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Turning Saccharomyces cerevisiae into a Frataxin-Independent Organism.

Yoon H, Knight SA, Pandey A, Pain J, Turkarslan S, Pain D, Dancis A - PLoS Genet. (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Division of Hematology-Oncology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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pgen.1005135.g003: 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.

Mentions: Aconitase (Aco1), an abundant [Fe4S4] protein of mitochondria, was evaluated. An in- gel assay showed the aconitase enzyme activities in mitochondrial lysates. The control strain YFH1 [ISU1] (Fig 3A, upper panel, lanes 1 and 7, Y-M) showed concentration dependent activity. The frataxin minus strain Δyfh1 [ISU1] (Fig 3A, upper panel, lanes 2 and 8, Δ-M) had no detectable activity regardless of the concentration of lysate in the assay. The various suppressors (Fig 3A, upper panel, Δ-C, Δ-I, Δ-L, Δ-V, lanes 3–6 and lanes 9–12) recovered significant activity, although not entirely to wild-type levels. Aconitase protein (Fig 3A, lower panel) as detected by immunoblotting was present in YFH1 [ISU1] control (lanes 1 and 7), and was decreased in the frataxin-minus Δyfh1 [ISU1] (lanes 2 and 8) mitochondria, perhaps because the apoprotein was turned over more rapidly by mitochondrial proteases. Aconitase protein levels were restored in the suppressor strains (Fig 3A, lower panel, lanes 3–6 and lanes 9–12), consistent with recovery of enzyme activity.


Turning Saccharomyces cerevisiae into a Frataxin-Independent Organism.

Yoon H, Knight SA, Pandey A, Pain J, Turkarslan S, Pain D, Dancis A - PLoS Genet. (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4440810&req=5

pgen.1005135.g003: 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.
Mentions: Aconitase (Aco1), an abundant [Fe4S4] protein of mitochondria, was evaluated. An in- gel assay showed the aconitase enzyme activities in mitochondrial lysates. The control strain YFH1 [ISU1] (Fig 3A, upper panel, lanes 1 and 7, Y-M) showed concentration dependent activity. The frataxin minus strain Δyfh1 [ISU1] (Fig 3A, upper panel, lanes 2 and 8, Δ-M) had no detectable activity regardless of the concentration of lysate in the assay. The various suppressors (Fig 3A, upper panel, Δ-C, Δ-I, Δ-L, Δ-V, lanes 3–6 and lanes 9–12) recovered significant activity, although not entirely to wild-type levels. Aconitase protein (Fig 3A, lower panel) as detected by immunoblotting was present in YFH1 [ISU1] control (lanes 1 and 7), and was decreased in the frataxin-minus Δyfh1 [ISU1] (lanes 2 and 8) mitochondria, perhaps because the apoprotein was turned over more rapidly by mitochondrial proteases. Aconitase protein levels were restored in the suppressor strains (Fig 3A, lower panel, lanes 3–6 and lanes 9–12), consistent with recovery of enzyme activity.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Division of Hematology-Oncology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus