GTP Is Required for Iron-Sulfur Cluster Biogenesis in Mitochondria
2007; Elsevier BV; Volume: 283; Issue: 3 Linguagem: Inglês
10.1074/jbc.m706808200
ISSN1083-351X
AutoresBoominathan Amutha, Donna M. Gordon, Yajuan Gu, Elise R. Lyver, Andrew Dancis, Debkumar Pain,
Tópico(s)Trace Elements in Health
ResumoIron-sulfur (Fe-S) cluster biogenesis in mitochondria is an essential process and is conserved from yeast to humans. Several proteins with Fe-S cluster cofactors reside in mitochondria, including aconitase [4Fe-4S] and ferredoxin [2Fe-2S]. We found that mitochondria isolated from wild-type yeast contain a pool of apoaconitase and machinery capable of forming new clusters and inserting them into this endogenous apoprotein pool. These observations allowed us to develop assays to assess the role of nucleotides (GTP and ATP) in cluster biogenesis in mitochondria. We show that Fe-S cluster biogenesis in isolated mitochondria is enhanced by the addition of GTP and ATP. Hydrolysis of both GTP and ATP is necessary, and the addition of ATP cannot circumvent processes that require GTP hydrolysis. Both in vivo and in vitro experiments suggest that GTP must enter into the matrix to exert its effects on cluster biogenesis. Upon import into isolated mitochondria, purified apoferredoxin can also be used as a substrate by the Fe-S cluster machinery in a GTP-dependent manner. GTP is likely required for a common step involved in the cluster biogenesis of aconitase and ferredoxin. To our knowledge this is the first report demonstrating a role of GTP in mitochondrial Fe-S cluster biogenesis. Iron-sulfur (Fe-S) cluster biogenesis in mitochondria is an essential process and is conserved from yeast to humans. Several proteins with Fe-S cluster cofactors reside in mitochondria, including aconitase [4Fe-4S] and ferredoxin [2Fe-2S]. We found that mitochondria isolated from wild-type yeast contain a pool of apoaconitase and machinery capable of forming new clusters and inserting them into this endogenous apoprotein pool. These observations allowed us to develop assays to assess the role of nucleotides (GTP and ATP) in cluster biogenesis in mitochondria. We show that Fe-S cluster biogenesis in isolated mitochondria is enhanced by the addition of GTP and ATP. Hydrolysis of both GTP and ATP is necessary, and the addition of ATP cannot circumvent processes that require GTP hydrolysis. Both in vivo and in vitro experiments suggest that GTP must enter into the matrix to exert its effects on cluster biogenesis. Upon import into isolated mitochondria, purified apoferredoxin can also be used as a substrate by the Fe-S cluster machinery in a GTP-dependent manner. GTP is likely required for a common step involved in the cluster biogenesis of aconitase and ferredoxin. To our knowledge this is the first report demonstrating a role of GTP in mitochondrial Fe-S cluster biogenesis. GTP is required for numerous cellular functions both inside and outside of mitochondria. Mitochondria have their own genome attached to the organellar inner membrane at the matrix side (1Chen X.J. Butow R.A. Nat. Rev. Genet. 2005; 6: 815-825Crossref PubMed Scopus (364) Google Scholar). Processes associated with mitochondrial DNA replication, repair, and transcription require GTP. Likewise, critical steps involved in mitochondrial protein synthesis (initiation, elongation, and termination) utilize GTP (2Vozza A. Blanco E. Palmieri L. Palmieri F. J. Biol. Chem. 2004; 279: 20850-20857Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Other GTP-requiring processes also likely exist in the mitochondrial matrix, as evidenced by the presence of several GTPases of unknown function in this compartment (3Sickmann A. Reinders J. Wagner Y. Joppich C. Zahedi R. Meyer H.E. Schönfisch B. Perschil I. Chacinska A. Guiard B. Rehling P. Pfanner N. Meisinger C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13207-13212Crossref PubMed Scopus (728) Google Scholar). GTPases outside the mitochondrial matrix and attached to the inner membrane or outer membrane function in fission and fusion of mitochondria, and these are required for morphologic changes of the organelle associated with the metabolic demand of the cell (4Okamoto K. Shaw J.M. Annu. Rev. Genet. 