JBC Papers in Press. Published on February 26, 2014 as Manuscript M113.

526665
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M113.526665
Role of ISCU in development of mitochondrial myopathy

Multiple Cellular Defects Associated with a Novel G50E ISCU Mutation leads to Development of
Mitochondrial Myopathy*

Prasenjit Prasad Saha1, Praveen Kumar S.K.1, Shubhi Srivastava1, Devanjan Sinha1, Gautam
Pareek1 and Patrick D Silva1#

1
Department of Biochemistry, Indian Institute of Science, Bangalore 560012, Karnataka, India.

The third and fourth authors should be regarded as joint Third Authors.

*Running Title: Role of ISCU in development of mitochondrial myopathy

#
To whom correspondence should be addressed: Dr. Patrick D Silva: Department of Biochemistry,
Indian Institute of Science, Biological Sciences Building, Bangalore 560012, Karnataka, India. Tel.: 91-
080-22932821; Fax: 91-080-23600814; E-mail: patrick@biochem.iisc.ernet.in.

Downloaded from http://www.jbc.org/ by guest on November 29, 2017

Keywords: Ironsulfur protein; Mitochondria; Electron transfer; Muscle; Proteinprotein interactions;
Reactive oxygen species (ROS)

Background: Muscle specific deficiency of iron- with severe myopathy have been identified to
sulfur (Fe-S) cluster scaffold protein ISCU, leads carry (c.149G>A) missense mutation converting
to myopathy. glycine 50 residue to glutamate. However, the
physiological defects and molecular mechanism
Results: Cells carrying myopathy-associated associated with G50E mutation have not been
G50E ISCU mutation, demonstrate impaired Fe-S elucidated. In this report, we uncover
cluster biogenesis and mitochondrial dysfunction. mechanistic insights concerning how the G50E
ISCU mutation in humans leads to development
Conclusion: Reduced mitochondrial respiration as
of severe ISCU myopathy, using human cell line
a result of diminished Fe-S cluster synthesis
and yeast as the model systems. The
results in muscle weakness in myopathy patients.
biochemical results highlight that, G50E
Significance: The molecular mechanism behind mutation results in compromised interaction
disease progression should provide invaluable with the sulfur donor NFS1 and J-protein
information to combat ISCU myopathy. HSCB protein, thus impairing the rate of Fe-S
cluster synthesis. As a result, electron transport
chain (ETC) complexes show significant
reduction in their redox properties leading to
Iron-sulfur (Fe-S) clusters are versatile loss of cellular respiration. Furthermore, the
cofactors involved in regulating multiple G50E mutant mitochondria display
physiological activities, including energy enhancement in iron level and reactive oxygen
generation through cellular respiration. species (ROS), thereby causing oxidative stress
Initially, the Fe-S clusters are assembled on a leading to impairment in the mitochondrial
conserved scaffold protein ISCU in functions. Thus, our findings provide
coordination with iron and sulfur donor compelling evidence that respiration defect due
proteins in human mitochondria. Loss of ISCU to impaired biogenesis of Fe-S clusters in
function leads to myopathy, characterized by myopathy patients, leads to manifestation of
muscle wasting and cardiac hypertrophy. In complex clinical symptoms.
addition to homozygous ISCU mutation
(g.7044G>C), compound heterozygous patients

Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
Role of ISCU in development of mitochondrial myopathy

INTRODUCTION ISU2, double deletion mutant is inviable thus

signifying its central importance in the Fe-S
Iron-sulfur (Fe-S) clusters are indispensable cluster biogenesis (12). The overall biogenesis
and ubiquitous cofactors that are involved in a process can be broadly categorized into two
variety of regulatory processes, including catalysis critical events: a) the de novo assembly of a Fe-S
and electron carrier activity (1). Although, the Fe- cluster on a scaffold protein and b) the transfer of
S clusters are relatively simple inorganic the Fe-S cluster from the scaffold to target
cofactors, their synthesis, assembly and successive apoproteins (13). A cysteine desulfurase, Nfs1
incorporation into apoproteins are highly intricate assists in the sulfur transfer process to the scaffold
process in living cells (2-3). Fe-S clusters are protein (14). This reaction is aided by direct
usually integrated into proteins through interaction between Nfs1 and the scaffold protein
coordination of the iron atoms by cysteine or (11,15). Upon transfer of iron and sulfur to the
histidine residues, though in more complex Fe-S scaffold; the Fe-S cluster is formed by an
clusters alternative ligands like Asp, Arg, Ser and unknown mechanism (13,16). The iron-binding
CO, CN functional groups have been reported (4). protein frataxin (Yfh1 in yeast and CyaY in
Coordination of Fe-S clusters to electron transport bacteria) is believed to function as an iron donor
chain complexes is indispensable for respiratory (17). The terminal transfer process is assisted by a

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function within the cell. For example, complex I of dedicated chaperone system comprising the
bacteria contain nine Fe-S clusters whereas mtHsp70 (Ssq1 in yeast) and the DnaJ-like
eukaryotic complex I harbor eight Fe-S clusters, cochaperone, HSCB (Jac1 in yeast) (18-19). The
which are anchored to domains exposed to the interaction of J-protein cochaperone such as Jac1
cytosol or mitochondrial matrix, respectively (5- (HSCB in human) with Fe-S scaffold Isu1 is
6). On the other hand; mammalian complex II conserved in evolution and indispensable in vivo
possesses three distinct Fe-S clusters of the [2Fe- (15,20-21).
2S], [3Fe-4S] and [4Fe-4S] type (7). Besides their
importance in electron transfer, Fe-S proteins also Owing to Fe-S proteins play a critical role in a
play a pivotal role in enzyme-substrate reactions. wide-range of cellular activities, a mutation in
Eukaryotic Fe-S cluster containing enzymes such different components of the synthesis machinery
as succinate dehydrogenase and aconitase play a disrupts the process of Fe-S cluster biogenesis and
critical role in TCA cycle metabolism (8-9). is thus associated with multiple pathological
Therefore, the biogenesis of these functionally conditions in humans. For instance, one mutation
important Fe-S clusters in mitochondria is an identified in human mitochondrial iron-sulfur
indispensable process for mitochondrial function assembly enzyme, ISCU is known to cause severe
as well as cell survival. myopathy (ISCU myopathy; OMIM *611911).
ISCU Myopathy is a recessively inherited disorder
Mitochondria are the major cellular characterized by lifelong exercise intolerance
compartment for Fe-S cluster biogenesis in where minor exertion causes pain of active
eukaryotes, including the mammalian system. The muscles, shortness of breath, fatigue and
central part of Fe-S protein biogenesis in human tachycardia (22-23). The disease is non-
mitochondria is the de novo synthesis of Fe-S progressive, but in certain cases metabolic
cluster on a highly conserved scaffold protein, acidosis, rhabdomyolysis and myoglobinuria have
ISCU, before its transfer to apoproteins (10). also been reported (24-25). Myopathy as a result
Mammalian ISCU is a nuclear encoded protein, of ISCU deficiency was found to have high
predominantly localized in the mitochondrial incidence rates in individuals of northern Europe
matrix compartment and comprises of 167 amino ancestry with a carrier rate of 1:188 in Northern
acids with an N-terminal targeting signal. Swedish population (23). Most affected
However, presence of cytosolic ISCU has also individuals are homozygous for a mutation in
been reported in humans (11). In Saccharomyces intron 4 (g.7044G>C) of ISCU that results in
cerevisiae, there are two orthologs of human synthesis of aberrantly spliced ISCU mRNA,
ISCU, namely Isu1 and Isu2, which are localized successively causing accumulation of truncated
in the matrix compartment (12). In yeast, ISU1 and non-functional ISCU protein (22,26-27). Recently,

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Role of ISCU in development of mitochondrial myopathy

a progressive myopathy associated with early ~90% confluency. The cells were trypsinized,
onset of severe muscle weakness, extreme exercise washed with ice-cold 1X PBS (phosphate-buffered
intolerance and cardiomyopathy have been saline, pH 7.4) and utilized for different
reported in some patients. Interestingly, these experiments and mitochondria isolation.
patients were compound heterozygous for the
common intronic splice mutation (g.7044G>C) on Yeast strains, genetic analysis, plasmid
one allele leading to truncated protein and a novel construction and mutagenesiss For genetic
(c.149G>A) missense mutation in exon 3 on the analysis in yeast, full-length human WT ISCU and
other allele. The missense mutation in exon 3, yeast WT ISU1 were amplified from HeLa cell
changes a completely conserved glycine residue to cDNA library (Stratagene) and W303 yeast
a glutamate at the 50th position (G50E) in the genomic DNA, respectively. ISCU and ISU1 with
amino acid sequence (28). The transmission of the a C-terminal FLAG-tag were cloned in pRS414
G50E mutation alone was found to be recessive, yeast expression vector under TEF or GPD
since carrier population did not show significant promoter containing a Trp marker for selection.
symptoms of the disease. However, the exact The isu1/isu2 double deleted yeast haploid strain,
molecular mechanisms of disease development as W303 (trp11 ura31 leu23, 112 his311, 15
a result of G50E mutation in ISCU in conjunction ade21 can1100 GAL2+ met2-1 lys2-2

