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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

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.
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pgen.1005135.g001: 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.

Mentions: In order to evaluate this genetic dominance further, matched Δyfh1 strains were compared, one expressing a single copy of substituted ISU1 and deleted ISU2, called Δyfh1 [ISU1-Ile] (Table 1) and another expressing both ISU1 and ISU2 in addition to the plasmid-borne substituted Isu1-M141I, called Δyfh1 [ISU1-Ile] ISU1 ISU2 (Table 1). As assessed by colony formation, both strains showed improved growth compared with the Δyfh1 control (Fig 1A, compare rows 3 and 4 to row 2), although the single copy [ISU1-Ile] showed slightly better Δyfh1 suppression activity as assessed by growth (Fig 1A, compare rows 3 and 4), and other phenotypes such as iron uptake and Fe-S cluster levels. Thus, a high degree of genetic dominance of the Isu1 Met to Ile substitution was observed in producing reversal of Δyfh1 phenotypes.


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)

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.
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Related In: Results  -  Collection

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

pgen.1005135.g001: 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.
Mentions: In order to evaluate this genetic dominance further, matched Δyfh1 strains were compared, one expressing a single copy of substituted ISU1 and deleted ISU2, called Δyfh1 [ISU1-Ile] (Table 1) and another expressing both ISU1 and ISU2 in addition to the plasmid-borne substituted Isu1-M141I, called Δyfh1 [ISU1-Ile] ISU1 ISU2 (Table 1). As assessed by colony formation, both strains showed improved growth compared with the Δyfh1 control (Fig 1A, compare rows 3 and 4 to row 2), although the single copy [ISU1-Ile] showed slightly better Δyfh1 suppression activity as assessed by growth (Fig 1A, compare rows 3 and 4), and other phenotypes such as iron uptake and Fe-S cluster levels. Thus, a high degree of genetic dominance of the Isu1 Met to Ile substitution was observed in producing reversal of Δyfh1 phenotypes.

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