2005; 39: 503-536Crossref PubMed Scopus (601) Google Scholar). These GTP-requiring processes in mitochondria are apparently conserved from yeast to humans. However, the compartmentalization of GTP synthesis and transport are quite different between mammalian and yeast mitochondria. In mammalian mitochondria, GTP is synthesized in the matrix by two different enzymes, a GTP-specific isoform of succinyl-CoA ligase and a nucleoside diphosphate kinase. Succinyl-CoA ligase converts succinyl-CoA to succinate with the generation of GTP in the tricarboxylic acid cycle (5Kibbey R.G. Pongratz R.L. Romanelli A.J. Wollheim C.B. Cline G.W. Shulman G. Cell Metab. 2007; 5: 253-264Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Nucleoside diphosphate kinases catalyze the transfer of a γ phosphate from nucleoside triphosphates (NTPs) to nucleoside diphosphates (6Lascu I. Gonin P. J. Bioenerg. Biomembr. 2000; 32: 237-246Crossref PubMed Scopus (160) Google Scholar). The high energy phosphate is usually supplied by ATP, and the enzyme regulates the crucial balance between ATP and GTP or other NTPs. The human Nm23/nucleoside diphosphate kinase family consists of eight related genes and widely expressed proteins termed Nm23-H1 to Nm23-H8. One of these isoforms, Nm23-H4, contains a mitochondrial targeting signal and is localized to the mitochondrial matrix (7Lacombe M.-L. Milon A. Munier A. Mehus J.G. Lambeth D.O. J. Bioenerg. Biomembr. 2000; 32: 247-258Crossref PubMed Scopus (298) Google Scholar, 8Gordon D.M. Lyver E.R. Lesuisse E. Dancis A. Pain D. Biochem. J. 2006; 400: 163-168Crossref PubMed Scopus (36) Google Scholar). Unlike in the case of mammalian mitochondria, GTP is not made in the matrix of Saccharomyces cerevisiae mitochondria, and therefore, yeast mitochondria are dependent on cytosolic GTP supply (2Vozza A. Blanco E. Palmieri L. Palmieri F. J. Biol. Chem. 2004; 279: 20850-20857Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). In yeast, succinyl-CoA ligase produces ATP instead of GTP (9Przybyla-Zawislak B. Gadde D.M. Ducharme K. McCammon M.T. Genetics. 1999; 152: 153-166Crossref PubMed Google Scholar). Furthermore, yeast nucleoside diphosphate kinase is encoded by a single nuclear gene YNK1 (10Fukuchi T. Nikawa J. Kimura N. Watanabe K. Gene (Amst.). 1993; 129: 141-146Crossref PubMed Scopus (59) Google Scholar), and the corresponding protein (Ynk1p) and activity are present in the cytosol and mitochondrial intermembrane space but not in the matrix (11Amutha B. Pain D. Biochem. J. 2003; 370: 805-815Crossref PubMed Google Scholar). How cytosolic GTP is transported across the inner membrane into the matrix of yeast mitochondria remained elusive until recently (see below). For decades, the mitochondrial inner membrane was considered to be impermeable to GTP, and this explains why the possible role of matrix GTP in various metabolic processes has not received much attention. Mitochondrial inner membrane contains a family of carrier proteins that allow exchange of various substrates across this membrane (12Robinson A.J. Kunji E.R.S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 2617-2622Crossref PubMed Scopus (203) Google Scholar). Palmieri and co-workers (2Vozza A. Blanco E. Palmieri L. Palmieri F. J. Biol. Chem. 2004; 279: 20850-20857Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) identified one such protein in yeast mitochondria as the GTP/GDP carrier and named the protein Ggc1p. They demonstrated that Ggc1p allows exchange of cytosolic GTP for matrix GDP across the inner membrane. Ggc1p-deficient (Δggc1) mitochondria lack this exchange activity, and the matrix contains greatly reduced levels of GTP and increased levels of GDP. Surprisingly, in an earlier study the same Δggc1 yeast mutant (previously called Δyhm1) was found to exhibit abnormal iron regulation such as increased surface ferric reductase and high-affinity ferrous transport activities and accumulation of excess iron (∼30-fold) in