Mitochondrial Iba57p is required for Fe/S cluster formation on aconitase and activation of radical SAM enzymes

Abstract

A genome-wide screen for Saccharomyces cerevisiae iron-sulfur (Fe/S) cluster assembly mutants identified the gene IBA57. The encoded protein Iba57p is located in the mitochondrial matrix and is essential for mitochondrial DNA maintenance. The growth phenotypes of an iba57Delta mutant and extensive functional studies in vivo and in vitro indicate a specific role for Iba57p in the maturation of mitochondrial aconitase-type and radical SAM Fe/S proteins (biotin and lipoic acid synthases). Maturation of other Fe/S proteins occurred normally in the absence of Iba57p. These observations identify Iba57p as a novel dedicated maturation factor with specificity for a subset of Fe/S proteins. The Iba57p primary sequence is distinct from any known Fe/S assembly factor but is similar to certain tetrahydrofolate-binding enzymes, adding a surprising new function to this protein family. Iba57p physically interacts with the mitochondrial ISC assembly components Isa1p and Isa2p. Since all three proteins are conserved in eukaryotes and bacteria, the specificity of the Iba57/Isa complex may represent a biosynthetic concept that is universally used in nature. In keeping with this idea, the human IBA57 homolog C1orf69 complements the iba57Delta growth defects, demonstrating its conserved function throughout the eukaryotic kingdom.

FIG. 1.

The mitochondrial matrix protein Iba57p is required for lysine and glutamate prototrophy and mitochondrial genome maintenance. (A) The indicated S. cerevisiae strains either carrying the empty vector pRS413 or plasmid pIBA57 were spotted onto SC plates lacking histidine and containing glutamate or lysine as indicated. The plates were incubated either in air (+O₂) or in an anaerobic jar (−O₂) for 2 days at 30°C. (B) BY4742 WT and BY4742 iba57Δ cells were each mated with a [rho°] tester strain. Diploid colonies were selected and patched onto rich medium containing either glucose or glycerol. (C) W303-1A WT, iba57Δ, Gal-IBA57, and Gal-IBA57-Myc cells were grown on SC galactose medium lacking both lysine and glutamate. (D) Gal-IBA57-Myc cells expressing Myc-tagged Iba57p from a GalL promoter were grown in rich galactose medium, and mitochondria (Mito) and postmitochondrial supernatant (PMS) were isolated. Immunostaining was performed with antisera raised against the mitochondrial matrix protein Mge1p, the cytosolic 3-phosphoglycerate kinase (Pgk1p) and monoclonal anti-Myc (A14; Santa Cruz). (E) Mitochondria from panel C were either left intact, hypotonically swollen, or lysed with 0.5% Triton X-100 detergent. Each sample was incubated in the presence or absence of 100 μg of proteinase K/ml for 20 min on ice, and the proteinase K was inactivated by using phenylmethylsulfonyl fluoride (14). The resulting samples were analyzed by immunostaining for the Myc-tag of Iba57p, the mitochondrial intermembrane space protein cytochrome b2 (Cyb2p), the inner membrane protein Tim44p, and the matrix protein Mge1p. (F) Mitochondria from panel C were lysed by sonication, separated by ultracentrifugation into supernatant (SN) and pellet fractions, and analyzed by immunostaining as described above.

FIG. 2.

Depletion of Iba57p results in diminished de novo Fe/S cluster formation on aconitase. (A) Whole-cell extracts from the indicated strains grown overnight in SC medium were assayed for activities of aconitase, cytochrome c oxidase, and alcohol dehydrogenase. Gal-IBA57 and Gal-IBA57-Myc strains were depleted of Iba57p by growth in SC glucose medium for 40 h prior to analysis. (B) Gal-IBA57-Myc cells were grown in SC medium containing either galactose (Gal) or glucose (Glc), and Mge1p and Iba57p-Myc were detected by immunostaining. (C) W303-1A (WT), Iba57p-depleted Gal-IBA57, iba57Δ, and aco1Δ cells were grown overnight in iron-poor SD medium. Cells were labeled with 10 μCi of ⁵⁵Fe for 2 h, and Aco1p was immunoprecipitated from crude extracts with polyclonal antiserum. The amount of coimmunoprecipitated radioactivity was quantified by scintillation counting, and the total amount of Aco1p was assessed by immunostaining. Por1p was used as a loading control. (D) Mitochondria were isolated from W303-1A (WT) and iba57Δ cells grown in rich glucose medium, and Aco1p and Mge1p were detected by immunostaining. (E) Mitochondria from WT and iba57Δ cells were lysed by sonication and separated into supernatant (SN) and pellet fractions by ultracentrifugation. The fractions were analyzed by immunostaining of the indicated proteins. (F) Mitochondria from the indicated strains corresponding to 1 mg of protein each were lysed in buffer containing 0.1% dodecyl-maltoside and 0.6 mM H₂O₂ and frozen in liquid helium, and EPR spectra were recorded at 10K (EPR conditions: microwave power, 2 mW; microwave frequency, 9.46 GHz; modulation amplitude, 1.25 mT; modulation frequency, 100 kHz).

FIG. 3.

Depletion of Iba57p results in diminished de novo Fe/S cluster formation on homoaconitase (Lys4p). The indicated strains carrying p426LYS4-HA were grown in iron-poor SC medium containing the indicated carbon source. Cells were radiolabeled and Lys4p immunoprecipitated with anti-HA (Santa Cruz) immunobeads as described in Fig. 2. Lys4-HA was detected by immunostaining with anti-HA. Por1p was used as a loading control.

FIG. 4.