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with g.7044G>C allele in compound heterozygous isu2::HIS3 isu1::LEU2), containing the plasmid
patients have not been elucidated. pRS316-ISU1 for the maintenance of viability. For
in vivo phenotype analysis, the haploid isu1/isu2
Owing to the critical function played by ISCU strain was transformed with pRS414-ISCU,
scaffold protein in Fe-S cluster biogenesis process pRS414-G50E ISCU, pRS414-ISU1 and pRS414-
in humans, the G50E mutant is expected to isu1G50E. The transformants were selected on
contribute significantly towards ISCU myopathy. tryptophan omission plates at 30 C. The
In this report, we delineate the impact of G50E transformants (Trp+) were then subjected to spot
mutation in mitochondrial function by utilizing the test analysis using serial dilutions of the cells on
HeLa cell line and yeast as a model system. Our different media like Trp-, Trp- containing H2O2
findings highlight that G50E mutation leads to and medium containing 5-FOA (US Biological) to
severe growth defects, compromised Fe-S cluster eliminate the WT ISU1 containing the plasmid
containing enzyme activity, sensitivity to oxidative pRS316.
stress, increased cellular ROS, elevated iron level
and reduced interaction of scaffold protein with its For purification of ISCU and Isu1 using a
interacting partners thus contributing significantly bacterial expression system, the ORFs without N-
toward mitochondrial myopathy. Moreover, at the terminal signal sequences of ISCU (35-167 aa) and
protein level, the G50E mutation was found to ISU1 (37-165 aa) were cloned in between into the
form a higher-order oligomeric structure that BamHI-SalI restriction sites of pRSFDuet-1
probably reduces the functionality of the protein. vector, carrying an N-terminal vector backbone
his6-tag. The GST fusion constructs of hNFS1 (56-
457 aa into BamHI-SalI), yNFS1 (37-497 aa into
BamHI-XhoI), HSCB (22-235 aa into BamHI-
Experimental procedures SalI) and JAC1 (1-184 aa into BamHI-XhoI) were
generated by introducing the respective coding
Cell culture and transfection HeLa cells sequences downstream of the GST-tag in the
were transfected with pCI-neo-ISCU and pCI-neo- pGEX-KG vector, respectively. For mammalian
G50E ISCU using Lipofectamine 2000 for transfection experiments, the full-length human
expression of wild type ISCU and G50E ISCU. ISCU and G50E ISCU were cloned downstream to
Cells were cultured in Dulbeccos modified a CMV-based promoter of pCI-neo vector
Eagles medium (Invitrogen) containing 10% fetal
(Promega) with an FLAG-tag at the 3 end of the
bovine serum (Gibco) and 1% penicillin-
open reading frame. G50E point mutation was
streptomycin (Sigma). The cells were incubated at
created through Quik Change site-directed
37 C in 5% CO2 for 48 h, prior to experiments.
mutagenesis, using high-fidelity Pfu Turbo DNA
The adherent cultures of HeLa cells were grown to

3
Role of ISCU in development of mitochondrial myopathy

Polymerase (Stratagene). All the clones were maxima of 580 nm. Transfected HeLa cells
verified by DNA sequencing reactions carried out cultured for 48 h and 0.1 OD (A600) of yeast cells
at Eurofins Inc. and Macrogen Inc. from the early log phase were harvested and
incubated with the dye (0.5 M for HeLa and 2.5
Protein expression and purification For M for Yeast) for 10 min, following which they
purification of N-terminal histidine6-tagged human were washed with 1X PBS and subjected to FACS
WT ISCU and G50E ISCU or yeast WT Isu1 and analysis using a 488 nm argon laser for excitation
Isu1G50E, the proteins were expressed in E. coli (BD FACS Canto II). The respiratory inhibitor
BL21(DE3) strain by allowing growth at 30 C to rotenone (1 mM) was used as a positive control for
an A600 of 0.6, followed by induction using 0.5 generating higher superoxide levels. The MFI
mM IPTG for 6 h. Cells were harvested by values of 10,000 events were recorded per sample
centrifugation and then lysed in buffer A (50 mM and plotted to compare the relative ROS levels
Tris-Cl, pH 7.5, 150 mM NaCl, 1 mM using the WinMDI 2.9 software (29)
Dithiothreitol, 20 mM imidazole, 1 mM PMSF
and 10% glycerol) along with 0.2 mg/ml of Fluorescence imaging in cell lines HeLa cells
lysozyme, followed by incubation at 4 C for 1 h. harboring WT ISCU and G50E ISCU were seeded
The samples were gently treated with 0.2% on a cover slip (5000 cells per well in 12 well

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deoxycholate, followed by DNase I (10 g/ml) plates) and cultured for 48 h. Media was removed,
treatment for 15 min at 4 C. The cells were washed with 1X PBS and cells were stained with
further lysed by sonicating 3 times (15 s each) at 0.1 M of MitoSOX Red and 250 ng/ml Hoechst
25% amplitude using an Ultrasonic processor with 33342 for 15 min in 300 l 1X PBS, at 37 C in a
2 min intervals, in ice. The cell lysates were 5% CO2 incubator. Prior to analysis, cells were
clarified by centrifuging at 22,000g for 30 min at 4 twice washed with 1X PBS. Images were acquired
C. The supernatant was incubated with Ni-NTA using 63X objective lens on a Zeiss apotome
Sepharose (GE Healthcare) for 2 h at 4 C. fluorescence microscope. For MitoSOX Red
Unbound proteins and nonspecific contaminants fluorescence imaging of yeast, 0.1 OD (A600) of
were removed by multiple washes of buffer A cells from mid log phase grown at 37 C, were
alone followed by sequential single washes of incubated with 5 M dye for 15 min. The cells
buffer B (buffer A along with 0.05% Triton X- were washed twice with 1X PBS before analysis.
100), buffer C (buffer A along with 1 mM ATP, Images were acquired using 100X objective lens
10 mM MgCl2), buffer D (buffer A along with 1 M on a Leica fluorescence microscope. Similar
NaCl), and buffer E (buffer C along with 40 mM strategy was used for H2DCF-DA and calcein blue
imidazole). Finally, the bound proteins were eluted staining. HeLa cells were treated with 1 M
with buffer A containing 250 mM imidazole. H2DCF-DA for 15 min and yeast cells with 5 M
Purification of other proteins was also achieved by H2DCF-DA for 20 min and for calcein blue
the same strategy as mentioned above with minor staining, HeLa cells were treated with 3 M
modifications. For GST-tagged proteins, the calcein blue for 20 min. To test the specificity of
purification was performed in a conventional calcein blue staining, the mutant cells were treated
manner using the GST Sepharose beads (GE with 1 mM iron specific chelator deferoxamine
Healthcare). The bound proteins were stored in (DFO) (30). Images of HeLa cells were taken
buffer containing 50 mM Tris-Cl, pH 7.5 and 100 using 63X objective lens of Zeiss apotome
mM NaCl at 4 C. fluorescence microscope and that of yeast cell
with 100X objective lens on a Leica fluorescence
Measurement of mitochondrial ROS levels by microscope.
FACS In order to analyse the extent of
superoxide generation by the mitochondria; we In vitro GST pull down analysis Purified
utilized MitoSOX Red dye (Molecular Probes). GST-hNFS1 (1.5 M)/GST-yNfs1 (1.5 M) and
This dye is specifically targeted to the GST-HSCB (1 M) / GST-Jac1 (2.5 M) were
mitochondria, where it undergoes oxidation by the incubated with a 10 l bed volume of glutathione-
mitochondrial superoxide radicals and fluoresces agarose beads in 200 l of GST buffer A (50 mM
upon binding to mtDNA, with an emission Tris-Cl, pH 7.5, 100 mM NaCl, 40 mM Imidazole,

4
Role of ISCU in development of mitochondrial myopathy

50 M Pyridoxal phosphate, 10% Glycerol) and to yeast cell mitochondria was resuspended in 50
GST buffer B (50 mM Tris-Cl, pH 7.5, 100 mM mM potassium phosphate buffer (pH 7). Reaction
NaCl, 40 mM Imidazole, 0.2% Triton X-100, 5% was started by adding 60 M reduced cytochrome
Glycerol) for interaction study with either WT or c and decrease in absorbance at 550 nm for 3 min
mutant ISCU/Isu1, respectively. Unbound-proteins was observed. To check specificity of complex IV
were removed by washing the beads three times activity this experiment was performed in the
with the respective GST buffers. After removing presence and in the absence of complex IV
unbound-proteins, the samples were blocked with inhibitor NaN3 (300 M). Aconitase activity was
0.1% BSA for 20 min at 20 C, followed by also measured following similar protocol as
washing two times with GST buffer to remove described earlier (32). Briefly, 25 g of protein
excess unbound BSA. The beads were corresponding to HeLa cells mitochondria and 50
resuspended in 200 l of GST binding buffer and g of protein corresponding to yeast cell
incubated with increasing concentration of either mitochondria were dissolved in buffer A (50 mM
WT or mutant ISCU/Isu1 for 30 min at 20 C. The Tris-Cl, pH 8.0, 50 mM NaCl) containing 1%
GST beads were washed three times with GST deoxycholic acid (DOC) and incubated for 10 min.
buffer and resolved on SDS-PAGE (sodium Reaction was started by addition of sodium citrate
dodecyl sulfate-polyacrylamide gel dihydrate and absorbance increase at 235 nm for 2

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electrophoresis) followed by Coomassie dye min was followed in a quartz cuvette.
staining. Mitochondrial ATP was quantified using
Mitochondrial ToxGlo Assay kit (Promega).
Enzymatic activity assay and mitochondrial Briefly, 1-2 g of mitochondria were resuspended
ATP-level Quantification Enzymatic assays of in 80 l of SEM buffer (250 mM sucrose, 1 mM
complex I, complex II and complex IV were EDTA, 10 mM MOPS-KOH, pH 7.2) and mixed
performed according to previously described with 20 l of 5X Cytotoxicity Reagent (bis-AAF-
protocol (31). Briefly, 15 g of protein R110) for 1 min by orbital shaking (700 rpm). The
corresponding to mitochondria isolated from HeLa reaction mixture was incubated at 37 C for 30
cells were subjected to three cycles of freeze- min followed by equilibration of assay plate at
thawing before complex I activity was assayed. room temperature (25 C) for 5 min.
Mitochondria were resuspended in 50 mM of Subsequently, 100 l of ATP-detection reagent
potassium phosphate buffer (pH 7.5) containing was added to each well and mixed by orbital
100 M NADH, 3 mg/ml fatty acid free BSA, 300 shaking (700 rpm) for 5 min. The luminescence
M sodium azide (NaN3). Reaction was started by was measured using 2450-microplate counter
adding 60 M of Ubiquinone and decrease of (Perkin Elmer).
absorbance at 340 nm was monitored for 2 min.
To check for specificity of reaction, the Analysis of mitochondrial mass and membrane
experiment was performed both in the presence potential The total mitochondrial masses of the
and absence of specific inhibitor rotenone (10 transfected HeLa cells and transformed yeast
M). For complex II activity analysis, 2 g of strains were determined using 10-N-nonyl acridine
protein corresponding to HeLa cells mitochondria orange (NAO) (Molecular Probes). Briefly, 0.2
and 10 g of protein corresponding to yeast cell OD (A600) of log phase yeast cells were harvested
mitochondria were resuspended in 25 mM and washed with 1X PBS followed by incubation
potassium phosphate buffer (pH 7.5) containing 20 with 10 mM NAO for 30 min at room temperature.
mM succinate, 1 mg/ml fatty acid-free BSA, 300 Subsequently, the cells were washed and
M NaN3, 10 M rotenone, 10 g/ml Antimycin resuspended in 1X PBS, and FACS analysis was
A and 80 M of 2, 6-dichlorophenolindophenol performed using the BD FACS Canto II flow
(DCPIP) followed by incubation at 37 C for 10 cytometer. An argon laser was used for the
min. Reaction was started by adding 60 M of excitation at wavelength 488 nm, while the
Ubiquinone and decrease in the absorbance at 600 emission was recorded at 520 nm. For each
nm was recorded for 3 min. For complex IV analysis, 10,000 events were recorded and the data
activity 2 g of protein corresponding to HeLa cell were analyzed based on three independent
mitochondria and 10 g of protein corresponding experiments, using the WinMDI 2.9 software. In