mitochondria (13Lesuisse E. Lyver E.R. Knight S.A.B. Dancis A. Biochem. J. 2004; 378: 599-607Crossref PubMed Google Scholar). How Ggc1p participates in mitochondrial iron homeostasis was not clear from these studies. We recently investigated whether the role of Ggc1p in mitochondrial iron metabolism is mediated by its effects on GTP/GDP levels in the mitochondrial matrix. We showed that in vivo targeting of the human nucleoside diphosphate kinase Nm23-H4 to the mitochondrial matrix of Δggc1 rescues high cellular iron uptake and mitochondrial iron accumulation. The Nm23-H4 enzyme exhibits activity capable of converting GDP to GTP using ATP as the phosphate donor in the mitochondrial matrix, and it is this activity that is responsible for rescuing the iron phenotypic defects of the mutant (8Gordon D.M. Lyver E.R. Lesuisse E. Dancis A. Pain D. Biochem. J. 2006; 400: 163-168Crossref PubMed Scopus (36) Google Scholar). These results suggest that GTP in the mitochondrial matrix plays an important role in organellar iron homeostasis. In most eukaryotic cells iron (Fe) primarily exists in complex with porphyrin (heme) or with sulfur (Fe-S clusters). Iron-sulfur clusters are critical cofactors of proteins that participate in numerous important cellular processes. Cluster biogenesis occurs in the mitochondrial matrix; it is a multistep process and requires multiple components (for review, see Ref. 14Lill R. Mühlenhoff U. Annu. Rev. Cell Dev. Biol. 2006; 22: 457-486Crossref PubMed Scopus (286) Google Scholar). Various yeast mutants that lack components involved in Fe-S cluster biogenesis exhibit abnormal cellular iron uptake and mitochondrial iron overload (15Li J. Kogan M. Knight S.A.B. Pain D. Dancis A. J. Biol. Chem. 1999; 274: 33025-33034Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 16Kispal G. Csere P. Prohl C. Lill R. EMBO J. 1999; 18: 3981-3989Crossref PubMed Scopus (595) Google Scholar, 17Knight S.A.B. Sepuri N.B.V. Pain D. Dancis A. J. Biol. Chem. 1998; 273: 18389-18393Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 18Kim R. Saxena S. Gordon D.M. Pain D. Dancis A. J. Biol. Chem. 2001; 276: 17524-17532Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 19Li J. Saxena S. Pain D. Dancis A. J. Biol. Chem. 2001; 276: 1503-1509Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 20Garland S.A. Hoff K. Vickery L.E. Culotta V.C. J. Mol. Biol. 1999; 294: 897-907Crossref PubMed Scopus (169) Google Scholar, 21Kaut A. Lange H. Diekert K. Kispal G. Lill R. J. Biol. Chem. 2000; 275: 15955-15961Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar) similar to the phenotypes of the Δggc1 mutant. Based on these similarities, we tested whether GTP in the mitochondrial matrix plays a role in Fe-S cluster biogenesis. Several Fe-S cluster-containing proteins, such as aconitase [4Fe-4S] and ferredoxin [2Fe-2S], reside in mitochondria. Aconitase reversibly catalyzes the conversion of citrate to isocitrate in the tricarboxylic acid cycle and requires a [4Fe-4S] cluster for this activity. Ferredoxin itself is known to participate in Fe-S cluster biogenesis (22Lange H. Kaut A. Kispal G. Lill R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1050-1055Crossref PubMed Scopus (251) Google Scholar). Here we show a requirement for nucleotides, particularly GTP, in Fe-S cluster biogenesis of aconitase (Aco1p) and ferredoxin (Yah1p). Yeast Strains—No significant difference was noted in cluster biogenesis in mitochondria isolated from D273-10B (ATCC 24657) and BY4741 (Invitrogen) wild-type strains. D273-10B mitochondria were used for experiments in Table 1 and Figs. 2, 4, 5, and 7; these experiments involved the use of only wild-type mitochondria. BY4741 was used as the wild-type strain for experiments in Figs. 1, 3, and 6; these experiments were performed to compare cluster biogenesis in wild-type and various mutant mitochondria. The Δggc1 mutant strain in the BY4741 background was generated by plasmid shuffling. A similar strain was constructed carrying the high copy number plasmid pRS425 with a strong glyceraldehyde phosphate dehydrogenase