Biochem. J. (2015) 471, 231–241
Human mitochondrial MIA40 (CHCHD4) is a component of the Fe–S cluster export machinery Anjaneyulu Murari*, Venkata Ramana Thiriveedi*, Fareed Mohammad*, Viswamithra Vengaldas*, Madhavi Gorla*, Prasad Tammineni*, Thanuja Krishnamoorthy* and Naresh Babu V. Sepuri*1 *Department of Biochemistry, University of Hyderabad, Gachibowli, Hyderabad 500046, India
Mitochondria play an essential role in synthesis and export of iron–sulfur (Fe–S) clusters to other sections of a cell. Although the mechanism of Fe–S cluster synthesis is well elucidated, information on the identity of the proteins involved in the export pathway is limited. The present study identifies hMIA40 (human mitochondrial intermembrane space import and assembly protein 40), also known as CHCHD4 (coiled-coil–helix–coiled-coil–helix domain-containing 4), as a component of the mitochondrial Fe–S cluster export machinery. hMIA40 is an iron-binding protein with the ability to bind iron in vivo and in vitro. hMIA40 harbours CPC (Cys-Pro-Cys) motif-dependent Fe–S clusters that are sensitive to oxidation. Depletion of hMIA40 results in accumulation of
iron in mitochondria concomitant with decreases in the activity and stability of Fe–S-containing cytosolic enzymes. Intriguingly, overexpression of either the mitochondrial export component or cytosolic the Fe–S cluster assembly component does not have any effect on the phenotype of hMIA40-depleted cells. Taken together, our results demonstrate an indispensable role for hMIA40 for the export of Fe–S clusters from mitochondria.
that are exported through the ISE machinery that is present in the mitochondria. Atm1, an inner membrane protein of yeast mitochondria, and its mammalian homologue ABCB7 (ATPbinding cassette transporter B7), and Erv1, an intermembrane space thiol oxidase, and its mammalian homologue ALR have been identified as components of the ISE machinery of mitochondria [13,15,16]. The phenotype that is associated with deficiency of Atm1 or Erv1 includes accumulation of iron in mitochondria and defects in maturation of cytosolic proteins that contain Fe–S clusters. Although there are reports suggesting that it may be exporting sulfur to the cytosol, the exact component that is exported by Atm1 or Erv1 and the export mechanism are not known [17,18]. Interestingly, Erv1 is also a member of the mitochondrial protein import machinery. Erv1 is specially required for the import of intermembrane space proteins of mitochondria. Erv1, along with Mia40, an intermembrane space protein of mitochondria, functions in the import of numerous cysteine-rich intermembrane space proteins by an oxidative folding mechanism [19,20]. Mia40 harbours six conserved cysteine residues that are clustered in the form of one CPC (Cys-Pro-Cys) and two CX9 C (Cys-Xaa9 -Cys) motifs. Mia40, through its CPC motif, promotes the oxidative folding of precursor proteins. In contrast, the CX9 C motifs are involved in creating intramolecular disulfide bonds for stabilizing the structure of Mia40 [21,22]. hMIA40 (human mitochondrial intermembrane space import and assembly protein 40), also known as CHCHD4 (coiled-coil–helix–coiled-coil–helix domain-containing 4), a homologue of yeast Mia40 contains similar cysteine motifs and is also involved in the import of intermembrane space-targeted proteins [23,24].
Fe–S (iron–sulfur) clusters are essential inorganic structures required by all organisms across evolution from bacteria to humans to perform various cellular functions . Several enzymes that are involved in a variety of biological processes such as electron transfer, redox catalysis, DNA replication and repair, and regulation of gene expression contain Fe–S clusters as prosthetic groups [2–6]. The synthesis, maturation and transfer of Fe–S clusters to apoproteins are very complex processes requiring multiple protein components present in mitochondria and cytosol. Several studies in yeast suggest the existence of three different assembly systems for the biogenesis of Fe–S clusters. These are the ISC (iron–sulfur cluster) assembly system, the ISE (iron–sulfur exporter) system and the CIA (cytosolic iron–sulfur assembly) system [7–10]. The first two systems are present in the mitochondria, whereas the last-named system is present in the cytosol. Homologues of yeast proteins that have been implicated in the biogenesis of Fe–S clusters exist in higher eukaryotes as well. The Fe–S cluster systems in mammals are functionally equivalent to the yeast systems despite some of the components of ISC system being found in cellular organelles other than the mitochondria . The ISC system is required for the maturation and functioning of mitochondrial enzymes that contain Fe–S clusters . The components required for the ISC system are present in the mitochondrial matrix. Besides the assembly and insertion of Fe– S clusters, the ISC system of mitochondria is also essential for the maturation of cytosolic and nuclear proteins that contain Fe– S clusters [13,14]. This process probably uses the Fe–S clusters
Key words: CIA, GPAT, hMIA40, iron export, iron–sulfur (Fe–S) cluster, mitochondria.
Abbreviations: ABCB7, ATP-binding cassette transporter B7; CIA, cytosolic iron–sulfur assembly; DCPIP, 2,6-dichlorophenol-indophenol; DMEM, Dulbecco’s modified Eagle’s medium; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GPAT, glutamine phosphoribosylpyrophosphate amidotransferase; HEK, human embryonic kidney; hMIA40, human mitochondrial intermembrane space import and assembly protein 40; ISC system, iron–sulfur cluster system; ISE system, iron–sulfur exporter system; mtHsp70, mitochondrial heat-shock protein 70; Ni-NTA, Ni2 + -nitrilotriacetate; NUBP1, nucleotide-binding protein 1. 1 To whom correspondence should be addressed (email [email protected]
or [email protected]
). c 2015 Authors; published by Portland Press Limited
A. Murari and others
An in vitro study from Colin Thorpe’s group reported that hMIA40 harbours an Fe–S cluster . A more recent study by Spiller et al.  confirms that, consistent with the hMIA40, yeast Mia40 also contains a Fe–S cluster. It has been assumed that the Fe–S cluster is required for the oxidoreductase activity of both hMIA40 and yeast Mia40. However, the precise physiological significance of the Fe–S cluster in hMIA40/Mia40 is yet to be understood. In the present study, we found that hMIA40 harbours Fe–S clusters and functions in the cellular iron homoeostasis pathway in addition to its involvement in protein import. We find that hMIA40 binds iron in vivo and in vitro and contains oxidationsensitive Fe–S clusters. Depletion of hMIA40 by RNAi increases the iron load in mitochondria and concomitantly reduces very specifically the activity and stability of cytosolic enzymes that contain Fe–S clusters, albeit with no effect on mitochondrial Fe–S-containing proteins. Our study suggests that hMIA40 is an important component of the ISE machinery of mitochondria. EXPERIMENTAL Plasmid construction
The cDNA encoding hMIA40 was generated using total RNA from HeLa cells (Bangalore GeNei) as a template. The hMIA40 ORF was amplified from cDNA by using primers MIA40-Fwd1 (5 -CCCAGAATTCACCATGTCCTATTGCCGGCAGGAA-3 ) and MIA40-Rev1 (5 -CCACTCGAGTTAACTTGATCCCTCCTCTTCTTT-3 ). The hMIA40 ORF was cloned as an EcoRI– XhoI (recognition sequences are underlined in the primers) fragment into pET28 (a + ) vector to generate plasmid pNB130 carrying hMIA40 with a histidine tag at the N-terminus. Plasmid pNB130 harbouring the wild-type hMIA40 was subjected to site-directed mutagenesis as described in the manufacturer’s protocol (Fermentas) to create plasmids pNB309 (hMIA40 C53S&C55S), pNB310 (hMIA40 C64S) and pNB311 (hMIA40 C87S). For expression in mammalian cells, hMIA40 CPC motif cysteine mutants were cloned into pcDNA3.1 Myc-His vector with EcoRI and XhoI restriction sites to generate plasmids pNB202 (hMIA40) and pNB314 (hMIA40 C53S&C55S). Expression and purification of recombinant proteins
Escherichia coli Rosetta gami strain was transformed with plasmids pNB130, pNB309, pNB310 and pNB311. All recombinant cysteine mutant proteins were expressed and purified as described in [27,28]. Briefly, expression of recombinant proteins was induced by addition of 1 mM IPTG to bacterial cultures when they attained OD600 of 0.8. The expressed soluble recombinant proteins were purified on a Ni-NTA (Ni2 + nitrilotriacetate) affinity column (Clontech) using buffer A (50 mM Tris/HCl, pH 7.5, 100 mM NaCl and 2 mM DTT). The column was washed with buffer A containing 10 mM imidazole. Elution was carried out with buffer A containing 400 mM imidazole. UV absorption spectroscopy analysis
UV–visible spectra in the range 250–700 nm for recombinant proteins were recorded in a Hitachi U-2910 spectrophotometer at room temperature using a quartz cuvette of 1 cm pathlength . Recombinant proteins were analysed spectroscopically at 410 and 460/280 nm to calculate the metal/protein ratio as described in . c 2015 Authors; published by Portland Press Limited
Atomic emission spectroscopy analysis
For atomic emission spectroscopy analysis, recombinant hMIA40 protein was initially affinity purified on a Ni-NTA column as described above except that 5 mM sodium dithionite was used instead of 2 mM DTT in buffer A. The protein was converted into ash and analysed using atomic emission spectroscopy (GSI).
Reconstitution of Fe–S clusters in vitro
Reconstitution of Fe–S clusters in purified hMIA40 was carried out in an anaerobic chamber as described in . In brief, purified hMIA40 (∼30 μM) was incubated for 1 h in an anaerobic chamber to remove dissolved oxygen before the addition of 2 mM freshly prepared DTT. The sample was mixed gently for 30 min. To this sample, 10-fold excess of ferrous ammonium sulfate (300 μM) and sodium sulfide (300 μM) were added and gently stirred for 3 h. The insoluble material was removed by centrifugation at 20 817 g for 30 min at 4 ◦ C. The supernatant was dialysed against the storage buffer (50 mM Tris/HCl, pH 7.5, 100 mM NaCl and 2 mM DTT) and used for spectroscopic measurement for assembly of Fe–S. Three independent reconstitution studies were carried out to test the reproducibility and analysis.
Cell culture and transfection
HEK (human embryonic kidney)-293T cells were cultured in DMEM (Dulbecco’s modified Eagle’s medium) (Invitrogen) containing 10 % (v/v) FBS at 37 ◦ C under an atmosphere of 5 % CO2 . Cells growing in 175-mm-high flasks were transfected with either 20 μg of scrambled shRNA or hMIA40 shRNA plasmid (OriGene) by using LipofectamineTM transfectant agent (Invitrogen). For overexpression of hMIA40, HEK-293T cells were transfected with mammalian expression vector pCDNA3.1 Myc vector harbouring hMIA40 ORF (pNB202) and pNB314 (hMIA40 C53S&C55S). For co-expression studies, HEK-293T cells were co-transfected with 15 μg of each plasmid hMIA40 shRNA/Myc–ALR–Myc and hMIA40 shRNA/Myc–NUBP. To demonstrate the specificity and rescue of shRNA effects, 10 μg of shRNA (OriGene) targeted to the non-coding region of hMIA40 (5 -GGTACTACCACAGAGCTGGAGCTGAGGAA-3 ) was cotransfected with or without pNB202 (hMIA40) and pNB314 (hMIA40 C53S&C55S) plasmids into HEK-293T cells.