5
Role of ISCU in development of mitochondrial myopathy

order to detect changes in mitochondrial described methods (33). Protein determinations

membrane potential, isolated mitochondria (50 g) were performed by using the Bio-Rad protein
from HeLa cells and yeast strains were incubated assay kit with BSA as a standard.
with the JC-1 dye (5 mg / ml) for 5 min in the
dark. Subsequently, they were excited at 490 nm Gel filtration chromatography For
and subjected to an emission scan between 500 oligomerization analysis 500 l of protein samples
and 620 nm in a JASCO FP-6300 (2 mg / ml) were filtered through a 0.22 m filter
spectrofluorometer. The ratios of the peaks at 590 and subjected to Gel Filtration analysis using
(aggregate form of the JC-1 dye) and 530 nm Superdex 200 10/300 GL column (GE Healthcare)
(monomeric form) were used as an indicator of in buffer G (50 mM Tris-Cl, pH 7.5, 50 mM NaCl,
membrane polarization. The WT mitochondria 50 mM Imidazole). The column was calibrated
were incubated in 100 M valinomycin for 15 min prior to experiment using standard molecular
prior to dye staining, as a control for complete weight marker from BioRad [thyroglobulin (670
depolarization. kDa), -globulin (158 kDa), ovalbumin (44 kDa),
Coimmunoprecipitation (CoIP) in myoglobin (17 kDa) and vitamin B12 (1.35 kDa)]
mitochondrial lysate Mitochondria isolated form and void volume (V) was calculated using blue
HeLa cells or yeast (1 mg of protein) were lysed dextran (2000 kDa). The elution volume (Ve) of

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for 3 min on ice in 1 ml of buffer A [50 mM Tris- standard markers, WT and mutant ISCU proteins
Cl, pH 7.5, 150 mM NaCl, 0.2% Tween 20, 10 M was determined under similar buffer and pH
leupeptin, 10 M pepstatin, 2 mM PMSF conditions. The molecular mass was calculated
(phenylmethylsulphonylfluoride), 50 M from the standard plot of Ve/ V versus log of
pyridoxal phosphate and 1 mM ascorbic acid]. molecular mass of standard molecular weight
Membrane debris was removed by centrifugation markers.
for 10 min at 18,000g and the supernatant was
incubated with 5 l of anti-FLAG antibody (0.5 g Miscellaneous Cell proliferation was
/ l) for 2 h on a rotary shaker at 4 C. This assessed by MTT-assay according to the
supernatant was added to 30 l of protein G beads manufacturers recommendations (Invitrogen).
pre-equilibriated with buffer A and kept for 2 h on MTT (3-[4,5-dimethylthiazol-2-yl]-2,5
a rotary shaker at 4 C. Beads were collected by diphenyltetrazolium bromide) was added directly
centrifugation for 5 min at 800g and washed three to the 72 h grown culture medium and was
times with 800 l of buffer A. Bound proteins reduced by metabolically active cells to insoluble
were subjected to SDS-PAGE and identified by purple formazan dye crystals. The absorbance of
immunostaining. the sample was read directly in the wells at an
optimal wavelength of 570 nm. For calcein blue
Iron quantification analysis Assessment of FACS analysis, HeLa cells were cultured for 72 h
total cellular iron levels by atomic absorption and treated with 1 M of calcein blue dye for 5
spectroscopy (AAS) was performed using 1 min prior to FACS analysis. Oligomerization of
million HeLa cells and 0.2 OD (A600) of yeast ISCU in the mitochondrial lysate was analysed by
cells. Mitochondrial iron estimation by AAS was blue-native PAGE (BN-PAGE) similar to the
performed using 50 g of protein corresponding to previous published protocols (34). Briefly,
HeLa cell and yeast mitochondria. Samples were mitochondrial lysate was prepared in buffer
digested with concentrated HCl, diluted and containing 0.5% Tween 20 to disrupt the ISCU-
subjected to AAS (Atomic Absorption bound subcomplexes with interacting partner
Spectrometer AA 200) according to previously proteins. The supernatant was subjected to BN-
described procedures with minor modifications PAGE separation followed by immunostaining
(12). For colorimetric cellular iron quantification, using anti-ISCU antibodies. One-way ANOVA
HeLa cells (~1 million) and 0.2 OD (A600) of yeast (and non-parametric) was performed using
cells were sonicated prior to analysis. For GraphPad Prism. P-value of <0.05 was defined as
colorimetric mitochondrial iron estimation, 0.2 significant, and asterisks were used to denote
0.4 mg of mitochondrial protein from HeLa cells significance as follows: *, P< 0.05; **, P< 0.01;
and yeast was determined following previously ***, P< 0.001.

6
Role of ISCU in development of mitochondrial myopathy

Results Human G50E ISCU mutant exhibits decreased

Fe-S cluster containing enzyme activity and
Myopathy associated glycine 50 residue of ISCU reduced cellular respiration Mitochondria are
is critical for cell viability In humans, the the major site for the synthesis of Fe-S clusters in
synthesis of Fe-S cluster utilizes the mitochondrial eukaryotic cells (11). Since ISCU forms the key
scaffold protein ISCU as a platform for the component for Fe-S assembly, we investigated
complex interactions between various intervening whether the reduced cell viability in G50E mutant
proteins (Fig. 1A). ISCU is among the most is due to impaired Fe-S cluster biogenesis. To
conserved proteins of Fe-S cluster biogenesis assess the biogenesis of functional Fe-S cluster,
machinery throughout eukaryotic evolution (33). we monitored the enzyme activity of Fe-S cluster
Recent studies have reported that a missense containing proteins using purified mitochondria
mutation changes the conserved glycine 50 residue from HeLa cells cultured for 48 h. Before
to glutamate (G50E) and contributes significantly enzymatic analysis, the purity of isolated
towards the development of myopathy (28). mitochondria was assessed using
Multiple protein sequence alignment of predicted immunodecoration with ISCU-specific antibody
ISCU orthologs suggested significant conservation for the ed mitochondrial fraction. As a positive
of the glycine 50 residue across different species, control, components of the mitochondrial inner

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highlighting its importance in the protein function. membrane translocon mechinary, such as Tim23
Additionally, ISCU orthologs also showed a and Tim44 proteins were probed. To detect the
significant sequence homology throughout the other organellar contamination, immunoblotting
length of the protein across genera. Human ISCU was performed for other markers such as cathepsin
revealed a 71% sequence identity at the amino D (lysosomes), catalase (peroxisomes) and
acid level with Saccharomyces cerevisiae, Isu1 superoxide dismutase (nuclear) as negative
and 75% sequence identity with Escherichia coli, controls. Negligible amounts of non-mitochondrial
IscU (Fig. 1B). In the three-dimensional modelled proteins were detected in the enriched
structure of ISCU (generated using 1wfz PDB mitochondrial fraction thus confirming the purity
template), glycine 50 is located at the N-terminal of the mitochondria utilized for the enzymatic
exposed random coil region, which forms a lid analysis (Fig. 2A).
over the core -strands of the protein (Fig. 1C).
Although the G50E mutation is associated We chose three model iron-sulfur proteins
with clinical cases of myopathy patients, the namely, membrane associated complex I and
cellular defects and the molecular mechanisms that complex II of electron transport chain, and matrix
leads to the disease condition is still elusive. The localized TCA cycle enzyme aconitase. Activity
importance of glycine 50 residue in the of complex I (NADH: ubiquinone oxidoreductase)
maintenance of cell viability was analyzed by containing eight Fe-S clusters was found to be
MTT-assay. Wild type (WT) or G50E mutant significantly reduced in case of G50E mutant as
constructs of ISCU were transfected into HeLa compared to mitochondria isolated from WT ISCU
cells and were allowed to be expressed for 72 h and untransfected HeLa cells. The specificity of
(Fig. 1D). The relative cell viability was the reaction was further verified by inhibiting the
determined by MTT-assay based on the complex I with its specific inhibitor, rotenone. As
conversion of MTT into formazan crystals in expected, rotenone treated mitochondria showed
living cells. We observed more than two-fold considerably reduced enzymatic activity as
reduction in cell viability in case of cells compared to the untreated controls. A cumulative
overexpressing the mutant copy of ISCU as reduction in complex I function was observed in
compared to that of cells expressing WT copy of case of rotenone treated G50E mitochondria, thus
protein or untransfected (UT) controls (Fig. 1E), highlighting the importance of the residue for
thus highlighting the critical role of glycine 50 in normal protein function (Fig. 2B). Similarly, a
normal protein function required for the marked reduction in the activity of complex II
maintenance of cell growth. (succinate dehydrogenase) composed of three Fe-S
centers was observed in G50E mutant
mitochondria (Fig. 2C). We further validated our