promoter for expression of the human nucleoside diphosphate kinase Nm23-H4 targeted to the mitochondrial matrix (8Gordon D.M. Lyver E.R. Lesuisse E. Dancis A. Pain D. Biochem. J. 2006; 400: 163-168Crossref PubMed Scopus (36) Google Scholar). A congenic BY4741 haploid yeast strain that expresses Aco1p with a C-terminal TAP (tandem affinity purification) tag from the ACO1 promoter in the genome was purchased from Open Biosystems. An aco1 mutant strain, which expresses aconitase with a mutation in a cysteine residue involved in iron binding (C448S), was a generous gift of Dr. Ronald A. Butow (23Chen X.J. Wang X. Kaufman B.A. Butow R.A. Science. 2005; 307: 714-717Crossref PubMed Scopus (9) Google Scholar). Expression of the cysteine desulfurase Nfs1p from the regulated galactose-inducible GAL1 promoter has been described elsewhere (15Li J. Kogan M. Knight S.A.B. Pain D. Dancis A. J. Biol. Chem. 1999; 274: 33025-33034Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). For the experiment in Fig. 8, the scaffold protein Isu1p with a C-terminal His6 tag was constitutively overexpressed in a wild-type strain (BY4741) from the plasmid pRS426 with glyceraldehyde phosphate dehydrogenase promoter essentially as described (24Mühlenhoff U. Gerber J. Richhardt N. Lill R. EMBO J. 2003; 22: 4815-4825Crossref PubMed Scopus (343) Google Scholar).TABLE 1Nucleotide-dependent reconstitution of aconitase activityMitochondriaNucleotidesTotal aconitase activityμmol cis-aconitate formed/mg protein/minWild type-1.21 ± 0.01Wild type+1.32 ± 0.02aco1 (C448S)+0 Open table in a new tab FIGURE 4Iron-dependent insertion of Fe-35S clusters into endogenous apoaconitase. As indicated, mitochondria (200 μg of proteins) were treated with 5 mm o-phenanthroline (o-Phe) or 5 mm EDTA for 15 min on ice, diluted with HSB buffer, and centrifuged. Mitochondrial pellets were resuspended in HSB buffer and incubated at 30 °C for 20 min with [35S]cysteine (10 μCi; 0.19 μm), ATP (4 mm), and GTP (1 mm) in the absence or presence of ferrous ascorbate (Fe2+; 10 μm). Samples were analyzed by native PAGE followed by autoradiography.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 5Hydrolysis of both GTP and ATP is required for cluster biogenesis of endogenous apoaconitase. Mitochondria (100 μg of proteins) were preincubated at 25 °C for 5 min. After the addition of different concentrations of GTPγS (A) or AMP-PNP (B), mitochondria were incubated with [35S]cysteine (10μCi; 0.19μm) and ATP at 30 °C for 15 min. Samples were analyzed by native PAGE followed by autoradiography.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 7Requirement of GTP hydrolysis for the cluster biogenesis of imported ferredoxin. Mitochondria (200 μg of proteins) with (lanes 2 and 3) or without (lane 1) imported apoferredoxin (apoYah1p) were incubated with ATP (1 mm) and [35S]cysteine (10 μCi; 0.19 μm) in the absence or presence of GTPγS (1 mm) at 30 °C for 15 min. Samples were analyzed by native PAGE followed by autoradiography.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 1The Fe-35S-labeled endogenous protein in isolated wild-type mitochondria is aconitase. Mitochondria were incubated with [35S]cysteine (10 μCi; 0.19 μm), ATP (4 mm), and GTP (1 mm) at 30 °C for 15 min. Samples were diluted with HSB buffer, and mitochondria were reisolated by centrifugation. Mitochondrial membranes were ruptured, and soluble proteins in duplicate were separated by native PAGE. The gel was divided into two halves. One was dried and exposed to film for autoradiography (A). The other half was processed for the measurement of in-gel aconitase activity (B). In a separate experiment, mitochondrial proteins were directly analyzed by SDS-PAGE followed by immunoblotting using anti-Aco1p antibodies (C). Samples used were wild-type mitochondria (WT, 100 μg of proteins), mitochondria that contained an enzymatically inactive form of Aco1p but no wild-type and active aconitase (aco1, 100 μg of proteins), and mitochondria that contained aconitase with a C-terminal TAP tag but no wild-type and untagged Aco1p (Aco1p-TAP, 200 μg of