SUPPORTING ONLINE MATERIAL MATERIALS AND

SUPPORTING ONLINE MATERIAL
MATERIALS AND METHODS
Reagents and Purified Proteins -- All chemicals were purchased from Sigma. Buffers and
solutions were made with milli-Q-deionized water. Stock solutions of ferrous ammonium sulfate
(10 mM) were freshly prepared in water purged with N2. Human frataxin in the assembled form
were obtained from the laboratory of G. Isaya (Mayo Clinic and Foundation, Rochester,
Minnesota) and were prepared as previously described (S1). Purified bovine heart mitochondrial
[3Fe-4S]+1 aconitase was from the laboratory of M.C. Kennedy (Gannon University, Erie
Pennsylvania) and was purified as previously described (S2). Bovine serum albumin (Sigma)
was desalted into the appropriate buffer using a PD-10 column (Amersham Biosciences). The
mitochondrial iron content was measured by inductively coupled plasma mass spectrometry
(ICP-MS) at the Mayo Metals Laboratory.
Isolation of Subsarcolemmal Mitochondria from Rat Heart -- Male Sprague-Dawley rats
(Harlan, 250-300 g) were anesthetized with sodium pentobarbital and decapitated. Hearts were
removed and immediately rinsed in ice cold homogenization buffer (210 mM mannitol, 70 mM
sucrose, 1.0 mM EDTA, and 5.0 mM MOPS at pH 7.4). Hearts (0.9-1.1 g) were then minced
and homogenized in 20 ml of homogenization buffer/g of tissue with a Polytron homogenizer
(low setting, 2 s). The homogenate was centrifuged at 500 x g for 5 min and the supernatant
filtered through cheese cloth. The mitochondrial pellet was obtained upon centrifugation of the
supernatant at 10,000 x g for 10 min. After two rinses, the mitochondria were resuspended in
homogenization buffer prepared without EDTA to a final concentration of 40.0 mg/ml. Protein
Bulteau, A.-L., et al.
determinations were made using the BCA method (Pierce), with bovine serum albumin as a
standard.
Yeast Saccharomyces Cerevisiae Strains and Growth Conditions --
Yeast lacking the yeast
homologue of frataxin, yfh1∆ (as shown by PCR and Western blot analysis), but complemented
by human frataxin expressed from either a low copy (YC-FRDA) or a high copy (pG3-FRDA)
plasmid or complemented with yeast frataxin expressed from a low copy (YC-YFH1) plasmid
were utilized in this study (S3, S4). Cells were grown at 30°C on YPEG (Yeast extract 1%,
Peptone 2%, Ethanol 2 %, Glycerol 3%).
Isolation of Mitochondria from Yeast Saccharomyces cerevisiae -- Cells were disrupted by
using glass beads in the presence of protease inhibitors.
Mitochondria were prepared as
described (S5, S6).
Evaluation of Mitochondrial Respiration
--
The rate of mitochondrial oxygen consumption
was monitored using a Clark-style oxygen electrode (Instech). Mitochondria diluted to 0.25
mg/ml in 125 mM KCl and 5.0 mM KH2PO4 at pH 7.25 were placed in a sealed chamber and
respiration was initiated by the addition of 10 mM pyruvate and 2.5 mM malate. Respiration
was allowed to proceed for 1.0 min and then state 3 respiration was initiated upon addition of
100 µM ADP. State 4 respiration was determined following conversion of ADP to ATP.
Incubation of Intact Mitochondria with H2O2 -- Mitochondria were diluted to 0.25 mg/ml in
125 mM KCl and 5.0 mM KH2PO4 at pH 7.25 in the absence or presence of exogenously added
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Bulteau, A.-L., et al.
citrate (2.0 mM). Mitochondria were then incubated for 2.0 min followed by addition of 100 µM
H2O2. All incubations were performed at room temperature.
Western Blot Analysis -- Prior to analysis, mitochondrial extracts were incubated in Laemmli
sample buffer for 5.0 min at 100°C. Mitochondrial proteins (40 µg/lane) were resolved on a 420% SDS-PAGE gel and electrotransferred onto a Hybond nitrocellulose membrane (Amersham
Pharmacia Biotech).
The following primary rabbit antibodies were employed: anti-yeast
aconitase [provided by R. Lill (Institute fur Zytobiologie, Philipps-Universitat, Marburg,
Germany)], polyclonal antibody specific to human aconitase, and polyclonal antibodies against
the yeast and human frataxin protein prepared by G. Isaya (Mayo Clinic and Foundation,
Rochester, Minnesota).
Primary antibody binding was visualized utilizing peroxidase-
conjugated secondary antibody and an appropriate chemiluminescence substrate (Supersignal
West from Pierce).