7
Role of ISCU in development of mitochondrial myopathy

results by measuring the activity of a in the cytoplasm as a green fluorescent monomeric

mitochondrial matrix Fe-S cluster enzyme, form with emission maxima at 530 nm. The ratio
aconitase that reversibly converts citrate to of red to green fluorescence serves as an indicator
isocitrate in the second step of TCA cycle. for functional mitochondria membrane potential
Supporting the above observation, the activity of (38). Indeed, WT mitochondria showed decreased
aconitase was found substantially lessened in the fluorescence at 530 nm indicating the presence of
G50E mutant mitochondria, suggesting intact membrane potential. In contrast, the G50E
impairment in the Fe-S cluster biogenesis (Fig. mutant showed a significant increment in the
2D). In order to address the specificity in the fluorescence intensity at 530 nm and reduction at
impairment of Fe-S biogenesis by the loss of 590 nm as compared to mitochondria from
function of ISCU as a result of the mutation, we untransfected HeLa cells (Fig. 2G; compare peaks
checked activity of complex IV or cytochrome c represented in red with green and black colors).
oxidase that contains two heme centers instead of Upon quantification of red to green ratio, a 21%
Fe-S centers. Notably, complex IV showed a reduction in mitochondrial membrane potential
marginal declined activity in mutant mitochondria was estimated for the G50E mutant (Fig. 2H).
inconsequence to decreased activity of Fe-S Valinomycin which is known to disrupt the
cluster containing ferrochelatase, which is membrane potential of intact mitochondria was

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involved in the heme synthesis (Fig. 2E). In lieu used as a positive control (39). Secondly, we
with the loss of electron transport chain complex assessed the overall mitochondrial mass using a
activity, we checked for the mitochondrial fluorescent dye 10-N-nonyl acridine orange
respiratory activity in HeLa cells. A defect in (NAO), which binds to the cardiolipin of
mitochondrial respiration results in diminished mitochondrial membrane, thus giving an estimate
ATP synthesis (35-36). In consistent with the of the total mitochondrial mass present inside the
above data; we observed that G50E mutant cell (40). Interestingly, the G50E mutant showed a
mitochondria harbored substantially lesser amount significant decrease in mitochondrial mass as the
of ATP levels as compared to mitochondria fluorescence signal peak was shifted towards the
isolated from WT ISCU and untransfected HeLa lower region as compared to WT ISCU and
cells (Fig. 2F). Together, these results highlights untransfected HeLa cells (Fig. 2I; compare peaks
that G50E ISCU mutation impairs the Fe-S cluster represented in red with green and black colors).
biogenesis leading to decreased activity of Fe-S Upon quantification of mean fluorescence
cluster containing ETC-enzymes thereby causing a intensity values, a 52% reduction in the overall
defect in the cellular respiration in the myopathy mitochondrial mass was observed in the G50E
patients. mutant in comparison to the WT ISCU (Fig. 2J).
These findings indicate that G50E mutation in
G50E mutation in ISCU results in loss of ISCU results in the reduced biogenesis of
mitochondrial membrane potential and mitochondria along with loss of membrane
mitochondrial mass Previous reports have potential.
suggested that impairment of Fe-S cluster
biogenesis often leads to mitochondrial G50E mutation in ISCU results in increased
dysfunction (37). Therefore, we embarked upon to iron load and ROS level in mitochondria Defect
analyze whether loss of Fe-S cluster biogenesis as in Fe-S cluster biogenesis is often associated with
a result of G50E mutation results in impairment of impairment of mitochondrial iron homeostasis. It
mitochondrial function. First, we assessed the has been reported earlier in a neurodegenerative
maintenance of mitochondrial inner membrane disorder, Friedreichs ataxia that deprivation of
potential, using the membrane potential sensitive Fe-S biogenesis results in accumulation of iron
cationic dye, JC-1. In presence of an intact inside mitochondria (41). Considering this fact, we
membrane potential, the JC-1 dye accumulates investigated the cellular and mitochondrial iron
inside the mitochondrial matrix to form J- levels in WT ISCU HeLa cells and G50E mutant
aggregates, which fluoresces at the red region with cells. The overall cellular free iron was measured
emission maxima of 590 nm. However, upon using an iron sensitive probe, calcein blue, through
dissipation of membrane potential; JC-1 remains flow cytometry and fluorescence imaging. Calcein

8
Role of ISCU in development of mitochondrial myopathy

blue is a cell permeable metalo-fluorochromic flow cytometric analysis and corelated with the
indicator dye which upon binding with free iron amount of ROS present in the mitochondria. We
results in reduction in fluorescence intensity (42- observed that ISCU G50E mutation led to an
43). Flow cytometric analysis of G50E mutant elevation in the mitochondrial superoxide levels
cells stained with calcein blue showed a reduction indicated by a significant shift in the fluorescence
in fluorescence intensity (shift in the fluorescence signal peak towards higher ROS levels as
peak towards a lower region) as compared to WT compared to WT HeLa cells (Fig. 4A; compare
ISCU and untransfected HeLa cells, suggesting the peaks represented in red with green and black
presence of higher free iron content in mutant cells colors whereas blue represents rotenone a
(Fig. 3A and B; compare peaks indicated in red control). Upon quantification of flow cytometric
with green and black colors). The flow cytometric data, greater than the 6-fold increase in MitoSOX
results were further validated by fluorescence staining was observed in G50E HeLa cells (Fig.
imaging of WT and mutant HeLa cells, which 4B). As a positive control, cells were treated with
revealed the diminished fluorescence intensity in 1 mM rotenone, which elevates the mitochondrial
G50E mutant thus confirming an overall enhanced superoxide levels by inhibiting respiratory
cellular unbound/free iron levels. To demonstrate complex I. In support to the flow cytometric data,
the specific quenching of calcein by iron, the live cell imaging of G50E mutant cells treated

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G50E HeLa cells were treated with 1 mM of iron- with MitoSOX Red dye revealed robust
chelator, deferoxamine (DFO). Upon DFO enhancement in the fluorescence intensity as
treatment, an elevated fluorescence in G50E HeLa compared to WT (Fig. 4C and D) suggesting
cells was observed suggesting that the quenching elevation in the mitochondrial superoxide levels.
of calcien fluorescence observed in G50E HeLa To address whether elevation of mitochondrial
cells as the consequence of enhanced unbound/free superoxide levels alters the overall cellular ROS
iron levels and not due to interference from other levels, the WT and mutant cells were stained with
metal ions (Fig. 3C and D). The increment in the H2DCF-DA dye followed by fluorescence
cellular iron load as observed in G50E mutant microscopic analysis. H2DCF-DA is a chemically
HeLa cells through fluorescence based assays was reduced form of fluorescein and upon modification
additionally validated by iron specific colorimetric by peroxide species present in the cells produces a
assay and atomic absorption spectroscopy analysis green fluorescence signal. Consistent with the
using whole cell or mitochondrial lysates. Both the MitoSOX data, G50E HeLa cells showed
colorimetric assay and AAS analysis showed a significantly increased green fluorescence as
substantial increment in the mitochondrial iron compared to WT, further validating that the G50E
load in G50E mutant as compared to WT ISCU mutation results in enhancement of the overall
mitochondria indicating that impaired Fe-S cellular ROS levels (Fig. 4E and F). In summary,
biogenesis leads to accumulation of iron levels in above findings indicate that an enormous
the organelle (Fig. 3E to H). increment in the superoxide and overall ROS
Increased cellular iron load and loss of levels in G50E ISCU mutant induces a severe
respiratory complex activity is well known to oxidative stress thus inflicting multiple cellular
promote reactive oxygen species (ROS) damages, including impairment in the
production (44). In retrospect, higher ROS levels mitochondrial functions.
also promote accumulation of iron inside cell (45-
47). Based on our observations of enhanced Analogous G50E mutation in yeast Isu1
mitochondrial iron load and respiratory defect in protein leads to inviability and mitochondrial
G50E mutant, we further assessed for the dysfunction Our observations in the mammalian
maintenance of mitochondrial ROS levels in HeLa system highlighted that the glycine 50 residue is
cells. The accumulation of mitochondrial ROS crucial for proper functioning of ISCU. G50E
was determined by measuring the superoxide mutation in ISCU results in mitochondrial
levels with MitoSOX Red dye, which is dysfunction that facilitates the development of
specifically targeted to mitochondria in live cells. myopathy in humans. Therefore, to understand
Oxidation of MitoSOX Red reagent by superoxide whether the glycine 50 residue plays a similar
produces red fluorescence, which is quantified by conserved role in other ISCU orthologs across

9
Role of ISCU in development of mitochondrial myopathy

kingdom, we performed analogous amino acid antibodies (Fig. 5C). This infers that, at higher
substitution (G50E) in Saccharomyces cerevisiae, expression levels of isu1G50E mutant in presence of
ISU1 gene. Yeast mitochondrial Isu1 is an a WT copy of ISU1 gene leads to a semi-dominant
ortholog of human ISCU and is essential for the negative phenotype at 37 C. We utilized isu1G50E
biogenesis of Fe-S clusters in mitochondria. In overexpressing under the GPD promoter in yeast
yeast mitochondria, ISU1 has a second paralog strains harboring a WT copy of ISU1 for further in
ISU2 believed to be a result of whole genome vivo experiments.
duplication. However, Isu1 is the predominant
form that is expressed under all growth conditions To address whether the deleterious effect
and isu1/isu2 double deletion mutant is inviable of analogous G50E mutation in Isu1 is due to
(12). functional defects in yeast mitochondria, we first
We first assessed whether WT human ISCU measured the mitochondrial mass of isu1G50E yeast
could rescue the inviability of the isu1/isu2 cells using NAO staining. Similar to G50E ISCU
strain. To test, a centromeric plasmid pRS414 TEF HeLa cells mutant, isu1G50E yeast strains also
carrying a WT copy of human ISCU was showed a significantly reduced mitochondrial
transformed into a isu1/isu2 strain harboring a mass as compared to WT (Fig. 5D and E).
WT copy of ISU1 on pRS316 plasmid having Likewise, assessment of mitochondrial membrane