proteins).View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3Cluster biogenesis of aconitase both in vivo and in isolated mitochondria requires cysteine desulfurase (Nfs1p) activity. The NFS1 open reading frame was placed under the control of GAL1 promoter (15Li J. Kogan M. Knight S.A.B. Pain D. Dancis A. J. Biol. Chem. 1999; 274: 33025-33034Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). The resulting strain was grown to mid-logarithmic phase under inducing conditions in YPR galactose (yeast extract (1%), peptone (2%), raffinose (2%), and galactose (0.2%)). Cells were harvested, resuspended in YPR containing no galactose, and allowed to grow for 22 h. Under these non-inducing conditions, Nfs1p was no longer synthesized from the GAL1 promoter. Mitochondria were isolated from these Nfs1p-depleted (nfs1) cells. Mitochondrial proteins (100 μg) were analyzed by SDS-PAGE followed by immunoblotting using anti-Nfs1p (A), anti-Aco1p (C), and anti-Tom40p (D) antibodies. B, mitochondrial proteins (100 μg) were separated by native PAGE, and aconitase activity was measured by an in-gel assay. E, mitochondria (100 μg of proteins) were incubated at 30 °C for 15 min with [35S]cysteine (10 μCi; 0.19 μm), ATP (4 mm), and GTP (1 mm), and insertion of Fe-35S clusters into endogenous apoaconitase was examined by native PAGE followed by autoradiography. Wild-type (WT) mitochondria served as control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6In vivo targeting of Nm23-H4 to the matrix of Δggc1 mitochondria restores cluster biogenesis of aconitase. A, cells were treated with Zymolyase to generate spheroplasts. After lysis of spheroplasts with Triton X-100, proteins were separated by native PAGE, and aconitase activity was evaluated by an in-gel assay (top panel). Each lane corresponds to equal number of starting cells; volume (ml) of cell suspension × A600 was kept constant at 0.25 for each lane. Identical samples were also analyzed by SDS-PAGE followed by immunoblotting using anti-Aco1p antibodies (bottom panel). WT, wild type; Δg, Δggc1; Δg + Nm, Δggc1 mutant with the human Nm23-H4 targeted in vivo to the matrix. B and C, as indicated, mitochondria (200 μg of proteins) were supplemented with ATP (4 mm) plus GTP (1 mm) (panel B) or ATP (4 mm) alone (panel C). After the addition of [35S]cysteine (10 μCi; 0.19 μm), samples were incubated at 30 °C for 15 min, and radiolabeling of endogenous aconitase was analyzed by native PAGE followed by autoradiography. D, a schematic of matrix GTP-dependent cluster biogenesis in mitochondria. IMS, intermembrane space; IM, inner membrane; AAC, ADP/ATP carriers.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 8Efficient assembly of cluster intermediates on Isu1p requires GTP hydrolysis. Nucleotide-depleted wild-type mitochondria (500 μg of proteins) with or without overexpressed Isu1p-His6 were incubated with [35S]cysteine (20 μCi; 0.19 μm) and ferrous ascorbate (10 μm) in the absence or presence of GTPγS (2 mm). Mitochondria were reisolated, solubilized with buffer containing Triton X-100 (0.2%) and imidazole (10 mm), and incubated with Ni-NTA agarose. Proteins bound to Ni-NTA-agarose were eluted with imidazole (0.4 m), and fractions corresponding to 250 μg of starting mitochondrial proteins were analyzed. A, radioactive counts associated with samples corresponding to mitochondria lacking Isu1p-His6 were considered as nonspecific background counts and were deducted from those associated with samples corresponding to mitochondria containing Isu1p-His6. Specific counts thus obtained are shown. B, imidazole-eluted fractions that contained Isu1p-His6 were analyzed by immunoblotting using anti-Isu1p antibodies.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Insertion of Radiolabeled Clusters Into Apoproteins—Mitochondria were isolated from various strains and purified on Percoll gradients as described (25Murakami H. Pain D. Blobel G. J. Cell Biol. 1988; 107: 2051-2057Crossref PubMed Scopus (299) Google Scholar). Experiments were performed with or without prior depletion of endogenous nucleotides in isolated mitochondria. To deplete endogenous nucleotides, mitochondria were preincubated at 25 °C for 5–10 min (26Stuart R.A. Gruhler A. van der Klei I. Guiard B. Koll H. Neupert W. Eur. J. Biochem. 