

Fig. 5. Cys201 and Cys204 of the CX₂CX₂C element are required for Cfd1p function. AR27/IRP1 was transformed with empty vector (none) or with vector carrying His₆-tagged wild-type, CFD1^C201S, CFD1^C204S or CFD1^C207S fusion genes (see Materials and methods). (A) Glutamate-dependent growth. Approximately 10⁴ cells were spotted in 10 µl onto media with or without glutamate, as indicated. Photographs were taken after 4 days of growth at 30°C. (B) Cytosolic aconitase activity. Aconitase assays were performed as described in the legend to Figure A using extracts from logarithmically growing AR27/IRP1 transformed with the indicated CFD1 genes.

Fig. 4. Comparative alignment of yeast Cfd1p. Yeast Cfd1p (Sc Cfd1p) was aligned with human Cfd1p (Hu Cfd1p), mouse Cfd1p (Mu Cfd1p), Salmonella enterica serovar typhimurium ApbC (St Mrp) and Archaeoglobus fulgidis MinD (Af MinD) using CLUSTAL_X. The human and mouse proteins are also called NuBP2 (). Dark shading in the alignment show identities and light shading shows conserved residues. Residues that form the Walker P-loop, Switch I and Switch II (Walker B motif) elements, which comprise important nucleotide binding and intramolecular signaling domains in P-loop ATPases, are indicated beneath the alignment (; ). The location of the CX₂CX₂C motif (residues 201–207) is also indicated beneath the alignment as CXXCXXC. Regions that form β-strands (black rectangles) or α-helices (gray rectangles) in the MinD protein are aligned below its sequence ().

Fig. 8. Cfd1p localizes to the cytoplasm. Mid-log phase yeast were subjected to subcellular fractionation as described in Materials and methods. The presence of individual proteins in each fraction was assessed by protein immunoblotting. S100, soluble cytoplasmic material after 100 000 g centrifugation; P100, material pelleted from crude cytoplasmic fraction by 100 000 g centrifugation; N1, floating material from Nycodenz gradient; N2, light membrane vesicles; M, mitochondria-enriched fraction; N4, large aggregates and heavy membrane vesicles. The fractionation patterns shown are from NS1/CFD1 (Cfd1p), 0615d/myc tagged-IRP1 (IRP1) and AR27/ACO1 transformed with 6HisCFD1 (Aco1p and porin; note that 6HisCfd1p showed a similar fractionation pattern to native Cfd1p). Relative to the fraction of S100 material loaded, ∼10-fold more material from P100, N1 and N2, and 100-fold more material from M and N4 were loaded. The results shown are representative of five independent experiments.

Fig. 2. AR27/IRP1 has reduced c-aconitase activity. Strains 0615d/IRP1 and AR27/IRP1 were grown in SD-ura medium to an OD₆₀₀ of 0.8, and cells were harvested and extracts prepared as described previously (). Extracts were used to measure endogenous c-aconitase activity (A), IRP1 protein by immunoblotting (B), or were incubated with 0.5 mM ferrous ammonium sulfate and 50 mM DTT at room temperature for 1 h to activate c-aconitase followed by aconitase assay (C). Unit activity is in µmol cis-aconitate produced per min per mg total extract protein using 20 mM isocitrate as substrate (). The results of the enzyme assays are representative of three independent experiments.