Isolation of mitochondria from HEK-293T cells
Mitochondria were isolated from HEK-293T cell lines as described in . Briefly, HEK-293T cells were grown as monolayers and harvested in mitochondria isolation buffer (20 mM Hepes, pH 7.5, 1.5 mM MgCl2 , 1 mM EDTA, 1 mM EGTA, 210 mM sucrose and 70 mM mannitol). The cell suspension was subjected to homogenization using a Polytron 1600 instrument with two 5 s pulses at 15 rev./min. The lysate was subjected further to Dounce homogenization. The homogenate was centrifuged at 1000 g for 10 min at 4 ◦ C to separate the nucleus, and the supernatant was again centrifuged at 10 000 g for 15 min at 4 ◦ C. The resultant mitochondrial pellet was washed twice and suspended in a buffer containing 250 mM sucrose, 5 mM magnesium acetate, 40 mM potassium acetate, 10 mM sodium succinate, 1 mM DTT and 20 mM Hepes/KOH, pH 7.4.
Role of hMIA40 in iron homoeostasis Measurement of iron in mitochondria
The non-haem iron present in the mitochondria was measured using the bathophenonthroline method . Briefly, mitochondria were suspended in medium containing 10 mM Mes, pH 4.5, 1 % SDS and 0.5 mM dithionite followed by addition of 50 μM bathophenonthroline. Fe(II)–chelate formation was measured using a dual-wavelength Hitachi spectrophotometer at 540 nm and 575 nm. To measure the uptake of radioactive 55 Fe (American Radiolabeled Chemicals) by mitochondria, 50 % confluent HEK-293T cells were incubated with 500 nM 55 Fe for 48 h before isolation of mitochondria. Isolated mitochondria were washed with ice-cold bathophenonthroline (500 μM) to remove membrane-bound 55 Fe. The amount of radioactivity present in the mitochondrial fraction was measured using a Beckman scintillation counter. Measurement of cytosolic iron
The cytosolic iron content was measured using radioactive 55 Fe. To measure the 55 Fe present in cytosol, 50 % confluent HEK-293T cells were incubated with 500 nM 55 Fe for 48 h before isolation of mitochondria. The amount of radioactivity present in the cytosol (post-mitochondrial supernatant) was measured using a Beckman scintillation counter. Immunoprecipitation
HEK-293T cells were transfected with mammalian expression vector pcDNA3.1 Myc (His) or vector (Invitrogen) harbouring hMIA40 ORF. After 36 h, cells were allowed to grow in serumfree DMEM for 6 h and thereafter incubated with radioactive 55 Fe and 1 mM sodium ascorbate for 2 h. Next, cells were lysed with NP40 buffer (20 mM Tris/HCl, pH 8.0, 137 mM NaCl, 10 % glycerol and 1 % Nonidet P40) and lysates were immunoprecipitated with anti-hMIA40 antibody. The amount of 55 Fe radioactivity in the immunoprecipitate was measured using a Beckman scintillation counter. For the hMIA40 cysteine mutant experiment, wild-type or mutant cell lysates were incubated with Ni-NTA beads under reduced conditions (1 mM DTT) at room temperature for 2 h. Ni-NTA beads were washed three times with NP40 buffer and the 55 Fe present in Ni-NTA beads was measured directly using a Beckman scintillation counter.
with 5 mM NADH in a buffer containing 25 mM potassium phosphate, pH 7.4, 5 mM MgCl2 and 0.25 % BSA for 1 min at 37 ◦ C. The activity assay was initiated by addition of 3 mM decylubiquinone and the decrease in absorbance at 340 nm was measured. Complex I activity was calculated by using the velocity of reaction (absorbance/min) and the molar absorption coefficient of NADH (3.4 mM − 1 ·cm − 1 at 340 nm with reference wavelength). Mitochondrial Complex II activity was measured at 600 nm using a spectrophotometer as described in . Briefly, isolated mitochondria (100 μg) were solubilized in 0.05 % Triton X100 and divided into two. One part was used as blank and contained mitochondrial lysate and 50 mM potassium phosphate buffer. The second sample contained mitochondrial lysate, 50 mM potassium phosphate buffer and 5 mM succinate. Both samples were incubated at 37 ◦ C for 10 min and then DCPIP (2,6-dichlorophenol-indophenol) was added as a chromophore. Oxidized DCPIP is blue in colour and becomes colourless upon acceptance of electrons from Complex II. Complex II activity is calculated by using the velocity of reaction and the molar absorption coefficient of DCPIP (16.9 mM − 1 ·cm − 1 ) at 600 nm. Western blotting
Cell lysate (100 μg) and recombinant proteins were separated by SDS/PAGE and transferred on to the nitrocellulose membrane (Pall). The blots were probed with primary antibodies against hMIA40, GAPDH (glyceraldehyde-3-phosphate dehydrogenase), GPAT (glutamine phosphoribosylpyrophosphate amidotransferase), Myc, ALR, NUBP1 (nucleotide-binding protein 1) and mtHsp70 (mitochondrial heat-shock protein 70) (Abcam) followed by HRP (horseradish peroxidase)-coupled rabbit or mouse secondary antibodies and developed using ECL Prime (GE Healthcare) and a Bio-Rad Laboratories imaging system. Statistical analysis
Data were analysed by one-way ANOVA with Bonferroni’s posthoc test. A P-value less than 0.05 was considered significant. RESULTS AND DISCUSSION hMIA40 contains oxidative-sensitive Fe–S clusters
Xanthine oxidase activity resident in the cytosolic fraction was measured as described in . Briefly, the cytosolic fraction obtained from HEK-293T cells was incubated with 0.05 mM xanthine in a buffer containing 33 mM potassium phosphate (pH 7.5). The reaction mixture was mixed and equilibrated to 25 ◦ C. The increase in absorbance (0–5 min) at 290 nm was measured and the enzyme activity was calculated based on standard xanthine oxidase activity (Sigma). Aconitase activity was measured in the cytosolic and mitochondrial fraction isolated from HEK-293T cells. The fraction was incubated with 1 mM isocitrate in a buffer containing 50 mM Tris/HCl, pH 7.5, and 5 mM MnCl2 . The conversion of isocitrate into cis-aconitate was measured as an increase in absorbance at 240 nm. Mitochondrial Complex I activity was determined by measuring the oxidation of NADH to NAD + at 340 nm with 380 nm as the reference wavelength at 37 ◦ C . A 50 μg amount of 0.05 % Triton X-100-treated mitochondria was incubated