1994; 220: 9-18Crossref PubMed Scopus (92) Google Scholar). Isolated mitochondria contain a stored pool of iron that can be used for the synthesis of Fe-S clusters. Unless otherwise indicated, experiments were performed with no added iron salt. The procedures for iron depletion and supplementation are described in Fig. 4 legend. Insertion of newly formed and radiolabeled clusters into an endogenous apoprotein in isolated intact mitochondria was examined as follows. The assay mixture (50 μl) contained mitochondria (100–200 μg of proteins) in HSB buffer (20 mm Hepes/KOH, pH 7.5, 0.6 m sorbitol, 0.1 mg/ml bovine serum albumin, 10 mm Mg(OAc)2, and 40 mm KOAc) containing 5 mm NADH and 1 mm dithiothreitol. Nucleotides (GTP and/or ATP) and poorly hydrolyzable nucleotide analogs (GTPγS 4The abbreviations used are: GTPγSguanosine 5′-3-O-(thio)triphosphateAMP-PNP5′-adenylyl imidodiphosphateNi-NTAnickel-nitrilotriacetic acid. or AMP-PNP) were included at various concentrations as indicated in the legends for Figs. 5, 7, and 8. After the addition of [35S]cysteine (10 μCi, 1075 Ci/mmol), samples were incubated at 30 °C for 15–20 min. Reaction mixtures were diluted 20-fold with ice-cold HSB buffer, and mitochondria were reisolated by centrifugation at 15,000 × g for 5 min at 4 °C. The pellet was resuspended in 35 μl of 50 mm Tris/HCl, pH 8.0, containing 0.5 mm phenylmethylsulfonyl fluoride. Mitochondrial membranes were ruptured by freezing the samples at –80 °C followed by thawing and bath sonication for 30 s at 4 °C. This process was repeated three times. Samples were centrifuged at 15,000 × g for 15 min at 4 °C, and supernatant fractions containing soluble proteins were subjected to native PAGE. The gel was fixed with 20% methanol in 50 mm Tris/HCl, pH 8.0, for 1 h at 4°C, dried, and exposed to film for autoradiography. Radiolabeled protein bands were quantitated using the software NIH Image. guanosine 5′-3-O-(thio)triphosphate 5′-adenylyl imidodiphosphate nickel-nitrilotriacetic acid. Insertion of newly formed and radiolabeled clusters into imported apoferredoxin was examined as follows. The precursor form of ferredoxin with a C-terminal His6 tag was expressed in bacteria and found to be sequestered in inclusion bodies. The protein was solubilized with 8 m urea in 50 mm Tris/HCl, pH 8.0, and centrifuged at 250,000 × g for 20 min at 20 °C to remove insoluble material. The supernatant fraction analyzed by SDS-PAGE followed by Coomassie Blue staining showed a single major protein band and was ∼95% pure (27Zhang Y. Lyver E.R. Knight S.A.B. Pain D. Lesuisse E. Dancis A. J. Biol. Chem. 2006; 281: 22493-22502Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Import assay was performed in HSB buffer containing 5 mm NADH, 1 mm dithiothreitol, and 1 mm ATP (27Zhang Y. Lyver E.R. Knight S.A.B. Pain D. Lesuisse E. Dancis A. J. Biol. Chem. 2006; 281: 22493-22502Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 28Sepuri N.B.V. Gordon D.M. Pain D. J. Biol. Chem. 1998; 273: 20941-20950Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 29Sepuri N.B.V. Schülke N. Pain D. J. Biol. Chem. 1998; 273: 1420-1424Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Import was initiated by adding urea-denatured ferredoxin precursor (400 ng) to mitochondria (200 μg of proteins) in the assay buffer (total volume 50 μl). The final urea concentration was 0.16 m. After incubation at 30 °C for 10 min, samples were diluted 20-fold with HSB buffer, and mitochondria were reisolated by centrifugation at 15,000 × g for 2 min at 4 °C. Mitochondrial pellet with ferredoxin imported into the matrix was resuspended in HSB buffer (50 μl) containing 5 mm NADH, 1 mm dithiothreitol, 10 μCi of [35S]cysteine (1075 Ci/mmol), and 1 mm ATP with or without 1 mm GTPγS. Reaction mixtures were incubated at 30 °C for 15 min and diluted with HSB buffer, mitochondria were reisolated, membranes