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URA3 as a selectable marker. The transformed potential using JC-1 dye staining exhibited 25%
strains were selected on tryptophan omitted media. lower membrane potential in isu1G50E yeast as
The resultant yeast transformants were scored for compared to that of WT suggesting impairment in
viability, by drop test analysis on media containing the biogenesis as well as mitochondrial functions
5-fluoroorotic acid (5-FOA) which selects for cells (Fig. 5F and G).
that have lost the pRS316 plasmid, thereby losing
the WT copy of ISU1 in this case. Interestingly, G50E mutation in yeast Isu1 leads to a similar
human WT ISCU rescued the inviability of reduction in Fe-S cluster containing enzyme
isu1/isu2 strain (Fig. 5A). Importantly, on the activity, ATP levels, mitochondrial iron
other hand, both human G50E ISCU and yeast accumulation and elevated mitochondrial ROS
isu1G50E resulted in failure to rescue inviability of The indispensability of glycine 50 residue of ISCU
isu1/isu2 strain on 5-FOA at all temperatures for cell viability and mitochondrial function has
tested, thus highlighting the critical nature of already been established using HeLa cells. In order
amino acid glycine 50 of Isu1 for in vivo function to ascertain the conservation of function of glycine
in yeast. 50th residue across genera, we performed a similar
cellular analysis in isu1G50E yeast cells pre-exposed
The isu1G50E mutant is inviable in haploid state to temperature stress at 37 C. To address whether
and hence found unsuitable for the in vivo the inviability of isu1G50E mutant yeast is due to
functional analysis. Therefore, to analyze the impaired Fe-S biogenesis process, we measured
effect of G50E Isu1 mutation in Fe-S biogenesis, the Fe-S cluster enzyme activity. Supporting our
the isu1G50E mutant was first overexpressed under results observed in human cell lines, isu1G50E yeast
the mild TEF promoter in yeast strains harboring a strains showed 2.6-fold reduced complex II
WT copy of the ISU1 gene, on pRS316 plasmid. activity (Fig. 6A) and 3-fold lower activity of
However, at similar expression levels, isu1G50E aconitase (Fig. 6C) as compared to WT Isu1
mutant did not exhibit any growth phenotype at all harboring yeast cells. In contrast, complex IV that
temperature conditions tested (Fig. 5B upper does not possess Fe-S clusters demonstrates a
panel). On the other hand, when isu1G50E mutant marginal decrease in the activity in mutant yeast
expressed under the control of strong GPD cells (Fig. 6B). These results provide evidence to
promoter, the cells were temperature sensitive at indicate G50E mutation in Isu1 leads to a similar
37 C as compared to WT (Fig. 5B lower panel). impairment in the Fe-S cluster biogenesis hence
Moreover, both WT Isu1 and isu1G50E decreasing activity of Fe-S cluster containing
overexpressing strains showed equal levels of enzymes. As a result of loss of ETC enzyme
protein expression in the mitochondrial lysate, complex activity, the isu1G50E overexpressing yeast
upon immunoblot analysis using anti-FLAG mitochondria showed 1.8-fold lower respiratory

10
Role of ISCU in development of mitochondrial myopathy

activity in terms of total mitochondrial ATP- 7D; upper left panels, compare untreated).
content as compared to WT Isu1 yeast Importantly, isu1G50E cells showed a robust
mitochondria (Fig. 6D). These observations increment in red fluorescence when treated with
provide compelling evidence to prove that, similar H2O2 as compared to WT Isu1 yeast strain,
to mammalian system, G50E mutation in yeast indicating its susceptibility to cellular
Isu1 also leads to impairment in Fe-S cluster redox alterations (Fig. 7D; right panels, compare
biogenesis and reduced cellular respiration. treated). A similar analysis was performed to
measure the overall cellular ROS production using
To check alterations in the cellular iron H2DCF-DA dye in response to oxidative stress. As
content due to defective Fe-S cluster biogenesis, shown in Fig. 7E, isu1G50E yeast cells displayed
we checked for the iron accumulation in mutant higher basal peroxide levels as compared to WT
isu1G50E yeast cells by a colorimetric based assay Isu1 strain, in untreated condition (Fig. 7E; left
and AAS analysis. Colorimetric assay showed lower panel, compare untreated). However, a
17% higher cellular iron and 34% higher significant enhancement in green fluorescence was
mitochondrial iron levels in G50E mutant (Fig. 6E observed for the isu1G50E yeast strain as compared
and F). Similarly, AAS analysis revealed 22% to WT Isu1 yeast cells, after H2O2 treatment (Fig.
higher cellular iron, and 32% elevated 7E; Right panels, compare treated). These

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mitochondrial iron levels in the mutant as findings conclusively establish that isu1G50E strain
compared to WT Isu1 yeast strains thus harbors enhanced mitochondrial and overall
confirming our previous observations using HeLa cellular ROS levels due to defective Fe-S cluster
cells (Fig. 6G and H). biogenesis.
Previous reports and our observations
from the human cell lines have indicated that Human G50E ISCU and yeast isu1G50E mutant
mitochondrial respiratory defects and iron over proteins are defective in interaction with sulfur
load results in increased ROS levels in donor hNFS1/yNfs1 and J-protein, HSCB/Jac1
mitochondrial compartment. To check the ROS ISCU serves as a platform for the de novo
levels in the mitochondria of isu1G50E mutant yeast synthesis of a Fe-S cluster in a highly coordinated
cells, flow cytometric analysis was performed manner along with other key partner proteins of
using MitoSOX Red dye. As indicated in Fig. 7A the machinery. Previously, it has been
(compare the peak represented in red color with demonstrated that ISCU protein, specifically
green and blue) and Fig. 7B, isu1G50E yeast strains associates with hNFS1, a cysteine desulfurase
exhibited substantially higher mitochondrial ROS which is involved in the transfer of inorganic
as compared to WT Isu1 yeast strains. The sulfur to Fe-S cluster assembly (11). On the other
enhanced ROS levels in yeast usually associated hand, biochemical studies in yeast have
with the enhanced sensitivity towards the demonstrated that mitochondrial DnaJ-like
extraneous stress. To probe the sensitivity of cochaperone Jac1, interacts with Isu1 to enhance
isu1G50E mutant strain to extraneous oxidative the specific transfer of a scaffold bound Fe-S
stress, cells were subjected to hydrogen peroxide cluster to a bonafide Fe-S apoproteins (20,48). In
(H2O2) treatment. As shown in Fig. 7C, isu1G50E addition to that recently it has been reported in
strain was responsive to oxidative stress at 37 C, yeast that binding of yNfs1 and cochaperone Jac1
exemplified through a compromised growth to the iron-sulfur cluster scaffold Isu1 is mutually
phenotype in minimal media containing 1 mM exclusive (15). Therefore, we asked whether G50E
H2O2. On the other hand, WT Isu1 grew normally mutation in ISCU results in compromised
in the presence of 1 mM H2O2 containing media. interaction with sulfur donor NFS1 or with J-
The sensitivity of isu1G50E strain to extraneous protein, HSCB leading to defective Fe-S cluster
oxidative stress was further demonstrated by biogenesis. To evaluate whether G50E ISCU
fluorescence live cell imaging. The isu1G50E strain mutant protein is capable of forming a subcomplex
stained with MitoSOX Red dye showed an with hNFS1 or with HSCB, we performed in vitro
enhanced basal level of fluorescence in untreated glutathione S-transferase (GST) pull down
conditions as compared to the WT Isu1 strain analysis using purified proteins. The interaction
consistent with the flow cytometric analysis (Fig. with hNFS1 was investigated by incubating

11
Role of ISCU in development of mitochondrial myopathy

immobilized GST bound hNFS1 with increasing and IscU (ortholog of ISCU and NFS1 from
concentrations of WT ISCU or G50E ISCU Archaeoglobus fulgidus), suggesting that the
mutant. The GST pull down analysis showed an glycine residue plays a critical role to maintain
efficient interaction of hNFS1 with WT ISCU complex structure (Fig. 8K). In summary, our
(Fig. 8A and B). Interestingly, however, its results suggest that glycine 50 residue is critical
interaction with G50E mutant was significantly for ISCU function and G50E mutation in
compromised (Fig. 8A and B). As a control, GST ISCU/Isu1 results in compromised interaction with
alone did not interact with either WT or mutant its bonafide partner proteins, which are essential
ISCU protein (Fig. 8A, middle panel). In addition for the assembly process as well as transfer of Fe-s
to that, we assessed the interaction of mutant with cluster into apoproteins.
J-protein cochaperone by using a similar GST pull
down analysis. Interestingly, G50E ISCU mutant G50E mutant ISCU exhibits a semi-dominant
exhibits significantly reduced interaction with phenotype by forming oligomeric complex with
HSCB as compared to WT ISCU (Fig. 8C and D). WT protein It has been reported earlier that, in
Based on these findings, we further different organisms, ISCU homologs exist in
investigated the presence of similar defective variable oligomeric states. For example,
interactions between yeast isu1G50E mutant with Escherichia coli apo-IscU was reported to exist as

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orthologous protein partners such as Nfs1 and Jac1 a monomer or an S-S bridged dimer (49-50).
using GST pull down analysis. Similar to our However, IscU from Azotobacter vinelandii and
observations in humans, yNfs1 showed diminished Schizosaccharomyces pombe exists as a dimer,
interaction with isu1G50E while WT Isu1 exhibited retaining either one or two [2Fe-2S] centers or a
robust association with yNfs1 (Fig. 8E and F). single [4Fe-4S] cluster (51-52). In case of Aquifex
Likewise, Jac1 also showed a compromised aeolicus, IscU holo-protein forms a trimer
interaction with isu1G50E as compared to WT, containing sub stoichiometric [2Fe-2S] cluster,
similar to HSCB in humans (Fig. 8G and H). whereas its apo-form gets dissociated into a
smaller species, which is a mixture of monomeric
To further confirm our in vitro and predominant dimeric form (53). Therefore, to
observations, we assessed the subcomplex investigate whether there is any alteration in the
formation between G50E ISCU with hNFS1 or stoichiometry of G50E ISCU mutant protein, we
HSCB in HeLa cell mitochondrial lysates using in purified recombinant WT apo-ISCU and G50E
organellar coimmunoprecipitation (CoIP) apo-ISCU under aerobic condition from E. coli.
analysis. For this purpose, we isolated The purified mutant protein retained the structural
mitochondria from HeLa cells expressing FLAG- integrity similar to WT as confirmed by circular
tagged WT ISCU or G50E ISCU. The respective dichroism (Fig. 9 A), partial trypsin and
mitochondrial extracts were prepared in 0.2% chymotrypsin digestion analysis (Fig. 9B and C).
Tween 20 containing buffer and then subjected to The oligomeric state of WT and mutant proteins
CoIP analysis using anti-FLAG antibodies. Both were analyzed by subjecting them to gel filtration
hNFS1 and HSCB were coprecipitated along with chromatography using Superdex 200 10/300 GL
ISCU in extracts of WT mitochondria, indicating column. In case of WT ISCU, a single elution
that WT ISCU can form a stable subcomplex with peak was obtained, corresponding to molecular
hNFS1 and HSCB. However, CoIP analysis with mass of 30-33 kDa (Fig. 9D and E). This suggests
HeLa cell mitochondria expressing G50E ISCU, that apo-ISCU possibly exist as a dimer, based on
showed significantly reduced immunoprecipitation the calculated molecular mass of 14.5 kDa, from
of hNFS1 and HSCB, highlighting the importance ISCU primary sequence. Interestingly, gel
of glycine 50 residue for the subcomplex filtration analysis of G50E ISCU mutant resulted
formation in vivo (Fig. 8I). A similar CoIP in the appearance of two elution peaks of different
analysis in isu1G50E yeast mitochondrial lysates molecular weights; a smaller peak with molecular
also showed impaired interaction of Isu1 with mass of 30-33 kDa corresponding to the dimeric
yeast yNfs1 and Jac1 (Fig. 8J). Interestingly, in the form. On the other hand, the second elution peak
crystal structure, the position of glycine residue is with a molecular mass of 180-185 kDa, probably
located adjacent to the interaction interface of IscS corresponds to the higher-order oligomeric forms