Akin to its homologue in yeast, hMIA40 contains a redox-active CPC motif and two structural CX9 C motifs (Figure 1A). In addition, it was also reported that hMIA40 is an iron-binding protein in vitro . On the basis of the structural similarities and the in vitro results, we hypothesized that hMIA40 may indeed harbour Fe–S clusters in vivo. To test this hypothesis, we first wished to confirm that recombinant hMIA40 can bind iron in vitro. Towards this end, hMIA40 was expressed in bacteria as His– hMIA40 and affinity purified to more than 95 % homogeneity (Figure 1B). Purified protein migrates as a 18 kDa protein on an SDS/PAGE gel as predicted from its sequence. However, we also found a small additional, but significant, 70 kDa protein band in all our preparations of recombinant hMIA40 even under reducing conditions. We identified this 70 kDa band also as hMIA40 by MS/MS analysis (Supplementary Figure S1). Purified protein was further subjected to atomic emission spectroscopy analysis . As shown in Figure 1(C), recombinant hMIA40 indeed contains a small, but significant, amount of iron and a negligible amount of manganese. The molar ratio of metal to protein is approximately 0.068, indicating that, like its yeast homologue, c 2015 Authors; published by Portland Press Limited
A. Murari and others
hMIA40 contains oxidative-sensitive Fe–S clusters
(A) Schematic representation of hMIA40 showing one CPC redox-active motif and two CX9 C structural motifs. (B) The affinity-purified His–hMIA40 protein was resolved by SDS/PAGE under reducing conditions. hMIA40 migrates as a monomeric 18 kDa protein and as a small 70 kDa protein. The 70 kDa form was excised and subjected to MS/MS analysis to confirm hMIA40. Molecular masses are indicated in kDa. (C) AES analysis of purified recombinant His–hMIA40 to determine the metal content. (D) HEK-293T cells were transiently transfected for overexpression of Myc–His–hMIA40 followed by incubation with 55 Fe. Cell lysates were prepared using NP40 buffer and immunoprecipitation was carried out with either pre-immune serum (PI) or anti-hMIA40 antibody. 55 Fe present in the immunoprecipitate was measured using a scintillation counter and is shown as c.p.m./mg of protein. (E) Immunoprecipitated samples were separated by SDS/PAGE and probed with antibodies specific for hMIA40. (F) UV–visible spectra (250–700 nm) of purified recombinant His–hMIA40. Arrows indicate the characteristic peaks at 410 and 460 nm. (G) Time course of the absorption intensity changes of His–hMIA40 when exposed to air or (H) 1 mM H2 O2 or (I) in the absence and presence of 5 mM DTT. Results in histograms are means for three independent experiments. P < 0.001. OD, absorbance.
a certain fraction of hMIA40 has the ability to bind to iron in vitro. Furthermore, the observed low metal/protein ratio in the recombinant hMIA40 may be due to the loss of some metal during purification. We investigated further whether hMIA40 can bind to iron in vivo. To enrich for endogenous hMIA40 in vivo, HEK-293T cells were transiently transfected with plasmid containing pcDNA-hMIA40 and incubated with 55 Fe for 24 h. Thereafter, cells were harvested, lysed and the cell lysates were subjected to immunoprecipitation with hMIA40-specific antibody or pre-immune serum. The amount of radioactivity present in the immunoprecipitate was determined using a scintillation counter (Figure 1D). The immunoprecipitate of the cell lysate after using anti-hMIA40 antibody was more enriched in 55 Fe compared with the immunoprecipitate from pre-immune serum. We validated the immunoprecipitation of hMIA40 by probing the immunoprecipitated samples with the antibody specific for hMIA40 (Figure 1E). Taken together, our results indicate that c 2015 Authors; published by Portland Press Limited
hMIA40 binds to iron both in vitro and in vivo. Furthermore, the ability of hMIA40 to bind iron in vivo is found to be conserved from yeast to humans . To evaluate the presence of Fe–S clusters in hMIA40, we utilized the property of absorption of Fe–S in the UV– visible spectrum . Purified recombinant hMIA40 displayed absorption peaks at 410 nm and 460 nm in the visible spectrum, a characteristic spectral feature of a protein harbouring Fe–S clusters (Figure 1F). Similar to our result, bacterially expressed yeast Mia40 shows absorbance at 335, 400 and 460 nm . Furthermore, the yeast Mia40 was shown to carry a 2Fe–2S cluster. Although the 410 nm and 460 nm absorption peaks displayed by recombinant hMIA40 are a characteristic spectral feature of 4Fe–4S and 2Fe–2S respectively , EPR studies are required to confirm further the precise Fe–S clusters that are present in hMIA40. Fe–S clusters that are not an integral structural component of a protein are known to be more
Role of hMIA40 in iron homoeostasis
Depletion of hMIA40 results in accumulation of iron in mitochondria
HEK-293T cells were transfected with hMIA40 shRNA or scrambled shRNA vector control and the mitochondrial or cytosolic fraction was isolated. (A) Western blot analysis of whole-cell extracts of HEK-293T cells. The blot was probed with anti-hMIA40 (α-MIA40) and anti-mtHsp70 (α-mHSP70) antibodies. mtHsp70 was used as an internal loading control. Molecular masses are indicated in kDa. (B) Relative levels of hMIA40 in control and shRNA hMIA40 cell extracts obtained from three independent experiments. (C) Mitochondrial non-haem iron was measured using the bathophenonthroline method and is shown as nM/μg of mitochondrial protein. (D and E) 55 Fe present in mitochondria (D) or cytosol (E) was measured using a Beckman scintillation counter and is shown as c.p.m./mg. Results in histograms are means for three independent experiments. P < 0.001. The error bars represent the S.D.