were ruptured, and soluble proteins were analyzed by native PAGE followed by autoradiography as described above. Assembly of Cluster Intermediates on the Scaffold Protein Isu1p—Mitochondria were preincubated at 25 °C for 10 min to promote depletion of endogenous nucleotides just before use. The reaction mixture (100 μl) contained mitochondria (500 μg of proteins with or without overexpressed Isu1p-His6) in HSB buffer supplemented with [35S]cysteine (20 μCi, 1075 Ci/mmol) and ferrous ascorbate (10 μm). Reaction mixtures were incubated at 30 °C for 15 min in the absence or presence of GTPγS (2 mm). Samples were diluted 12-fold with HSB buffer, and mitochondria were reisolated by centrifugation at 15,000 × g for 5 min at 4 °C. Mitochondrial pellets were solubilized with 200 μl of buffer A (50 mm Tris/HCl, pH 7.5, 150 mm NaCl, 0.2% Triton X-100, and 1 mm phenylmethylsulfonyl fluoride) containing 10 mm imidazole and centrifuged at 15,000 × g for 15 min at 4 °C. Supernatant fractions were incubated with Ni-NTA-agarose (30 μl of settled beads) for 1 h at 4°C. After washing of the agarose beads with buffer A containing 10 mm imidazole, bound proteins were eluted with 100 μl of 0.4 m imidazole in buffer A. Aliquots of eluted fractions (50 μl) were analyzed by scintillation counting of radioactivity and also by SDS-PAGE followed by immunoblotting using anti-Isu1p antibodies. Aconitase Activity Measurement—Aconitase activity was determined using the whole cell extract or isolated mitochondria. For the whole cell extract, cells were treated with zymolyase to generate spheroplasts as described in the protocol for the isolation of mitochondria (25Murakami H. Pain D. Blobel G. J. Cell Biol. 1988; 107: 2051-2057Crossref PubMed Scopus (299) Google Scholar). Spheroplasts or isolated mitochondria were incubated with 50 mm Tris/HCl, pH 8.0, containing 50 mm NaCl, 1% Triton X-100, 10% glycerol, 2 mm sodium citrate, and 200 units/ml catalase in a total volume of 50 μl for 30 min on ice. Samples were centrifuged at 15,000 × g for 10 min at 4 °C. Supernatant fractions were subjected to native PAGE, and aconitase activity was measured by an in-gel assay as described (30Tong W.-H. Rouault T.A. Cell Metab. 2006; 3: 199-210Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). Bands displaying aconitase activity appeared blue and were quantitated using the software NIH Image. Data from the scanned gels are presented in black and white (Figs. 1B, 3B, and 6A). The nucleotide-dependent insertion of Fe-S clusters into endogenous apoAco1p was correlated with the activity of holo-Aco1p thus formed as follows. Briefly, mitochondria (500 μg of proteins) in HSB buffer containing 20 μm unlabeled cysteine, 10 μm ferrous ascorbate, 5 mm NADH, and 1 mm dithiothreitol were incubated at 30 °C for 15 min in the absence or presence of added nucleotides (1 mm GTP and 4 mm ATP) in a total volume of 250 μl. Reaction mixtures were diluted 5-fold with ice-cold 50 mm Tris/HCl, pH 7.5, containing 0.6 m sorbitol, and mitochondria were reisolated by centrifugation at 15,000 × g for 5 min at 4 °C. Mitochondrial pellets were treated with 125 μl of solubilization buffer (50 mm Tris/HCl, pH 7.5, containing 150 mm NaCl, 10% glycerol, 1 mm sodium citrate, 0.5% glucose, 1.5% n-octyl-β-d-glucopyranoside, and 1 mm phenylmethylsulfonyl fluoride) and kept on ice. Aliquots (10–20 μl) corresponding to 40–80 μg of proteins were assayed in triplicate for aconitase activity. The assay follows conversion of isocitrate to cis-aconitate over time measured by absorbance change at 240 nm (27Zhang Y. Lyver E.R. Knight S.A.B. Pain D. Lesuisse E. Dancis A. J. Biol. Chem. 2006; 281: 22493-22502Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 31Kennedy M.C. Emptage M.H. Dreyer J.L. Beinert H. J. Biol. Chem. 1983; 258: 11098-11105Abstract Full Text PDF PubMed Google Scholar). Insertion of Newly Formed Fe-35S Clusters into Endogenous Apoaconitase in Isolated Intact Mitochondria—We investigated Fe-S cluster biogenesis in isolated mitochondria using assays that
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