12
Role of ISCU in development of mitochondrial myopathy

of the G50E ISCU (Fig. 9F and G). Upon the stoichiometry of the protein association during
quantitation, we observed that the G50E ISCU the assembly process.
showed increased propensity to form larger
species with a ratio of 30% dimer to 70%
oligomeric form. To test the formation of
oligomeric species in G50E ISCU protein at in DISCUSSION
vivo conditions, the mitochondrial lysate was Following Friedreichs Ataxia, ISCU
subjected to BN-PAGE analysis followed by myopathy is the second most prevalent
immunostaining with ISCU-specific antibodies. pathophysiological condition, associated with
Besides dimeric forms, the G50E ISCU protein impairment of Fe-S cluster biogenesis in human
showed a distinct oligomeric species as compared mitochondria. However, the mechanistic insights
to wild type controls thus further supporting the into how mutations in the scaffold protein ISCU,
findings of the in vitro purified system (Fig. 9H). generates complex cellular traits, that contribute to
To unravel the mechanistic insights how the disease progression, is poorly understood. Our
overexpression of G50E ISCU or isu1G50E mutant experimental data provides the first compelling
leads to semidominant phenotype, we tested for evidence to highlight mitochondrial specific
etiology associated with ISCU myopathy

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their ability to form subcomplexes with WT
protein by CoIP analysis. For the mutation, G50E. The glycine 50 residue is highly
immunodetection, the wild type or G50E ISCU conserved in the ISCU class of proteins, across
was overexpressed as FLAG-tagged in HeLa cells phylogenetic boundaries. Owing to its conserved
and subjected for CoIP in the mitochondrial lysate nature, its importance in the Fe-S cluster
using anti-FLAG antibodies followed by scaffolding function is unanimous across species.
immunostaining with the ISCU-specific antibody. The G50E mutation in mammalian system causes
Both FLAG-tagged WT and G50E ISCU marked reduction in the cell viability, while the
displayed the ability to form the subcomplex with analogous mutation in yeast displays a lethal
endogenous WT ISCU (Fig. 9I, lower panel). As a phenotype, highlighting its critical nature for the
CoIP control, the blot was further probed for the protein function.
HSCB which showed a diminished interaction The Fe-S clusters are built in the ISCU/Isu1
(Fig. 9I, upper panel) with the G50E ISCU mutant platform through precise coordinated events
as compared to WT consistent with the previous involving iron and sulfur transfer process.
results (Fig. 8I). A similar CoIP analysis was Therefore, it is reasonable to predict that, the
performed in yeast mitochondrial lysate prepared mutations in ISCU scaffold protein can directly
from overexpressed either FLAG-tagged WT Isu1 influence the initial assembly process as well as
or isu1G50E using FLAG-specific antibodies. The the rate of Fe-S cluster synthesis in the
endogenous Isu1 protein was expressed as his6- mitochondrial matrix. In support of this
tagged and detected using an anti-His specific hypothesis, we observed that the G50E mutation in
antibody. Consistent with the findings of HeLa ISCU showed a significantly attenuated enzyme
cells, similar subcomplexes were detected between activity of proteins containing Fe-S clusters, both
FLAG-tagged WT or isu1G50E protein with in mammalian as well as in yeast system. Most
endogenous his6-tagged WT Isu1 (Fig. 9J, lower & notably, the activities of multi-enzyme complexes
middle panels). As CoIP control, the isu1G50E (I & II) of the electron transport chain (ETC) was
protein showed a diminished interaction with Jac1 considerably reduced due to compromised Fe-S
(Fig. 9J, upper panel) as compared to WT Isu1 cluster biogenesis, in G50E ISCU mitochondria.
coherent with the previous results (Fig. 8J). In This is in agreement with clinical symptoms of
conclusion, these findings highlights that the semi- ISCU myopathy patients showing marked
dominant phenotype associated with G50E ISCU impairment in the succinate oxidation to fumarate,
mutation is attributable to its tendency to form an due to decreased activity of succinate
oligomeric complex with endogenous wild type dehydrogenase and aconitase, in muscle
protein thus probably contributing towards the mitochondria (54). Besides, our experimental
impairment in the scaffolding function by altering evidence also suggests that the compromised

13
Role of ISCU in development of mitochondrial myopathy

activity of Fe-S cluster containing multi-enzyme neurodegenerative diseases (60-61). Importantly;

complex of ETC results in diminished pumping of our analyses demonstrate a significant up-
protons across the inner membrane leading to regulation of ROS levels in the G50E mutant
decreased membrane potential, ATP synthesis and mitochondria of mammalian as well as in yeast
thus overall reduced respiratory activity causes system. This is presumably due to a loss in ETC-
impaired mitochondrial function. Consequently, a complex activity in response to decreased
significant decrease in the overall mitochondria synthesis of Fe-S clusters thus enhancing the
mass was observed in the G50E ISCU mutant leakage of free electrons, and leading to
probably due to enhanced mitophagy. This is one production of superoxide radicals. Alternatively,
of the most important cellular etiologies that the mitochondrial overloading of reduced form of
contribute towards disease outcome, particularly in iron can also serve as a source for the production
skeletal muscles and cardiomyocytes that demand of potent hydroxyl radicals via Fenton's reaction
the highest energy requirement for their (62). These hydroxyl radicals are extremely
constitutive contractile functions. Therefore, the deleterious to the cellular components as it can
reduced respiratory activity associated with ISCU cause further damages to the pre-existing Fe-S
mutation may directly lead to the manifestation of clusters of the ETC-complexes, as well as other
disease symptoms such as severe muscle biomolecules, leading to severe oxidative stress

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weakness, muscle wasting and mild cardiac and mitochondrial dysfunction. Based on our
hypertrophy, as observed in affected individuals findings, we hypothesize that the free radical
(28). mediated damage may therefore significantly
contribute to the development of complex
Mitochondrial Fe-S cluster biogenesis secondary traits in myopathy patients.
impairment invariably associates with loss of iron
homeostasis inside cell (55). In a well- The initial Fe-S cluster assembly process is
characterized mitochondrial disease such as aided by a precise coordinated interaction of the
Friedreichs Ataxia, a defect in function of frataxin scaffold protein with putative iron donor such as
leads to decreased Fe-S cluster synthesis. Besides, frataxin/Yfh1 and cysteine desulfurase,
frataxin deficiency also results in increase in iron hNFS1/yNfs1, which is in complex with Isd11
levels in the mitochondrial matrix compartment protein. On the other hand, the molecular
(41). Our results highlight that, the reduction in chaperone J-protein, HSCB (Jac1 in yeast) and its
Fe-S cluster synthesis associated with myopathy partner mtHsp70 (Ssq1 in yeast) play a critical role
mutation in ISCU/Isu1 scaffold protein leads to in the transfer of Fe-S clusters from the ISCU/Isu1
accumulation of iron levels in the mitochondrial scaffold to recipient proteins (63). Both are
compartment similar to frataxin deficiency. Thus, indispensable processes for the homeostasis of
our study reconciles well with results of iron metabolism in the mitochondrial
histochemical analysis obtained from skeletal compartment. Our in vitro as well as ex vivo
muscle biopsy of myopathy patients that showed experiments reveal that the G50E mutation in
an intracellular iron overload (22). ISCU/Isu1 results in significant compromised
interaction with the sulfur donor, hNFS1/yNfs1
The reactive oxygen species (ROS) such as and J-protein cochaperone, HSCB/Jac1. As a
superoxide and peroxide radicals are primarily result, the myopathy associated ISCU mutant may
generated as a byproduct of oxidative have severe impairment in the initial synthesis of
phosphorylation, due to incomplete reduction of the cluster as well as in the rate of transfer into the
free electrons from the ETC-complexes (56-58). apoproteins, thereby affecting overall rate of Fe-S
However, the cellular redox balance is critically cluster biogenesis. Studies from the yeast model
regulated through multiple well-orchestrated system have earlier indicated that formation of the
pathways involving members of several Jac1-Isu1 complex as well as its targeting to the
antioxidant systems (59). The upregulation of Hsp70s is an essential process in the Fe-S cluster
ROS levels has been implicated in multiple biogenesis (15,20). Most notably, recent analysis
pathological conditions, including cardiovascular also revealed that yNfs1 and Jac1 both have
diseases, hypertension, atherosclerosis and mutually exclusive binding surface on Isu1, that

14
Role of ISCU in development of mitochondrial myopathy

amino-acid residues from the Isu1 core -strands system. Additionally, our findings delineate the
L63, V72 and F94 are involved in the interaction cellular mechanisms behind the impairment of
with Jac1 and yNfs1, in yeast (15). Strikingly, the mitochondrial function associated with G50E
G50E mutant also exhibits a similar compromised mutation leading to manifestation of pathological
interaction with both HSCB and hNFS1 indicating symptoms observed in ISCU myopathy. Although,
that they possess a mutually exclusive interaction our basic understanding about Fe-S cluster
surface on ISCU protein thus confirming the biogenesis is primarily obtained from a yeast
previous observation. Based on the severity of model system, a relevant complex process in the
phenotype observed with the myopathic mutation mammalian system is poorly understood.
in human as well as in the yeast system, it is Following Friedrichs ataxia, ISCU myopathy is
reasonable to predict that glycine 50 is one of the the second most prevalent mitochondrial disorder
critical residues required for mediating the hNFS1 identified with high penetrance rate in the
and HSCB interaction. European lineage and is connected with the
impairment in Fe-S cluster biogenesis. So, our
Though the functional oligomeric state of findings at the cellular as well as in molecular
human ISCU is not very clear, but the monomeric level should provide invaluable information to
form probably is not a functional entity in terms of understand the disease progression in both