sensitive to an oxidative environment . To test the stability of the Fe–S clusters detected in hMIA40, absorption of purified recombinant hMIA40 in the UV–visible spectrum was monitored after exposing it to air and oxidative conditions. A decrease in absorption can be directly correlated to a loss in stability of Fe– S clusters . We observed a gradual decrease in absorption when hMIA40 was either exposed to air (Figure 1G) or H2 O2 (Figure 1H) at room temperature. These results suggest that the Fe–S clusters are sensitive to oxidation. However, in the presence of a reducing agent, there is a small, but steady, increase in the absorption for 10 min followed by a decline (Figure 1I). The initial increase in absorption could reflect the increased stabilization of Fe–S clusters under reducing conditions. However, this could paradoxically increase the exposure of the increased Fe–S clusters to oxidation by air that is reflected in the fall in absorbance. The spectrophotometric studies under oxidizing and reducing conditions suggest that the Fe–S clusters are not integral structural
components of hMIA40 and are susceptible to oxidation and reduction.
hMIA40 regulates cellular iron homoeostasis
Our in vitro and in vivo studies clearly demonstrate that hMIA40 is an Fe–S protein. hMIA40 is known to interact with ALR , a component of the ISE machinery and protein import machinery of the intermembrane space. On the basis of these findings, we hypothesized further that hMIA40 may play a role in cellular iron homoeostasis. To determine whether hMIA40 has any effect on iron homoeostasis, we initially depleted hMIA40 in HEK-293T cells by transiently transfecting the cells with hMIA40 shRNA. In parallel, we also transfected HEK-293T cells with scrambled shRNA to serve as a control. To ascertain the level of hMIA40, SDS/PAGE followed by immunoblot analysis was carried out c 2015 Authors; published by Portland Press Limited
A. Murari and others
hMIA40 required for cytosolic Fe–S-containing enzyme functions
HEK-293T cells were transfected with hMIA40 shRNA or scrambled shRNA vector control and the mitochondria or cytosolic fraction was isolated. (A) Cytosolic aconitase activity was measured at 240 nm and is shown as μM/min per mg of protein. (B) XO activity was measured spectrophotometrically at 290 nm and the activity is shown as μM/min per mg of protein. (C) Western blot analysis of whole-cell extracts of HEK-293T cells treated with hMIA40 shRNA and vector control. The blot was probed with antibodies against hMIA40, GPAT and GAPDH. GAPDH was used as an internal loading control. Molecular masses are indicated in kDa. (D) Complex I activity was measured in hMIA40 shRNA cells and control cells spectrophotometrically at 340 nm and presented as μM/min per mg of protein. (E) Complex II activity was measured in hMIA40 shRNA cells and control cells spectrophotometrically at 600 nm and presented as μM/min per mg of protein. (F) Mitochondrial aconitase activity was measured at 240 nm and is represented as units/mg of protein. Results in histograms are means for three independent experiments. P < 0.001. The error bars represent the S.D.
