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. 2011 Nov 11;89(5):656-67.
doi: 10.1016/j.ajhg.2011.10.005.

A fatal mitochondrial disease is associated with defective NFU1 function in the maturation of a subset of mitochondrial Fe-S proteins

Aleix Navarro-Sastre  1 , Frederic Tort, Oliver Stehling, Marta A Uzarska, José Antonio Arranz, Mireia Del Toro, M Teresa Labayru, Joseba Landa, Aida Font, Judit Garcia-Villoria, Begoña Merinero, Magdalena Ugarte, Luis Gonzalez Gutierrez-Solana, Jaume Campistol, Angels Garcia-Cazorla, Julian Vaquerizo, Encarnació Riudor, Paz Briones, Orly Elpeleg, Antonia Ribes, Roland Lill
Affiliations

A fatal mitochondrial disease is associated with defective NFU1 function in the maturation of a subset of mitochondrial Fe-S proteins

Aleix Navarro-Sastre et al. Am J Hum Genet. .

Abstract

We report on ten individuals with a fatal infantile encephalopathy and/or pulmonary hypertension, leading to death before the age of 15 months. Hyperglycinemia and lactic acidosis were common findings. Glycine cleavage system and pyruvate dehydrogenase complex (PDHC) activities were low. Homozygosity mapping revealed a perfectly overlapping homozygous region of 1.24 Mb corresponding to chromosome 2 and led to the identification of a homozygous missense mutation (c.622G > T) in NFU1, which encodes a conserved protein suggested to participate in Fe-S cluster biogenesis. Nine individuals were homozygous for this mutation, whereas one was compound heterozygous for this and a splice-site (c.545 + 5G > A) mutation. The biochemical phenotype suggested an impaired activity of the Fe-S enzyme lipoic acid synthase (LAS). Direct measurement of protein-bound lipoic acid in individual tissues indeed showed marked decreases. Upon depletion of NFU1 by RNA interference in human cell culture, LAS and, in turn, PDHC activities were largely diminished. In addition, the amount of succinate dehydrogenase, but no other Fe-S proteins, was decreased. In contrast, depletion of the general Fe-S scaffold protein ISCU severely affected assembly of all tested Fe-S proteins, suggesting that NFU1 performs a specific function in mitochondrial Fe-S cluster maturation. Similar biochemical effects were observed in Saccharomyces cerevisiae upon deletion of NFU1, resulting in lower lipoylation and SDH activity. Importantly, yeast Nfu1 protein carrying the individuals' missense mutation was functionally impaired. We conclude that NFU1 functions as a late-acting maturation factor for a subset of mitochondrial Fe-S proteins.