Immunopurification of Aconitase -- After exposure of rat or yeast mitochondria (5.0 mg/ml) to
100 µM H2O2 in the presence or absence of citrate (2.0 mM), mitochondria were diluted to 1.0
mg/ml in 25 mM KH2PO4, pH 7.25, containing 0.05 % Triton X-100. Mitochondrial extracts
(500 µg of protein) were then incubated with 10 µl of anti-aconitase yeast or rat polyclonal
antibody at 4°C for 1 h. The mixture was then treated with 50 µl of protein G-Sepharose and
incubated for 16 h (4°C). The beads were then pelleted by centrifugation at 1,000 x g for 5 min,
washed three times with phosphate-buffered saline (PBS), pH 7.6 containing 1.0% NP-40, and
resuspended in gel-loading buffer containing SDS without added reductant prior to gel
electrophoresis/Western blot analysis.
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Bulteau, A.-L., et al.
Measurement of Aconitase Activity -- Mitochondria were diluted to 0.05 mg/ml in 25 mM
KH2PO4, pH 7.25, containing 0.05 % Triton X-100. Pure protein was diluted to 0.1 µM in
KH2PO4, pH 7.25. Aconitase activity was assayed as the rate of NADP+ reduction (340 nm,
6200 M-1 cm-1) by isocitrate dehydrogenase upon addition of 1.0 mM sodium citrate, 0.6 mM
MnCl2 (a cofactor of isocitrate dehydrogenase), 0.2 mM NADP+, and 1.0 U/ml isocitrate
dehydrogenase to solubilized mitochondria (0.05 mg/ml mitochondrial protein).
Isocitrate
dehydrogenase (Sigma) was exchanged into a buffer composed of 25 mM KH2PO4, pH 7.25 by
gel filtration (PD-10 column, Amersham-Pharmacia Biotech) prior to utilization in aconitase
assays.
Measurement of H2O2 Concentrations -- Mitochondria were diluted to 0.25 mg/ml in 125 mM
KCl and 5.0 mM KH2PO4 at pH 7.25, in the presence or absence of 2.0 mM sodium citrate,
followed by addition of H2O2 (100 µM). At appropriate time points, a 20 µl aliquot was
transferred to an assay mixture containing horseradish peroxidase (1.0 U/ml) and 500 µM
homovanillic acid (3-metoxy-4-hydroxyphenylacetic) in 25 mM KH2PO4, pH 7.25 (total volume
= 2.0 ml). The fluorescence of the sample was measured using a spectrofluorometer (Shimadzu)
at excitation/emission wavelengths of 320/425 nm. A standard curve was generated using
known concentrations of H2O2.
Kinetics of Aconitase Iron Sulfur Reconstitution -- Fe(II) (30 µM) was incubated in the absence
or presence of 3.0 µM purified human assembled frataxin or BSA in 10 mM HEPES-KOH, pH
7.3 at 30°C for 10 min. Aconitase reactivation was initiated by addition of 1.0 mM DTT and 3.0
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Bulteau, A.-L., et al.
µM purified aconitase in the [3Fe-4S]1+ form.
These experiments were performed in the
presence or absence of 500 µM sodium citrate. At indicated times, aliquots were removed from
the reaction mixture for measurement of aconitase activity. Total recoverable aconitase activity
was determined following reactivation of purified [3Fe-4S]1+ aconitase under anaerobic
conditions with 10 mM DTT and 0.5 mM FeSO4 in 100 mM Tris, pH 7.4 for 30 min at 0°C.
Electron Paramagnetic Resonance (EPR) Spectroscopy -- Highly purified aconitase in the
[3Fe-4S]1+ form was exposed to various experimental conditions.
At indicated times, the
incubation mixture was placed in quartz EPR tubes and frozen by immersion in liquid nitrogen.
EPR spectra were recorded by a Bruker ESP300 spectrometer operating at 9.45 GHz with 10
gauss field modulation at 100 kHz. Measurements were carried out at 10 K by use of an Oxford
liquid He flow cryostat. The microwave frequency was monitored by a frequency counter (HP5350) and the magnetic field strength was determined by an NMR gaussmeter (Brucker ER035M). The [3Fe-4S]1+ cluster of aconitase is characterized by a peak at g = 2.02 with a
shoulder at g = 2.014 (10Û.(DFKVSHFWUXPUHSUHVHQWVDQDYHUDJHRIVFDQV
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Bulteau, A.-L., et al.
REFERENCES
S1.
P. Cavadini, H. A. O’Neill, O. Benada, G. Isaya, Hum. Mol. Genet. 11, 217-27 (2002).
S2.
M. C. Kennedy, M. H. Emptage, J. L. Dreyer, H. Beinert, J. Biol. Chem. 258, 11098-105.
(1983).
S3.
P. Cavadini, C. Gellera, P. I. Patel, G. Isaya, Hum. Mol. Genet. 9, 2523-30 (2000).
S4.
P. Cavadini, J. Adamec, F. Taroni, O. Gakh, G. Isaya, J. Biol. Chem. 275, 41469-75
(2000).
S5.
K. Diekert, A. I. de Kroon, G. Kispal, R. Lill, Methods Cell. Biol. 65, 37-51 (2001).
S6.
G. Karthikeyan et al., Hum. Mol. Genet. 12, 3331-42 (2003).
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