on mitochondria samples that were isolated from transfected cells (Figure 2A). Transfection with shRNA specifically reduced hMIA40 levels compared with control cells, whereas the level of mtHsp70 was similar in both samples, indicating equivalent loading (Figure 2A). Three independent transfection studies were carried out with hMIA40 shRNA or scrambled shRNA in HEK293 cells and the relative levels of hMIA40 was quantified by Western blotting. We found that the reduction in hMIA40 levels are more than 50 % in hMIA40 shRNA-transfected cells compared with control shRNA-transfected cells (Figure 2B). The amount of iron present in mitochondria that were isolated from the transfected cells was measured using bathophenonthroline. Depletion of hMIA40 is correlated to increased accumulation of non-haem iron in mitochondria (Figure 2C). We repeated this experiment by performing transfection in the presence of radioactive iron. Consistent with the previous result, we observed the accumulation of 55 Fe in mitochondria that were isolated from cells depleted of hMIA40 (Figure 2D). We ruled out the possibility of cytosolic contamination as a negligible amount of β-actin, a cytosolic marker protein, was present in our mitochondrial preparations (Supplementary Figure S2). Furthermore, depletion of hMIA40 does not alter cytosolic 55 Fe levels compared with the control (Figure 2E). We conclude that hMIA40 is probably involved in export of Fe–S clusters from mitochondria to cytosol. Mitochondria form the main hub for synthesis and export of Fe–S clusters. Several cytosolic enzymes are dependent on Fe–S clusters that are exported from mitochondria for their activity . The enzyme components of the ISC synthesis pathway of the mitochondria have been well characterized genetically and biochemically in yeast and bacteria [11,41]. The cytoplasm and the nucleus in higher eukaryotes have their own Fe–S assembly machinery (CIA); nevertheless, perturbations in the c 2015 Authors; published by Portland Press Limited
mitochondrial ISC pathway causes maturation defects of nuclear and cytoplasmic enzymes that are dependent on Fe–S clusters. This underscores the importance of the mitochondrial ISC system in cellular iron homoeostasis. Yet, the molecular mechanism by which the mitochondrial ISE system exports iron to the cytoplasm is to be fully elucidated. As depletion of hMIA40 results in accumulation of iron in mitochondria, we additionally wished to probe whether this affected the function of cytosolic enzymes that are associated with Fe–S clusters. We monitored the activity of the cytosolic enzymes aconitase and xanthine oxidase in control cells and cells that were depleted of hMIA40 as described for the previous experiment. The activities of aconitase and xanthine oxidase are significantly lowered in the cytosolic fraction obtained from cells that were depleted for hMIA40 compared with control sample (Figures 3A and 3B). The stability of cytosolic GPAT is dependent on the presence of 4Fe–4S clusters and hence its stability could serve as a hallmark for the presence of cytoplasmic Fe–S assembly . The cell extracts of the hMIA40-depleted cells and control cells were resolved by SDS/PAGE and the steady-state level of GPAT was monitored by immunoblot analysis (Figure 3C). By analysing the whole-cell extracts of control and hMIA40 shRNA-treated cells, we found that depletion of hMIA40 reduces the steady-state levels of GPAT compared with control cells, although a comparable amount of GAPDH is present in both fractions. Furthermore, we assessed the mitochondrial Fe–S-containing enzyme activities that are dependent on the ISC machinery in hMIA40-depleted cell lines. We find no significant difference in mitochondrial Complex I, Complex II and aconitase enzyme activities in mitochondrial fractions of hMIA40-depleted cells and of scrambled shRNA-treated cells (Figures 3D–3F). These studies indicate that, although hMIA40 does not affect mitochondrial Fe–S cluster assembly or Fe–S-containing mitochondrial enzyme
Role of hMIA40 in iron homoeostasis
hMIA40 plays a critical role in maturation of cytosolic Fe–S cluster proteins
HEK-293T cells were co-transfected with hMIA40 shRNA/Myc–ALR or hMIA40 shRNA/Myc–NUBP1 and the mitochondria or cytosolic fraction was isolated. (A) Western blot analysis of whole-cell extracts of HEK-293T cells treated with hMIA40 shRNA (lane 2), vector control (lane 1), Myc–ALR (lane 3), hMIA40 shRNA/Myc–ALR (lane 4), Myc–NUBP1 (lane 5) and hMIA40 shRNA/Myc–NUBP1 (lane 6). The blot was probed with antibodies against GPAT, hMIA40, Myc and GAPDH. GAPDH was used as an internal loading control. (B and C) Cytosolic aconitase activity was measured from the cytosolic fraction at 240 nm and is shown as μM/min per mg of protein. (D and E) Mitochondrially accumulated 55 Fe was measured from the isolated mitochondrial fractions and is shown as c.p.m./mg of mitochondrial protein. Molecular masses are indicated in kDa. Results in histograms are means for three independent experiments. *P < 0.05, **P < 0.01. The error bars represent the S.D.
activities, it has a pronounced effect on the cytosolic Fe–S cluster assembly and on the activity and stability of cytosolic Fe–S-containing enzymes. Taken together, our results suggest that hMIA40 has an important function in the mitochondrial Fe–S export machinery and thereby affects cellular iron homoeostasis. Similar observations have also been reported when the dosage of Atm1 in yeast or ABCB7 in mammals , an inner
mitochondrial membrane protein, was reduced and therefore Atm1 was suggested to be a component of the ISE machinery of mitochondria . Although the precise component that is exported by Atm1 from mitochondria is debatable, deficiency of Atm1 leads to an iron overload in mitochondria accompanied by defects in Fe–S cluster biogenesis that occurs in cytosol and nucleus. However, no apparent effect was observed on c 2015 Authors; published by Portland Press Limited
A. Murari and others
The CPC motif in hMIA40 is required for binding to Fe–S clusters
(A) UV–visible spectra of purified recombinant His–hMIA40 wild-type, M1, M2 and M3 mutant proteins. (B) Metal/protein ratios detected in recombinant hMIA40 and cysteine mutants. (C and D) HEK-293T cells were transfected with vector, wild-type Myc–His–hMIA40 or Myc–His-tagged M1 hMIA40 plasmids. (C) The samples were processed through Ni-NTA beads. The 55 Fe present in beads was quantified using a scintillation counter as c.p.m./mg of protein. (D) Western blot analysis of vector- or Myc–hMIA40-transfected HEK-293T cell lysates probed with anti-Myc and anti-Hsp70 antibodies. Results in histograms are means for three independent experiments. *P < 0.05, **P < 0.01. ns, not significant. The error bars represent the S.D.
the mitochondrial ISC pathway or stability of mitochondrial enzymes associated with Fe–S clusters . Besides Atm1, the Fe–S export machinery probably includes Erv1 in yeast or ALR in mammalian cells, a thiol oxidase, as depletion of these proteins results in a phenotype similar to that of Atm1 deficiency.