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Figures

Figure 1
Figure 1
Biochemical and Genetic Characterization of Individuals (A) Characteristic urine organic acid profile of an affected individual compared with an age-matched control. Samples were analyzed by gas chromatography-mass spectrometry, and peaks were identified as lactic acid (LA), fumaric acid (FA), glutaric acid (GA), tyglylglycine (TG), 2-hydroxyglutaric acid (2HGA),2-ketoglutaric acid (2KGA), 2-hydroxyadipic acid (2HAA), and 2-ketoadipic acid (2KAA). IS is the internal standard (undecanoic acid). LA, FA, 2HGA, and TG were not always increased. Quantitative data from five individuals are presented in Table S1. (B) Sequence analysis of NFU1 in genomic DNA shows the homozygous and heterozygous c.622G>T nucleotide substitution. RFLP analysis of family 1 shows the c.622G>T substitution in two affected siblings (P1 and P2) and in their heterozygous parents (F1) (right panel). Arrows indicate restriction sites for BstNI in both wild-type (WT) and mutant NFU1. The length of the restriction fragments is indicated in the boxes in the top panel. (C) Multi-sequence alignment of the conserved part of NFU1 protein from man and other indicated eukaryotes. The c.622G>T substitution identified in ten NFU1 individuals replaces a conserved glycine with a cysteine at position 208 of the protein (p.Gly208Cys). The conserved Fe-S cluster binding motif CXXC is indicated.
Figure 2
Figure 2
The c.545+5G>A Mutation Generates a Defective Splicing of NFU1 (A) Left panel: RFLP analysis in genomic DNA shows the heterozygous c.622G>T substitution in the affected individual (P10) and his father (F); C indicates a control, and M indicates the mother. Right panel: sequence analysis identified a heterozygous c.545+5G>A nucleotide change in P10 and his mother. (B) Left panel: mRNA expression analysis by RT-PCR and RFLP in muscle tissue. P10 showed normal mRNA expression. RFLP analysis was negative for the allele carrying the c.545+5G>A mutation, but not for the allele with the c.622G>T mutation. P2 was used as a control. Right panel: immunoblot of muscle tissue extracts showed similar levels of NFU1 protein for P10, P2, and a control (C). GAPDH was used as a loading control. (C) Splicing assay incorporating wild-type and c.545+5G>A minigene Exontrap vector system (Mobitec, Göttingen, Germany). Abbreviatioins are as follows: Et1, Exontrap exon 1; Et2, Exontrap exon 2; NFU1 exon6, exon 6 and the flanking intronic regions (left panel). RT-PCR using vector-specific primers in COS7 cells transfected with these vectors showed a defective splicing as a consequence of c.545+5G>A mutation (right panel).
Figure 3
Figure 3
A Point Mutation in Human NFU1 Decreases the Amounts of Protein-Bound Lipoic Acid (A) Immunostaining of NFU1 in muscle tissue extracts from a control and individuals carrying a NFU1 mutation. GAPDH was used as a loading control. (B) Immunostaining of PDH- and KGDH-bound lipoic acid and PDH-E2 protein; muscle tissue extracts were used as in (A). GAPDH was used as a loading control. (C) Densitometric quantitation shows a marked decrease of lipoic acid bound to the E2 subunit of PDH in three NFU1 individuals (P1, P2, and P6), compared to four control (C1–C4) individuals. Control results are represented as the mean value ± 1 standard deviation (n = 4). The protein levels of the PDH-E2 subunit did not change. Anti-NFU1 antisera were raised in rabbits immunized with recombinant His6-tagged NFU1. Additional antibodies were directed against GAPDH, protein conjugated-lipoic acid (Calbiochem), and PDHc (kindly donated by W. Ruitenbeek, Netherlands). The densitometry program IMAGEJ was applied so that levels of total PDH-E2- and PDH-E2-bound lipoic acid could be quantified. All values were normalized to GAPDH.
Figure 4
Figure 4
Depletion of NFU1 in Human Cell Culture Diminishes the Function of Protein-Bound Lipoic Acid and Succinate Dehydrogenase, but Not That of Other Fe-S Proteins HeLa cells were transfected three times with a pool of four different NFU1-specific (3 μg each) or scrambled siRNA (12 μg) or were mock treated. Each transfection was followed by growth of cells for 3 days (d). (A and B) The efficiency of NFU1 silencing was analyzed by qRT-PCR (A) or SDS-PAGE and immunostaining of NFU1 (B). Actin served as a loading control. (C) The effect of NFU1 depletion on other cellular proteins was examined by immunostaining. Abbreviations are as follows: LA-PDH-E2, lipoic acid bound to pyruvate dehydrogenase E2 subunit; LA-KGDH-E2, lipoic acid bound to α-ketoglutarate dehydrogenase E2 subunit; lipoic acid bound to H protein (LA-H) of the glycine cleavage system; aconitase, mitochondrial aconitase; IRP1, iron regulatory protein 