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coordinating geometry to form Fe-S clusters. For homozygous and compound heterozygous affected
example, in Azotobacter vinelandii, and individuals.
Schizosaccharomyces pombe, IscU exists as a
dimer in which one or two [2Fe-2S] or [4Fe-4S]
clusters are constituted (53). In case of Aquifex
aeolicus, IscU exists in a trimeric form and the
[2Fe-2S] cluster is positioned at the interface
between two subunits, and the third subunit
provides additional stability for the interaction
(64). Results from gel filtration analysis
demonstrate that, WT apo-ISCU mostly exists in a
dimeric form whereas apo-G50E ISCU showed
higher tendency to form a larger oligomeric
species. Most notably, apo-G50E ISCU display a
tendency to form oligomeric subcomplexes with
wild type protein when overexpressed thus
imparting a semidominant negative phenotype.
The formation of such higher molecular weight
oligomers in G50E mutant in compound
heterozygous conditions may therefore impair the
precise co-ordination geometry required to
assemble the Fe-S cluster on ISCU. On the other
hand, the oligomeric forms of G50E mutant also
may hamper the physical interacting surface of
ISCU to its interacting partners hNFS1 or HSCB
resulting in their compromised interaction, thereby
abrogating the overall Fe-S cluster synthesis and
diminished mitochondrial function, in myopathy
patients.

In conclusion, our study elucidates insights

into the complex protein-protein interactions
involving the ISCU scaffold protein, in the process
of Fe-S cluster biogenesis, in the mammalian

15
Role of ISCU in development of mitochondrial myopathy

Footnotes

*This work was supported by research grant from Council of Scientific and Industrial Research
(CSIR-No. 37(1534)/12/EMR-II), India (to P.D.S).

Acknowledgments

We thank Dr Elizabeth A. Craig for providing the isu1/isu2 yeast strain and anti-Jac1 antibody. We
thank Dr. Roland Lill, Dr. Wing-Hang Tong for anti-yeast Nfs1 and anti-human NFS1 antibodies
respectively. We also thank the Flow Cytometry Facility of the Indian Institute of Science, Bangalore for
FACS experiments and Solid State and Structural Chemistry Unit (SSCU) of the Indian Institute of
Science, Bangalore for Atomic Absorption Spectroscopic analysis. The authors wish to thank Swarna
jayanthi Fellowship from DST, India (to PDS), Council of Scientific and Industrial Research, India for
Senior Research fellowship (to PPS, DS, GP), Department of Biotechnology for Research Associate
fellowship (to PKSK), India and DST, India for INSPIRE fellowship (to SS).

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Role of ISCU in development of mitochondrial myopathy

Figures legends

FIGURE 1. Comparative analysis of ISCU proteins and measurement of cellular viability. (A) A
proposed model of Fe-S cluster synthesis highlighting ISCU as a central scaffold in the biogenesis
process. Assembly of Fe-S cluster includes transfer of the sulfur atoms from NFS1-ISD11 complex and
iron from putative iron donor protein frataxin. The transfer and incorporation into recipient apoproteins
are facilitated by the ATP-dependent mtHsp70 chaperone GRP75 and the DnaJ-like cochaperone HSCB.
(B) Predicted orthologs of ISCU from different species; H. sapiens (Hs), M. musculus (Mm), S. cerevisiae
(Sc), A. thaliana (At) and E. coli (Ec) are aligned using ClustalW software. Identical, conserved and
semi-conserved residues are highlighted respectively in yellow, green and cyan color. The amino acid
positions corresponding to myopathy mutation (glycine 50) is boxed. (C) The structure of human ISCU
(44-162 aa) is modeled based on M. musculus Iscu (PDB ID 1wfz) using SWISS-MODEL program. The
N-terminal side chain depicting conserved residue glycine 50 is highlighted. The N- and C-terminal of
ISCU is abbreviated by N and C, respectively. (D) ISCU protein levels in the mitochondrial lysate of the
untransfected (UT) HeLa cells and cells carrying WT ISCU and G50E ISCU in a pCI-neo plasmid after
72 h of transfection. The mitochondrial protein Tim23 was used as a loading control. (E) MTT-assay for
the measurement of cellular viability (represented as bars) of UT, WT and G50E mutant in HeLa cells. (P-

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value of value of <0.05 was defined as significant, and asterisks are used to denote significance as
follows: *, P< 0.05; **, P< 0.01; ***, P< 0.001).

FIGURE 2. Measurement of Fe-S cluster enzyme activity, cellular respiration, mitochondrial mass
and membrane potential in HeLa cells. (A) The purity and enrichment of mitochondria obtained from
HeLa cells were analyzed by immunodecoration using antibodies against mitochondrial specific markers
as positive controls (Tim23, Tim44 and ISCU) and other organellar specific antibodies such as catalase
(peroxisomes), cathepsin (lysosomal) and nuclear (SOD) as negative controls. Equivalent amounts of cell
and mitochondrial lysates (100 g of protein) were loaded for the comparison. (B) The activity of
mitochondrial complex I of Electron Transport Chain (ETC) in untransfected (UT), HeLa cells
overexpressing WT ISCU and G50E ISCU in presence or absence of complex I inhibitor rotenone. (C)
Activity of ETC complex II in the presence of inhibitors of complex I, III and IV represented in bar
charts. (C) Assessment of the activity of mitochondrial matrix enzyme aconitase in HeLa cells denoted in
bar charts. (E) The activity of complex IV of ETC measured in the presence or absence of complex IV
inhibitor sodium azide (NaN3). (F) The relative ATP levels measured in mitochondria isolated from UT,
WT and mutant protein expressing HeLa cells using Mitochondrial ToxGlo Assay kit. (G, H) The
purified mitochondria isolated from HeLa cells were stained with the JC-1 dye and subjected for an
excitation at 490 nm followed by an emission wavelength scan ranging from 500 to 620 nm. The
fluorescence intensity values obtained were plotted against the wavelengths to calculate the relative
distribution of polarized versus depolarized mitochondria (G). A relative fluorescence intensity obtained
from the JC-1 dye (Fig. 2G) was quantified and plotted as a ratio of multimer (590 nm) to monomer (530
nm) (H). (I, J) The mean fluorescence intensity (MFI) histogram for UT, WT and G50E ISCU HeLa cells
stained with NAO followed by flow cytometric analysis for the measurement of overall mitochondrial
mass. The values were plotted based on three independent experiments (I). The total mitochondrial mass
for each HeLa cell lines obtained from (I) was quantitated, and normalized values were plotted in a bar
chart (J). (P-value of value of <0.05 was defined as significant, and asterisks are used to denote
significance as follows: *, P< 0.05; **, P< 0.01; ***, P< 0.001).

FIGURE 3. Estimation of total cellular and mitochondrial iron content of G50E ISCU in HeLa cells.
(A, B) Flow cytometric estimation of overall cellular free iron levels using calcein blue dye in
untransfected (UT) HeLa cells and cells harboring the WT ISCU and G50E ISCU. A total of 10,000

20
Role of ISCU in development of mitochondrial myopathy

events were analyzed for each case, and the fluorescence intensity values were plotted as a mean of three
independent experiments (A). The free iron content was quantitated from Fig. 3A FACS experiment and
represented in a bar chart (B). (C, D) Calcein blue fluorescence images of UT, WT ISCU, G50E ISCU
and G50E ISCU HeLa cells treated with 1 mM iron-chelator, deferoxamine (DFO (C). The calcein blue
fluorescence intensity obtained from Fig. 3C images was quantitated using ImageJ software and
represented in a bar chart (D). (E, F) The total cellular iron (E) and mitochondrial iron levels (F) in HeLa
cells were determined by colorimetric analysis and represented in a bar chart. The data is represented in a
bar chart after normalization of values obtained for G50E and WT ISCU against UT. (G, H) The total
cellular iron (G) and mitochondrial iron levels (H) in each HeLa cell type were determined using Atomic
Absorption Spectroscopy (AAS). The data is represented in a bar chart after normalization of values
obtained for UT and WT against G50E ISCU. (P-value of value of <0.05 was defined as significant, and
asterisks are used to denote significance as follows: *, P< 0.05; **, P< 0.01; ***, P< 0.001).

FIGURE 4. Estimation of mitochondrial and total cellular ROS of G50E ISCU in HeLa cells. (A, B)
The mitochondrial superoxide level in untransfected (UT) HeLa cells and cells harboring the WT ISCU

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and G50E ISCU were estimated flow cytometrically using fluorescent MitoSOX Red dye (A). The total
superoxide levels obtained from aforementioned experiment (A) was quantitated and represented in a bar
chart (B). The UT cells treated with 1 mM rotenone were used as positive control. For flow cytometry
experiments, 10,000 events were analyzed for each case, and the values were plotted based on three
independent experiments. (C, D) Untransfected HeLa cells and cells transfected with WT ISCU and G50E
ISCU were stained with mitochondrial superoxide indicator, MitoSOX Red (red fluorescence) and
nuclear counterstain Hoechst 33342 (blue fluorescence) and the images were recorded in a Ziess Apotome
fluorescence microscope using 63X objective lens (C). The total MitoSOX Red fluorescence intensity of
images from aforementioned experimental was quantitated using ImageJ software (D). (E, F) The
fluorescence images of UT HeLa cells and cells harboring WT ISCU and G50E ISCU stained with
H2DCF-DA dye to detect enhancement in overall cellular ROS levels (green fluorescence) (E). The
images were recorded in a Leica fluorescence microscope using 63X objective lens. The total H2DCF-DA
dye fluorescence intensity from the images (E) was quantitated using ImageJ software (F). (P-value of
value of <0.05 was defined as significant, and asterisks are used to denote significance as follows: *, P<
0.05; **, P< 0.01; ***, P< 0.001).