hMIA40 function is indispensable for cytosolic Fe–S biogenesis
We speculate that hMIA40 works in conjunction with Erv1/ALR to export Fe–S clusters from mitochondria as it has already been shown that Erv1/ALR and hMIA40 collaborate in the import of numerous cysteine-rich mitochondrial space proteins by an oxidative folding mechanism . Similarly, Fe–S proteins in the cytosol are dependent on the CIA machinery. Depletion of components of the CIA machinery leads to impaired maturation of cytosolic Fe–S proteins . To investigate the critical role of hMIA40 in Fe–S homoeostasis, we overexpressed either ALR or NUBP1 in hMIA40-depleted cell lines. Transient transfection of shMIA40 alone or co-transfection with ALR/NUBP1 was carried out and the whole-cell lysates were separated by SDS/PAGE and probed with antibodies specific for GPAT and Myc. As shown in Figure 4(A), steady-state levels of hMIA40 are reduced by 40 % in hMIA40 shRNA-treated cell lines compared with control shRNAtreated cells (lanes 1 and 2). However, 25–40 % reduced hMIA40 c 2015 Authors; published by Portland Press Limited
levels are present in cell extracts obtained from the cell lines that were co-transfected with hMIA40 shRNA and Myc-ALR/NUBP1 plasmids (Figure 4A, lanes 3–6). Nevertheless, we found reduced levels of GPAT in all hMIA40-depleted cell lines when compared with control shRNA or in ALR- or NUBP1-overexpressing cell lines (Figure 4A, compare lanes 2, 4 and 6 with lanes 1, 3 and 5). Furthermore, aconitase activity is significantly lowered in the cytosolic fraction obtained from cells that were overexpressing ALR or NUBP1 but depleted of hMIA40 (Figures 4B and 4C). In addition, we find an accumulation of 55 Fe in mitochondria samples that were depleted of hMIA40 irrespective of overexpression of ALR/NUBP1 (Figures 4D and 4E). These results collectively imply that hMIA40 function is non-redundant and essential for maturation of cytosolic Fe–S clusters.
The CPC motif of hMIA40 co-ordinates the binding of Fe–S
Most of the Fe–S clusters associated with proteins are normally co-ordinated by four cysteine residues resident in the protein. hMIA40 contains six conserved cysteine residues that are ordered in the form of a CPC motif and two CX9 C motifs. The CPC motif serves as a redox-active motif, whereas the CX9 C motifs are involved in providing the structural framework in the form of disulfide bonds [23,24]. We speculate that the CPC motif is involved in Fe–S binding and cellular iron homoeostasis. To
Role of hMIA40 in iron homoeostasis
The CPC motif in hMIA40 is essential for Fe–S cluster export from mitochondria
HEK-293T cells were transfected with hMIA40 shRNA specific to non-coding region or scrambled shRNA vector control or co-transfected with shRNA hMIA40/wild-type hMIA40 or shRNA hMIA40/mutant hMIA40, and the mitochondria or cytosolic fraction was isolated. (A) Western blot analysis of whole-cell extracts of HEK-293T cells. The blot was probed with antibodies against GPAT, hMIA40, Myc and GAPDH. GAPDH was used as an internal loading control. (B) Mitochondrially accumulated 55 Fe was measured from the isolated mitochondrial fractions of transfected cells and is shown as c.p.m./mg of mitochondrial protein. (C) Cytosolic aconitase activity was measured at 240 nm and is shown as μM/min per mg of protein. (D) Mitochondrial aconitase activity was measured at 240 nm and is represented as μM/min/mg of protein. (E) Complex I activity was measured in transfected cells spectrophotometrically at 340 nm and is presented as μM/min per mg of protein. Results in histograms are means for three independent experiments. *P < 0.05, **P < 0.01. The error bars represent the S.D.
determine the importance of the CPC motifs, we carried out site-directed mutagenesis of the cysteine resides within the CPC motif of recombinant hMIA40. We mutated both Cys53 and Cys55 (M1) to serine in the CPC motif. We also mutated the cysteine residues in the CX9 C structural motifs to determine the role of these cysteine residues in the binding of hMIA40 to Fe–S clusters. We mutated either Cys64 to serine (C64S, M2) or Cys87 to serine (C87S, M3) within the CX9 C motifs.
