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. 2015 May 21;11(5):e1005135.
doi: 10.1371/journal.pgen.1005135. eCollection 2015 May.

Turning Saccharomyces cerevisiae into a Frataxin-Independent Organism

Heeyong Yoon  1 , Simon A B Knight  1 , Alok Pandey  2 , Jayashree Pain  2 , Serdar Turkarslan  3 , Debkumar Pain  2 , Andrew Dancis  1
Affiliations

Turning Saccharomyces cerevisiae into a Frataxin-Independent Organism

Heeyong Yoon et al. PLoS Genet. .

Abstract

Frataxin (Yfh1 in yeast) is a conserved protein and deficiency leads to the neurodegenerative disease Friedreich's ataxia. Frataxin is a critical protein for Fe-S cluster assembly in mitochondria, interacting with other components of the Fe-S cluster machinery, including cysteine desulfurase Nfs1, Isd11 and the Isu1 scaffold protein. Yeast Isu1 with the methionine to isoleucine substitution (M141I), in which the E. coli amino acid is inserted at this position, corrected most of the phenotypes that result from lack of Yfh1 in yeast. This suppressor Isu1 behaved as a genetic dominant. Furthermore frataxin-bypass activity required a completely functional Nfs1 and correlated with the presence of efficient scaffold function. A screen of random Isu1 mutations for frataxin-bypass activity identified only M141 substitutions, including Ile, Cys, Leu, or Val. In each case, mitochondrial Nfs1 persulfide formation was enhanced, and mitochondrial Fe-S cluster assembly was improved in the absence of frataxin. Direct targeting of the entire E. coli IscU to ∆yfh1 mitochondria also ameliorated the mutant phenotypes. In contrast, expression of IscU with the reverse substitution i.e. IscU with Ile to Met change led to worsening of the ∆yfh1 phenotypes, including severely compromised growth, increased sensitivity to oxygen, deficiency in Fe-S clusters and heme, and impaired iron homeostasis. A bioinformatic survey of eukaryotic Isu1/prokaryotic IscU database entries sorted on the amino acid utilized at the M141 position identified unique groupings, with virtually all of the eukaryotic scaffolds using Met, and the preponderance of prokaryotic scaffolds using other amino acids. The frataxin-bypassing amino acids Cys, Ile, Leu, or Val, were found predominantly in prokaryotes. This amino acid position 141 is unique in Isu1, and the frataxin-bypass effect likely mimics a conserved and ancient feature of the prokaryotic Fe-S cluster assembly machinery.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Genetic manipulations of ISU1-Ile and other ISU1 alleles.
(A) Genetic dominance of ISU1-Ile conferring frataxin-bypass. Strains YFH1 [ISU1], Δyfh1 [ISU1], Δyfh1 [ISU1-Ile], and Δyfh1 [ISU1-Ile] ISU1 ISU2 (Table 1) were compared by spotting serial 5-fold dilutions of 105 cells on YPAD plates and photographing three days later. (B) Frataxin-bypass function and scaffold function of ISU1 alleles. A series of plasmids was constructed in the YCplac22 backbone. These plasmids carried native ISU1 or ISU1 alleles with substitutions of residue M141 to all 19 other standard amino acids. N123D or N123A substitutions were also constructed, predicted to give disordered or structured conformations, respectively [34]. Plasmid-borne YFH1 and empty vector (-) were included as controls. To test frataxin-bypass function the ISU1 alleles were transformed into the YFH1 shuffle strain, and the transformed cells were transferred to fluoroorotic acid (FOA) plates to remove the covering YFH1-URA3 plasmid and expose the Δyfh1 phenotype (plates 1–3). To test scaffold function the ISU1 alleles were transformed into the GAL1-ISU1/Δisu2 strain and plated on raffinose carbon source to repress genomic GAL1-ISU1. (C) Effects of cysteine mutations. Plasmids with various cysteine substitutions in Isu1 were tested for frataxin-bypass function by introducing them into the YFH1 shuffle strain and counterselecting on FOA (plates 1 and 2). The Isu1 substitutions were also tested for scaffold function by introducing them into the GAL1-ISU1/Δisu2 strain and shifting the carbon source from galactose to glucose (plates 3 and 4). Key to transformants. 1. Empty plasmid YCplac22, 2. ISU1, 3. ISU1-M141I, 4. ISU1-C69A-M141I, 5. ISU1-C96A-M141I, 6. ISU1-C139A-M141I, 7. ISU1-C139A-M141C. (D) Δyfh1 nfs1-14 double mutant does not support frataxin-bypass by ISU1-M141I. Shuffle strain 113–26 (Δyfh1 nfs1-14 [pRS416-YFH1]) was transformed with YCplac22, YCplac22-YFH1, or YCplac22-ISU1-Ile. Transformants were patched onto tryptophan drop-out medium without (plate 1) or with (plate 2) FOA.