1; and GPAT, glutamate phosphoribosylpyrophosphate amidotransferase. (D) Activities of the indicated enzymes were measured with cell extracts derived after three transfections (growth of 9 days). The values were normalized to the activities of citrate synthase (CS) or lactate dehydrogenase (LDH), and displayed as a fraction of the values obtained for mock-treated cells. Error bars indicate the relative error, rE (n = 3). COX indicates cytochrome c oxidase. Antibodies were directed against mitochondrial aconitase (kindly provided by L. Szweda), GPAT (kindly provided by H. Puccio), beta-actin (Santa Cruz Biotechnology), SDH (30 kDa Fe-S protein subunit, MitoSciences), and IRP1 (clone 295B, kindly provided by R. Eisenstein).
Figure 5
Figure 5
Depletion of ISCU Results in a General Defect of Fe-S Proteins and a Deficiency in Protein-Bound Lipoic Acid HeLa cells were transfected twice with a pool of four different ISCU-specific (3 μg each) or scrambled siRNA (12 μg) or were mock treated. Each transfection was followed by growth of cells for 3 days (d). Error bars indicate the relative error, rE (n = 3). (A and B) The efficiency of ISCU silencing was analyzed by qRT-PCR (A) or SDS-PAGE and immunostaining of ISCU (B). Actin served as a loading control. (C) The effect of ISCU depletion on protein-bound lipoic acid or various cellular proteins was examined by immunostaining. For abbreviations see Figure 3C. (D) The activities of the indicated enzymes were measured and evaluated as in Figure 4D after depletion of ISCU for 3 days. Anti-ISCU antisera were raised in rabbits immunized with recombinant ISCU.
Figure 6
Figure 6
The Gly208Cys Mutation of Yeast NFU1 Leads to Its Functional Impairment and Identifies NFU1 as a Late-Acting Fe-S Transfer Protein (A) Enzyme activities of mitochondrial aconitase (ACO), respiratory complexes II (SDH) and IV (COX), and pyruvate dehydrogenase (PDH) were determined relative to malate dehydrogenase (MDH) in mitochondria isolated from wild-type (WT) and nfu1Δ yeast cells cultivated in rich glucose medium. Deletion of NFU1 was confirmed by PCR (not shown) and immunostaining (inset). Nfu1 was detected between two unspecifically labeled bands. (B) Yeast nfu1Δ cells were transformed with overexpression vectors containing no gene (-), the wild-type gene, or the human-mutation-corresponding Gly194Cys variant of yeast NFU1 (Nfu1G194C). After growth in synthetic glucose-containing medium (SD) lacking uracil, mitochondria were isolated from these and wild-type (WT) cells. The indicated enzyme activities and the immunostaining were performed as in (A). Cells with a deletion of LIP5 (lip5Δ) encoding yeast LAS were used as a control. (C) Wild-type, Gal-NFS1, Gal-ISU1/isu2Δ (Gal-ISU1), Gal-SSQ1, Gal-GRX5 and isa1/2Δ cells overproducing wild-type Nfu1 or Nfu1G194C were grown in iron-poor SD media. Cells were radiolabeled with 10 μCi 55Fe for 2 hr, and the overproduced proteins were immunoprecipitated from cell extracts with polyclonal antibodies raised against Nfu1. The amounts of coprecipitated 55Fe were quantified by scintillation counting. The background obtained for samples with nonspecific antibodies was subtracted. Error bars indicate the SEM (n ≥ 4).
Figure 7
Figure 7
A Working Model for the Late-Acting Function of NFU1 in Mitochondrial Fe-S Protein Biogenesis The findings of this work suggest a late-acting function of NFU1 in the pathway of Fe-S (red and yellow circles) protein maturation. NFU1 is preferentially needed for Fe-S cluster assembly of succinate dehydrogenase (SDH) and lipoic acid synthase (LAS), but not of other proteins such as aconitase. An NFU1 functional defect due to a point mutation in the fatal mitochondrial disease described in this work results in defective SDH and LAS and hence in decreased synthesis of lipoic acid (LA) and a lack of lipoylation of the E2 subunits of PDH, α-KGDH, and BCDH and the H protein of GCS. In contrast to depletion of NFU1, depletion of the major Fe-S scaffold ISCU affects maturation of virtually all cellular Fe-S proteins, including cytosolic ones, which additionally need the CIA machinery for maturation. ISCU, together with the cysteine desulfurase Nfs1-Isd11 and frataxin, which is depleted in Friedreich ataxia, is also required for de novo synthesis of a transiently bound Fe-S cluster on NFU1. Chaperones and GRX5 may be involved in Fe-S cluster release from ISCU and transfer to both NFU1 and target apoproteins. Finally, NFU1 is needed for maturation of specific targets such as LAS and SDH. In this late step, NFU1 may cooperate with IBA57-ISCA1, yet these latter proteins appear to address most or all mitochondrial target proteins with a [4Fe-4S] cluster.
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