These recombinant mutants were purified to homogeneity from bacterial cells (Supplementary Figure S3) and equimolar protein samples were analysed for the presence of Fe–S by UV absorption spectroscopy. We find that the recombinant hMIA40 M1 double mutant did not have any absorption in the UV–visible spectrum (Figure 5A). In contrast, structural cysteine mutants M2 and M3 do not show any defect in absorption (Figure 5A). Consistent with the low Fe–S content result, the metal/protein c 2015 Authors; published by Portland Press Limited
A. Murari and others
ratio of the hMIA40 M1 mutant is also significantly lowered compared with wild-type hMIA40 and hMIA40 M2 and M3 mutants (Figure 5B). These results directly implicate the CPC motif in hMIA40 as being critical for binding to Fe–S clusters. Furthermore, these results suggest that the CPC motif and not the structural motifs are involved in co-ordinating Fe–S clusters in hMIA40 in vitro. However, we found a low metal/protein ratio in hMIA40, even with wild-type hMIA40 (Figures 1C and 5B), compared with other Fe–S proteins . Since hMIA40 was purified aerobically and contains oxidation-sensitive Fe–S, it is possible that a low metal/protein ratio is due to degradation of Fe–S clusters in hMIA40. Although we added DTT while purifying the protein to activate the cysteine residues, hMIA40 loses its Fe–S clusters when exposed to air (Figures 1G and 1H). We also reconstituted Fe–S in purified hMIA40 under anaerobic conditions and evaluated the metal/protein ratio. We found that there was no significant change in the metal/protein ratio in reconstituted hMIA40 even under anaerobic conditions (Supplementary Figure S4). It can be explained that the Fe– S exporter activity of hMIA40 probably requires a transient association of Fe–S clusters. To support this notion, no stable association of Fe–S with another ISE machinery component, Erv1 (ALR) has been observed even under anaerobic conditions . Since the oxidized form of hMIA40 is essential for import and folding of cysteine-rich proteins in the intermembrane space of mitochondria, it is possible that a small fraction of hMIA40 can exist in a reduced state for Fe–S binding and export. However, further work is required to elucidate the precise molecular function of hMIA40 in transferring Fe–S clusters and maturation of cytosolic Fe–S clusters. To evaluate further the role of the CPC motif in the ability of hMIA40 to bind iron in vivo, HEK-293T cells were transfected with plasmids pNB202 and pNB314, which express hMIA40 wild-type or M1 respectively. HEK-293T cells transfected with vector alone were used as internal control. The overexpressed wild-type and mutant hMIA40 have both His and Myc epitopes. The transfected cells were incubated with 55 Fe for 24 h and the cell lysates were subjected to Ni-NTA pull-down. After incubation with the lysates, the Ni-NTA beads were washed and measured for 55 Fe content. Ni-NTA beads that were incubated with wild-type hMIA40 lysates had relatively more radioactivity than Ni-NTA beads that were incubated with lysates from the hMIA40 double mutant (M1). Ni-NTA beads that were incubated with lysates from vector control were not enriched in 55 Fe (Figure 5C). All transfected lysates were probed with anti-Myc and anti-Hsp70 antibodies to confirm the presence of equivalent amounts of hMIA40 (in all samples except for vector control) and equal amounts of lysate (Figure 5D). To demonstrate the importance of the CPC motif in Fe–S export in vivo, we overexpressed either wild-type hMIA40 or hMIA40 CPC motif mutant (hMIA40 C53S&C55S) in cell lines that are depleted in endogenous hMIA40. We used hMIA40 shRNA that is specific to the non-coding region of hMIA40 to deplete the steady-state levels of endogenous hMIA40. Furthermore, the result from this experiment will explain the result from Figure 2. HEK-293T cells were transfected with either hMIA40 shRNA or co-transfected with hMIA40 shRNA/pNB202 (wild-type hMIA40) or hMIA40 shRNA/pNB314 (hMIA40 C53S&C55S) to overexpress wild-type and M1 respectively in the latter two hMIA40-depleted cell lines. In shRNA hMIA40-transfected samples, hMIA40 levels were reduced by 50 % compared with scrambled shRNA-transfected samples (Figure 6A). However, both wild-type and mutant hMIA40 protein levels are high in cotransfected samples as shRNA fails to inhibit the plasmid-borne hMIA40 translation. As expected, GPAT levels were reduced by c 2015 Authors; published by Portland Press Limited
50 % in cell lysates expressing shRNA. However, overexpression of wild-type hMIA40, but not mutant, rescues the steady-state levels of GPAT in hMIA40-depleted cell lines (Figure 6A). In addition, we found accumulation of 55 Fe in the mitochondria samples isolated from shRNA hMIA40 or shRNA hMIA40 co-transfected with mutant, but not with wild-type hMIA40, co-expressing cell lines (Figure 6B). Overexpression of wildtype, but not mutant, rescues the cytosolic aconitase activity in hMIA40-depleted cell lines (Figure 6C). The transfected samples were probed with Myc to confirm the equivalent overexpression of plasmid-borne wild-type and mutant proteins and GAPDH to confirm the equivalent amount of lysate in all samples (Figure 6A). Besides the comparable amount of wild-type and mutant hMIA40 proteins, we observed no significant difference in the mitochondrial Complex I and aconitase activity in all samples (Figures 6D and 6E). Taken together, our results irrefutably implicate the CPC motif in hMIA40 to be essential for efficient binding to iron both in vitro and in vivo. Since the CPC motif is required for protein import and Fe–S binding, it is possible that the microenvironment of the intermembrane space and its redox status may influence hMIA40’s function either in protein import or/and Fe–S export. The present study further addresses the importance of genetic mutations in proteins associated with Fe–S biogenesis that leads to several human disorders ranging from Friedreich’s ataxia to anaemia.
AUTHOR CONTRIBUTION Anjaneyulu Murari performed biochemical and cell culture experiments. Venkata Ramana Thiriveedi and Fareed Mohammad constructed the plasmids and purified recombinant proteins. Viswamithra Vengaldas, Madhavi Gorla and Prasad Tammineni helped in conducting the experiments. Naresh Babu Sepuri and Thanuja Krishnamoorthy designed the experiments and wrote the paper.
ACKNOWLEDGEMENT We thank Dr Krishnamoorthy (GSI, Hyderabad, India) for atomic emission spectroscopy analysis. We also thank members of the Sepuri laboratory for suggestions. We thank Professor Hema Balaram (JNCASR, Banagalore, India) for the anaerobic reconstitution experiment.
FUNDING We thank the Science and Engineering Research Board (SERB) of India [grant number SR/SO/BB-0067/2013] for funding to the laboratory. We also thank Department of Science and Technology Fund for Improvement of Science and Technology Infrastructure in Higher Educational Institutions (DST-FIST) and University Grants Commission Departmental Research Support (UGC-DRS) funding to the Department of Biochemistry. V.R.T. was supported by a Senior Research Fellowship (SRF) from the Indian Council of Medical Research (ICMR).
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Received 5 January 2015/5 August 2015; accepted 14 August 2015 Accepted Manuscript online 14 August 2015, doi:10.1042/BJ20150012
c 2015 Authors; published by Portland Press Limited