Depletion of Iba57p does not result in diminished de novo Fe/S cluster formation on ferredoxin (Yah1p) or isopropylmalate dehydratase (Leu1p). (A) The indicated strains carrying p426YAH1-HA were grown in iron-poor SD medium. Cells were radiolabeled and Yah1p-HA immunoprecipitated with anti-HA immunobeads (Santa Cruz) as described in Fig. 2. Yah1p was detected by immunostaining with anti-HA. Por1p was used as a loading control. (B) S288c WT and iba57Δ cells were spotted onto minimal medium plates containing only lysine and glutamate and either containing or lacking leucine. (C) W303-1A WT, depleted Gal-IBA57, and iba57Δ cells were grown overnight in glucose minimal medium, and cell lysates were prepared by the glass bead method. The Leu1p activity was assayed immediately. (D) W303-1A (WT), Iba57p-depleted Gal-IBA57, and iba57Δ cells were grown overnight in iron-poor SD medium, and ⁵⁵Fe binding to Leu1p was analyzed by the radiolabeling and immunoprecipitation assay described in Fig. 2. Leu1p and Por1p were detected with polyclonal antiserum. (E) Indicated strains carrying pFET3-GFP were grown overnight in SD medium supplemented with 200 μM ferric ammonium citrate (iron replete) or 50 μM bathophenanthroline disulfonic acid (iron depleted), and diluted to an OD₆₀₀ of 0.2. After an additional 4 h of growth the cells were harvested and then resuspended at an OD₆₀₀ of 1, and the fluorescence emission at 513 nm was determined. Gal strains were depleted of their respective gene product by growth in SD medium.

FIG. 5.

Iba57p is required for the function of but not Fe/S cluster assembly on biotin synthase. (A) The W303-1A (WT), iba57Δ, strain and bio2Δ carrying the empty pRS413 vectors and the iba57Δ strain expressing IBA57 from plasmid pIBA57 were grown overnight in biotin-free SC glucose medium lacking histidine. Each strain was then spotted onto SC glucose plates lacking histidine and biotin and supplemented with either 4 ng of biotin/ml, 50 mU of avidin/ml, or both 50 ng of desthiobiotin/ml and 50 mU of avidin/ml. (B) W303-1A (WT), iba57Δ, and isa1/2Δ cells were grown for 40 h in biotin-free SD medium and then diluted into fresh biotin-free SD medium either with or without 20 μg of desthiobiotin (DTB)/liter and grown for a further 24 h. Cells were harvested, cell lysates were prepared, and Arc1p was detected by immunostaining with peroxidase-linked streptavidin. The blots were developed immediately after decoration with streptavidin-peroxidase or after a subsequent incubation with 2 mM desthiobiotin for 16 h (DTB wash). Por1p served as a loading control. (C) W303-1A (WT), Iba57p-depleted Gal-IBA57, and iba57Δ cells carrying p426BIO2 were grown overnight in iron-poor SD medium, and ⁵⁵Fe binding to Bio2p was analyzed by radiolabeling and immunoprecipitation as described in Fig. 2. The Bio2p and Por1p levels were determined by immunostaining.

FIG. 6.

Iba57p is required for the function of but not Fe/S cluster incorporation into lipoic acid synthase. (A) The indicated strains were grown on SD plates containing amino acids as indicated. (B) Mitochondrial extracts were prepared from the indicated strains grown on lipoic acid-free SD medium. KDH and PDH activities were assayed and are presented relative to MDH activity to correct for general mitochondrial enzyme defects. (C) Mitochondrial extracts from panel B were probed with antibodies against Por1p and lipoic acid (46). The latter recognizes the lipoylated forms of the E2 subunits of PDH (lower band) and KDH (upper) and the H-subunit of GDC (Gcv3p). (D) Strains grown for 5 days in SD medium lacking lipoic acid were analyzed for lipoic acid content. (E) The indicated strains were transformed with plasmid p426-LIP5-HA, and ⁵⁵Fe binding to Lip5p-HA was analyzed by radiolabeling and immunoprecipitation as described in Fig. 3. Lip5p-HA and Mge1p were detected by immunostaining.

FIG. 7.

Iba57p interacts with Isa1p and Isa2p. (A) Cell extracts were prepared from W303-1A and iba57Δ strains grown overnight in YP glucose and immunostained for Isa1p and Isa2p. α-Tubulin (Serotec) served as a loading control. (B) W303-1A WT, depleted Gal-IBA57, and iba57Δ strains were transformed with p426-ISA2 and grown overnight in iron-depleted glucose minimal medium, and ⁵⁵Fe binding to Isa2p was analyzed by radiolabeling and immunoprecipitation as described in Fig. 3. Leu1p and Mge1p were detected with polyclonal antisera. (C) Mitochondria from WT cells and galactose-induced Gal-IBA57-Myc cells with or without overproduced Isa2p were lysed by detergent, and immunoprecipitations were carried out with antibodies to Isa2p and the Myc tag. The purified immunobeads were analyzed for the presence of Iba57p-Myc and Isa2p by immunostaining. (D) Mitochondria from WT, Gal-IBA57-Myc cells with or without overproduced Isa1p were lysed by detergent, and immunoprecipitations were carried out with antibodies to Isa1p and Myc. The purified immunobeads were analyzed by immunostaining.

FIG. 8.

Expression of human IBA57 complements the growth defect of iba57Δ cells. iba57Δ cells were transformed with the expression plasmids p-huIBA57a (encoding residues 26 to 357 of huIba57 fused to the mitochondrial targeting sequence of the ATPase subunit F1β) or p-huIBA57b [residue 26 of huIBA57 up to the poly(A) tail initiating at nucleotide 1942 from the Invitrogen human cDNA clone IMAGE:4589759, also fused to F1β]. These cells were grown on SD medium together with WT and iba57Δ cells in the presence or absence of lysine (Lys) and glutamate (Glu).