Fig 2
Fig 2. Characterization and comparison of frataxin-bypass substitutions of ISU1 amino acid 141: ISU1-Cys, ISU1-Ile, ISU1-Leu and ISU1-Val.
(A) Growth defects in Δyfh1 are corrected by the frataxin-bypass suppressors. The strains YFH1 [ISU1] (Y-M), Δyfh1 [ISU1] (Δ-M), and suppressor strains Δyfh1 [ISU1-Cys] (Δ-C), Δyfh1 [ISU1-Ile] (Δ-I), Δyfh1 [ISU1-Leu] (Δ-L), and Δyfh1 [ISU1-Val] (Δ-V), were grown in an argon-filled chamber, and serial dilutions were spotted on YPAD agar, without or with 4 mM hydrogen peroxide. (B) Mitochondrial protein levels by immunoblotting. Strains were inoculated from the argon-filled chamber into aerobic defined raffinose medium for 16–24 h prior to isolating mitochondria. Mitochondrial proteins (100 μg) were separated by SDS-PAGE and transferred to nitrocellulose. Strips were cut from a single blot and probed with the indicated antibodies against mitochondrial proteins (Aco1, Nfs1, Mir1, Yfh1, and Isu1). Cytochrome c (Cyc1) was analyzed using a duplicate gel. (C) Iron homeostasis in the frataxin-bypass suppressors. The indicated strains were grown in air for six doublings in defined raffinose medium in the presence of 10 μM 55Fe ascorbate. Cells were washed free of radioactive iron and separated into mitochondria and post-mitochondrial supernatant. An aliquot of mitochondria was lysed in the presence of 0.1% Triton X-100 for 10 min at room temperature, and supernatant (soluble) and pellet (insoluble) fractions were separated by centrifugation at 20,000 x g for 30 min at 4°C. Iron content was reported for whole cells (pmol per million cells), or various cellular fractions (pmol per micrograms protein).
Fig 3
Fig 3. Assessment of Fe-S cluster assembly in mitochondria isolated from frataxin-bypass mutants.
The same strains as in Fig 2 were assayed as follows: (A) Aconitase activity by in-gel assay. Mitochondria were lysed and 100 μg (lanes 1–6) or 200 μg (lanes 7–12) proteins were separated by native gel and developed using reagents that reflect aconitase activity (upper panel). The same lysates were separated by SDS-PAGE and analyzed by immunoblotting with anti-aconitase antibody to show the level of total aconitase protein (lower panel). (B) Persulfide formation on Nfs1 in mitochondria. Isolated and intact mitochondria were depleted of nucleotides and NADH by incubation at 30°C for 10 min. Either 50 μg or 100 μg protein equivalents were labeled for 15 min with 35S-cysteine. Samples were diluted with buffer, mitochondria were recovered by centrifugation, and total proteins were analyzed by non-reducing SDS-PAGE and autoradiography. The persulfide Nfs1-S-35SH is indicated by an arrow. Mitochondria from the nfs1-14 mutant was included as a negative control (lane 13), because this hypomorphic Nfs1 mutant has negligible persulfide-forming activity [16]. (C) New Fe-S cluster synthesis on apoaconitase in isolated mitochondria. Isolated and intact mitochondria, were labeled with 35S-cysteine in the presence of added 4 mM ATP, 1 mM GTP, 5 mM NADH and 10 μM ferrous ascorbate for 30 min at 30°C. Mitochondria were recovered and membranes were ruptured. After centrifugation, the supernatant fractions containing soluble mitochondrial proteins (corresponding to 25 or 50 μg for YFH1 [ISU1] and 100 or 200 μg for the other strains) were separated by native gel prior to autoradiography. The arrow indicates the aconitase protein containing newly made radiolabeled [Fe-35S] clusters.
Fig 4
Fig 4. Screening of randomized ISU1 in search of new frataxin-bypass suppressors reveals only amino acid substitutions of residue 141.
