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J Biol Chem. 2015 Oct 23;290(43):25876-90. doi: 10.1074/jbc.M115.678508. Epub 2015 Sep 4.

Mapping Key Residues of ISD11 Critical for NFS1-ISD11 Subcomplex Stability: IMPLICATIONS IN THE DEVELOPMENT OF MITOCHONDRIAL DISORDER, COXPD19.

Saha PP¹, Srivastava S¹, Kumar S K P², Sinha D¹, D'Silva P³.

Author information

1: From the Department of Biochemistry, Indian Institute of Science, Bangalore 560012, Karnataka and.
2: the Department of Biochemistry, Karnatak University, Dharwad 580003, Karnataka, India.
3: From the Department of Biochemistry, Indian Institute of Science, Bangalore 560012, Karnataka and patrick@biochem.iisc.ernet.in.

Abstract

Biogenesis of the iron-sulfur (Fe-S) cluster is an indispensable process in living cells. In mammalian mitochondria, the initial step of the Fe-S cluster assembly process is assisted by the NFS1-ISD11 complex, which delivers sulfur to scaffold protein ISCU during Fe-S cluster synthesis. Although ISD11 is an essential protein, its cellular role in Fe-S cluster biogenesis is still not defined. Our study maps the important ISD11 amino acid residues belonging to putative helix 1 (Phe-40), helix 3 (Leu-63, Arg-68, Gln-69, Ile-72, Tyr-76), and C-terminal segment (Leu-81, Glu-84) are critical for in vivo Fe-S cluster biogenesis. Importantly, mutation of these conserved ISD11 residues into alanine leads to its compromised interaction with NFS1, resulting in reduced stability and enhanced aggregation of NFS1 in the mitochondria. Due to altered interaction with ISD11 mutants, the levels of NFS1 and Isu1 were significantly depleted, which affects Fe-S cluster biosynthesis, leading to reduced electron transport chain complex (ETC) activity and mitochondrial respiration. In humans, a clinically relevant ISD11 mutation (R68L) has been associated in the development of a mitochondrial genetic disorder, COXPD19. Our findings highlight that the ISD11 R68A/R68L mutation display reduced affinity to form a stable subcomplex with NFS1, and thereby fails to prevent NFS1 aggregation resulting in impairment of the Fe-S cluster biogenesis. The prime affected machinery is the ETC complex, which showed compromised redox properties, causing diminished mitochondrial respiration. Furthermore, the R68L ISD11 mutant displayed accumulation of mitochondrial iron and reactive oxygen species, leading to mitochondrial dysfunction, which correlates with the phenotype observed in COXPD19 patients.

KEYWORDS:

electron transfer; iron-sulfur protein; mitochondria; mitochondrial disease; molecular chaperone; protein-protein interaction; reactive oxygen species (ROS); respiration

PMID:: 26342079
PMCID:: PMC4646244
DOI:: 10.1074/jbc.M115.678508

[Indexed for MEDLINE]

Free PMC Article

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FIGURE 3.

Interaction analysis of NFS1 with ISD11 mutants. A-P, glutathione-Sepharose bound WT GST-ISD11 (3 μm) or mutant GST-ISD11 (3 μm) was incubated with increasing concentrations of purified NFS1, as indicated. The unbound proteins were washed and analyzed by SDS-PAGE followed by Coomassie staining. GST alone (3 μm) was used as a negative control, and 50% input of NFS1 was taken as a loading control. The band intensities of bound NFS1 were analyzed by densitometry using ImageJ software. Interaction of NFS1 with WT ISD11 and F40A ISD11 (A and B), L63A ISD11 (C and D), R68A ISD11 (E and F), Q69A ISD11 (G and H), I72A ISD11 (I and J), Y76A ISD11 (K and L), L81A ISD11 (M and N), and E84A ISD11 (O and P) are represented. Q and R, interaction analysis of NFS1 with ts mutants in mitochondrial lysates. Mitochondria isolated from WT and ts mutant strains (Q69A, R68A, L63A, L81A, and Y76A) were subjected to lysis and the supernatant fractions were incubated with GST-NFS1 (2 μm). Unbound proteins were washed, and FLAG-tagged ISD11 was detected by Western blotting using the anti-FLAG antibodies. GST alone was taken as negative control and ISD11 as loading control (Q). The band intensities shown in Q were analyzed by densitometry using ImageJ software (R). S, mitochondria isolated from WT or R68L ISD11 expressing HeLa cells were lysed, and the supernatant fractions were incubated with GST-NFS1 (2 μm). Unbound proteins were removed by washing, followed by SDS-PAGE and immunostaining using an anti-FLAG antibody to detect the FLAG-tagged ISD11. GST alone was taken as negative control and ISD11 as loading control. Data are represented as mean ± S.E. The values were obtained from three independent experiments (n = 3). p value of <0.05 was defined as significant, and asterisks are used to denote significance, where: *, p < 0.05; **, p < 0.01; ***, p < 0.001. A.U., arbitrary unit.

Mapping Key Residues of ISD11 Critical for NFS1-ISD11 Subcomplex Stability

J Biol Chem. 2015 Oct 23;290(43):25876-25890.

FIGURE 6.

Analysis of mitochondrial iron, ROS levels and mitochondrial function in COXPD19 related ISD11_R68A mutant. A and B, representation of iron levels in yeast mitochondria expressing WT Isd11 or WT ISD11 or ISD11_R68A, measured by iron-specific colorimetric assay (A) and AAS (B). C and D, mitochondrial superoxide levels in WT and ISD11_R68A yeast strains were estimated using MitoSOX Red dye by flow cytometry and represented in the overlaying histograms (C) and mean fluorescence intensity obtained by flow cytometry was quantitated (D). WT Isd11 cells treated with 1 mm rotenone was utilized as positive control. E, yeast strains were treated with MitoSOX Red dye (red fluorescence) and subjected to microscopic analysis using DeltaVision Fluorescence Microscope (×100 objective). Left column, bright field; middle column, MitoSOX red fluorescence; and right column, merged. F, overall cellular peroxide levels were measured by staining using H₂DCFDA dye (green fluorescence), followed by fluorescence microscopy analysis using Delta Vision Fluorescence Microscope (×100 objective). Left, bright field; middle, H₂DCFDA fluorescence, and right columns, merged. G and H, mitochondrial mass in yeast strains expressing WT Isd11 or WT ISD11 or ISD11_R68A was assessed using NAO dye. The fluorescence was measured through flow cytometry and represented as histogram (G) or mean fluorescence intensity (MFI) obtained in flow cytomety was quantitated and represented as bar diagram (H). I, JC-1 staining was used for the estimation of mitochondrial membrane potential in yeasts strains. An emission scan from the 500 to 620 nm wavelength was performed in JC-1-stained yeast cells and the multimer (590 nm) to monomer (530 nm) ratio was quantified and represented. Mitochondria treated with valinomycin were used as a positive control. Data represented as mean ± S.E. The values were obtained from three independent experiments (n = 3). p value of <0.05 was defined as significant, and asterisks are used to denote significance, where: *, p < 0.05; **, p < 0.01; ***, p < 0.001. Scale bar, 10 μm. A.U., arbitrary unit.

Mapping Key Residues of ISD11 Critical for NFS1-ISD11 Subcomplex Stability

J Biol Chem. 2015 Oct 23;290(43):25876-25890.