FIGURE 5. Growth and functional defects associated with analogous G50E mutation in yeast Isu1.
(A, B) The isu1/isu2 yeast strain carrying a WT copy of ISU1 in pRS316 plasmid transformed with WT
ISU1 or isu1G50E mutant and subjected for serial drop dilution analysis on 5-FOA medium and incubated
at indicated temperatures for 96 h (upper panel). Similarly, Human WT ISCU and G50E ISCU mutant in
pRS414 TEF vector were transformed in isu1/isu2 yeast strain and subjected to serial drop dilution
analysis on 5-FOA medium as described in the aforementioned analysis (lower panel). (B) The isu1/isu2
yeast strain harboring a WT copy of ISU1 in pRS316 plasmid was transformed with WT ISU1 and mutant
isu1G50E in a centromeric plasmid pRS414 TEF (upper panel) or pRS414 GPD (lower panel). Cells were
spotted on Trp- plates followed by incubation for 72 h at indicated temperatures. (C) Immunoblot analysis
for Isu1 protein levels in the mitochondrial lysates of overexpressed strains (under pRS414 GPD vector as
indicated in B) prepared from WT and mutant yeast cells using anti-FLAG antibodies. The mitochondrial
protein Mge1 was used as a loading control. (D, E) The estimation of mitochondrial mass was determined
by flow cytometric analysis using NAO dye. For each flow cytometric analysis, 10,000 events were
analyzed and the relative fluorescence intensity values obtained from three independent experiments were
plotted (D). Relative mitochondrial mass in each yeast strain was determined from aforementioned FACS
analysis was quantitated (E). (F, G) Purified mitochondria from yeast strains were stained with JC-1 dye
to determine the mitochondrial membrane potential by scanning the emission wavelength ranging from

21
Role of ISCU in development of mitochondrial myopathy

500 to 620 nm and the fluorescence intensity was plotted against the wavelength (F). Mitochondria
treated with the valinomycin were used as a positive control. The ratio of fluorescence intensities between
multimer (590 nm) to monomer (530 nm) was quantitated and represented in a bar chart (G). (P-value of
value of <0.05 was defined as significant, and asterisks are used to denote significance as follows: *, P<
0.05; **, P< 0.01; ***, P< 0.001).

FIGURE 6. Measurement of enzymatic activity, respiration and iron levels in isu1G50E mutant yeast
strains. (A) Activity of mitochondrial complex II of Electron Transport Chain (ETC) in yeast strains
carrying either vector alone control (pRS414), WT ISU1 and isu1G50E in pRS414 GPD plasmid in the
presence or absence of inhibitors of complex I, III and IV. (B) Bar chart indicating the activity of complex
IV of ETC in yeast cells in the presence or absence of complex IV inhibitor sodium azide (NaN3). (C)
Activity of mitochondrial matrix enzyme aconitase in aforementioned yeast strains. (D) Quantification of
ATP levels in the mitochondria isolated from WT, and mutant yeast strains were measured using
Mitochondrial ToxGlo Assay. (E, F) Estimation of total cellular iron content (E) and mitochondrial iron
levels (F) in WT and mutant strains were determined by colorimetric analysis. The normalized values

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against the mutant strain are plotted in a bar chart. (G, H) Measurement of total cellular iron content (G)
and mitochondrial iron levels (H) in aforementioned yeast strains obtained using AAS analysis. The
normalized values against mutant strain are plotted in a bar chart. (P-value of value of <0.05 was defined
as significant, and asterisks are used to denote significance as follows: *, P< 0.05; **, P< 0.01; ***, P<
0.001).

FIGURE 7. Estimation of cellular ROS levels and oxidative stress sensitivity of isu1G50E. (A, B) Flow
cytometric analysis of mitochondrial superoxide levels in yeast strains carrying WT ISU1 and isu1G50E in
pRS414 GPD plasmid is estimated by MitoSOX Red fluorescence (A). Relative superoxide level
obtained from the aforementioned analysis was quantitated and represented in a bar chart (B). WT cells
treated with 1 mM rotenone was set as positive control. For flow cytometry experiments, 10,000 events
were analyzed for each case, and the values were plotted based on three independent experiments. (C)
Assessment of oxidative stress sensitivity of WT and mutant strains performed by drop test analysis on
selective medium with and without treatment of 1 mM H2O2 for 2 h. Equivalent numbers of yeast cells
from the WT Isu1 and isu1G50E strains were spotted by serial dilution and allowed them to grow at 37 C
for 72 h. (D) Fluorescence imaging analysis of WT Isu1 and isu1G50E yeast strains stained with
mitochondrial superoxide indicator MitoSOX Red (red fluorescence). The images obtained for untreated
strains are represented in left panels [Bright Field (BF); MitoSOX (middle) and Merge column]. The
images acquired for the strains after treatment with 1 mM H2O2 for 2 h are indicated in right panels. (E)
Fluorescence images of WT Isu1 and isu1G50E yeast strains stained with cellular peroxide indicator
H2DCF-DA (green fluorescence). The images obtained for untreated strains are represented in left panels
and after treatment with 1 mM H2O2 for 2 h is indicated in right panels. The images were recorded in
Leica fluorescence microscope using 100X objective lens. (P-value of value of <0.05 was defined as
significant, and asterisks are used to denote significance as follows: *, P< 0.05; **, P< 0.01; ***, P<
0.001).

FIGURE 8. Interaction of G50E mutant (ISCU/Isu1) with hNFS1/yNfs1 and HSCB/Jac1 by GST
pull down analysis. (A-D) Pre-bound 1.5 M of GST-hNFS1 (A) and 1.0 M of GST-HSCB (C) were
incubated with increasing concentrations of purified WT and mutant human ISCU protein as indicated.
The unbound proteins were washed with buffer and analyzed by SDS-PAGE followed by Coomassie dye
staining. GST alone (2.5 M) was used as a negative control and 25% input of ISCU served as a loading

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Role of ISCU in development of mitochondrial myopathy

control. The band intensities were densitometrically quantitated using ImageJ software for hNFS1 (B) and
HSCB (D). (E-H) Pre-bound 1.5 M of GST-yNfs1 (E) and 2.5 M of GST-Jac1 (G) were incubated with
increasing concentrations of purified WT and mutant yeast Isu1 protein as indicated. The unbound
proteins were washed with buffer and analyzed by SDS-PAGE followed by Coomassie dye staining. The
band intensities were densitometrically quantitated using ImageJ software for yNfs1 (F) and Jac1 (H). (I)
Mitochondria lysates were prepared from HeLa cells expressing either FLAG-tagged WT or G50E ISCU
in buffer A containing 0.2% Tween 20 and subjected for immunoprecipitation using anti-FLAG
antibodies. Fractions were analyzed for the presence of hNFS1 and HSCB by immunostaining. 10% of
total mitochondrial extract was used as input CoIP control (Right panel). (J) FLAG-tagged WT Isu1 or
isu1G50E mutant mitochondrial lysates were subjected to immunoprecipitation using anti-FLAG antibodies
and analysed for the presence of yNfs1 and Jac1 by immunostaining. 10% of total mitochondrial extract
was used as input CoIP control (Right panel). (K) Surface representation of (IscS-IscU)2 complex
showing the two IscS molecules (green and cyan) and the two IscU molecules (purple and yellow).
Close-up view: IscU surface highlights the Glycine position in red, located adjacent to the interface of
IscS and IscU.

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FIGURE 9. Secondary structure analysis and oligomerization study of G50E ISCU. (A) UV CD
spectra of WT ISCU (solid line) and G50E ISCU (dashed line) in 20 mM phosphate buffer (pH 8) were
recorded at 10 C. (B, C) 5 g of WT ISCU (left panel) or G50E ISCU (right panel) protein was
preincubated in cleavage buffer for 20 min at 10 C. The proteolysis was initiated by addition of 1 l
(1:50 dilution of 1g/l stock) of trypsin (B) and/or chymotrypsin (C) as indicated. The reaction was
stopped at the indicated time intervals using 2 mM PMSF. The samples were boiled in SDS sample buffer
and analyzed using SDS-PAGE followed by Coomassie dye staining. (D, E) Gel filtration
chromatography elution profile of WT ISCU protein separated on Supedex-200 column with a single peak
corresponding to the molecular size of ~30-33 kDa is indicated (D). The elution fractions from the gel
filtration of WT ISCU was analyzed on 15% SDS-PAGE and stained with Coomassie dye (E). (F, G) Gel
filtration elution profile of G50E ISCU mutant separated under similar conditions. The elution profile
indicating two major peaks corresponding to molecular sizes ~30-33 kDa and ~180-185 kDa are
highlighted (F). The eluted fractions from the gel filtration of G50E ISCU mutant were separated on 15%
SDS-PAGE and stained with Coomassie blue dye (G). (H) Mitochondrial lysate (500 g of the protein)
prepared from HeLa cells expressing either WT or G50E ISCU mutant was separated on BN-PAGE
followed by immunostaining with anti-ISCU specific antibody. The position of dimer and oligomers are
highlighted with reference to BN-PAGE markers. (I) Mitochondria lysates were prepared from HeLa
cells expressing either FLAG-tagged WT or G50E ISCU in buffer A containing 0.2% Tween 20 and
subjected for immunoprecipitation using anti-FLAG antibodies. The presence of endogenous WT ISCU
and HSCB were detected by immunostaining using either anti-ISCU or HSCB specific antibodies (left
panel). 10% of total mitochondrial extract used as input loading controls for CoIP (Right panel). (J) CoIP
was performed using mitochondrial lysate prepared from yeast cells expressing either FLAG-tagged WT
Isu1 or isu1G50E and subjected to immunodetection. The blot was detected for the presence of endogenous
his6-tagged WT Isu1 and Jac1 using anti-His, Jac1 specific antibodies (left panel). 10% of total
mitochondrial extract was used as input CoIP control (Right panel).

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 Multiple Cellular Defects Associated with a Novel G50E ISCU Mutation leads to
Development of Mitochondrial Myopathy
Prasenjit Prasad Saha, Praveen Kumar S. K., Shubhi Srivastava, Devanjan Sinha, Gautam
Pareek and Patrick D'Silva
J. Biol. Chem. published online February 26, 2014

Access the most updated version of this article at doi: 10.1074/jbc.M113.526665

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