(A) A library of linear ISU1 sequences with random mutations was created by error prone PCR and co-transformed with a gapped plasmid (pRS416-ISU1) into a YFH1 (Δisu1) shuffle strain. The transformants contained recombined ISU1 sequences with random mutations, and appeared uniform on defined uracil drop-out medium (plate 1). The transformants were replicated to cycloheximide and glucose containing plates, removing the covering pRS318-YFH1 plasmid (plate 2). The transformants were also replicated to cycloheximide and raffinose containing plates (plate 3), selecting against the Δyfh1 phenotype. (B) Summary table of screening results indicating starting template, number of colonies screened, number of hits conferring frataxin-bypass, and types of hits.
Fig 5
Fig 5. E. coli iscU or iscU-Met targeted to yeast mitochondria.
(A) Growth phenotypes in argon or in air. Strains with mitochondrial targeted E. coli IscU proteins indicated in the figure were grown on YPAD in argon or in air for 3 days and photographed. Strain YFH1 [ISU1] was included as a control. (B) Mitochondrial protein levels by immunoblotting. The following strains were evaluated: 1) YFH1 [ISU1] or Y-M, 2) YFH1 [iscU] or Y-eI, 3) YFH1 [iscU-Met] or Y-eM, 4) Δyfh1 [iscU] or Δ-eI clone 1, 5) Δyfh1 [iscU] or Δ-eI clone 2, 6) Δyfh1 [iscU-Met] clone 1 or Δ-eM, and 7) Δyfh1 [iscU-Met] clone 2 or Δ-eM. Cultures were inoculated into defined raffinose medium bubbled with argon. After an initial growth period the cultures were shifted to air. The doubling time of the Δyfh1 [iscU-Met] clones in air started at 2.5 h but prolonged to greater than 8 h, necessitating a longer growth period prior to harvesting these cells. Mitochondrial proteins were separated by SDS-PAGE, and transferred to nitrocellulose, which was cut horizontally into segments and probed with various antibodies against various mitochondrial proteins (Aco1, Nfs1, Mir1, Yfh1, and Cyc1). The segment used for anti-Cyc1 was stripped and reprobed with anti-Isu1. The antibody to the yeast Isu1 was used to detect the E. coli IscU. The two bands attributed to E. coli IscU are indicated by a double arrow, and yeast Isu1 is indicated by a single arrow. (C) Iron homeostasis. The indicated strains were precultured in argon-bubbled raffinose medium and then shifted to air in the presence of 10 μM 55Fe ascorbate. Following growth to a density of 2 x 107 cells/ml, cells were harvested. Total cellular iron and iron in cellular fractions (post-mitochondrial supernatant, mitochondria, and soluble or insoluble fractions of mitochondria) were measured as in Fig 2C. (D) Aconitase activity. Mitochondria from the indicated strains were lysed, and 100 μg (lanes 1–7) or 200 μg (lanes 8–14) of proteins were separated by native gel and analyzed for aconitase activity using the in-gel assay.
Fig 6
Fig 6. Cosegregation of Isu1/IscU-M and frataxin in global taxonomy.
Distribution of species containing M (red letter) versus C I L V (blue letters) at the X position of the motif LPPVK LH CSX LA and correlation with the presence of frataxin. The presence of frataxin is indicated by a red colored box next to each species in the taxonomic tree. The depicted species were chosen to maximize diversity. The species are displayed using the NCBI Taxonomy browser according to their positions in the NCBI Taxonomy Database. The arrow connecting shaded areas in Proteobacteria and Eukaryota represents the direction of possible horizontal gene transfer occurring during mitochondrial creation.
Fig 7
Fig 7. The Fe-S cluster assembly complex of E. coli.
The Protein Data Bank (PDB) file of the bacterial Fe-S cluster assembly complex [36] was kindly provided by Annalisa Pastore (King’s College London). The image was drawn using the Stanford Chimera program. The cysteine desulfurase is IscS for E. coli or Nfs1 for yeast; frataxin is CyaY for E. coli or Yfh1 for yeast; the Fe-S scaffold is IscU for E. coli or Isu1/Isu2 for yeast. Highlighted features of IscU/Isu1 are the suppressor amino acid Ile (red), the Fe-S cluster cysteine ligands (blue), and the PVK frataxin-binding site (green). The reciprocal interaction site on frataxin includes a tryptophan (green). The PLP cofactor of IscS/Nfs1 is shown as yellow spheres, and a dotted purple line is used to indicate the approximate position of the active site mobile loop, which is not seen in the structures.
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