Missense mutations linked to friedreich ataxia have different but synergistic effects on mitochondrial frataxin isoforms

Abstract

Friedreich ataxia is an early-onset multisystemic disease linked to a variety of molecular defects in the nuclear gene FRDA. This gene normally encodes the iron-binding protein frataxin (FXN), which is critical for mitochondrial iron metabolism, global cellular iron homeostasis, and antioxidant protection. In most Friedreich ataxia patients, a large GAA-repeat expansion is present within the first intron of both FRDA alleles, that results in transcriptional silencing ultimately leading to insufficient levels of FXN protein in the mitochondrial matrix and probably other cellular compartments. The lack of FXN in turn impairs incorporation of iron into iron-sulfur cluster and heme cofactors, causing widespread enzymatic deficits and oxidative damage catalyzed by excess labile iron. In a minority of patients, a typical GAA expansion is present in only one FRDA allele, whereas a missense mutation is found in the other allele. Although it is known that the disease course for these patients can be as severe as for patients with two expanded FRDA alleles, the underlying pathophysiological mechanisms are not understood. Human cells normally contain two major mitochondrial isoforms of FXN (FXN(42-210) and FXN(81-210)) that have different biochemical properties and functional roles. Using cell-free systems and different cellular models, we show that two of the most clinically severe FXN point mutations, I154F and W155R, have unique direct and indirect effects on the stability, biogenesis, or catalytic activity of FXN(42-210) and FXN(81-210) under physiological conditions. Our data indicate that frataxin point mutations have complex biochemical effects that synergistically contribute to the pathophysiology of Friedreich ataxia.

FIGURE 1.

Structures of frataxin monomer and trimer. A, shown is x-ray structure of FXN^81–210 monomer (PDB ID 3S4M) (27). B, shown is x-ray structure of the yeast frataxin trimer (Y73A variant) (PDB ID 3OEQ) (34). Amino acid residues Ile-154 and Trp-155 (Ilr-130 and Trp-131 in the yeast protein) are shown as sticks. Both structures show the typical α/β sandwich fold of frataxin with two α helices packed against a five-strand β sheet. The N-terminal α helix has seven turns in the monomer (A), whereas in the trimer (B) the first two turns of this helix unwind, enabling the interaction between the N terminus of one subunit and the β sheet of an adjacent subunit.

FIGURE 2.

I154F alters the solubility of FXN^42–210, not FXN^81–210, in E. coli. A and C, each of the indicated FXN isoforms was expressed in E. coli at the indicated temperatures. Upon lysis of bacterial cells at 4 °C, solubility was determined from the amounts of FXN isoform present in the total cell lysate (T) and the soluble fraction (S) after centrifugation of total cell lysate as described under “Experimental Procedures.” B and D, protein bands were quantified by densitometry. In each case, shown are the mean ± S.D. of two independent experiments; the asterisk denotes p ≤ 0.008 as determined by Student's t test.

FIGURE 3.

I154F has mild effects on the ability of FXN^81–210 to activate NFS1 and catalyze Fe-S cluster synthesis in vitro. A, cysteine desulfurase assays were performed anaerobically in the presence of 1 eq of the NFS1-ISD11 complex, 3 eq of wild type (WT) or mutant (I154F) FXN^81–210, and 3 eq of ISCU in the absence of iron essentially as described in Tsai and Barondeau (16). Activity was determined by the conversion of generated sulfide to methylene blue. Shown are the mean ± S.D. of at least four independent experiments with different FXN protein preparations. The asterisk denotes p ≤ 0.005 as determined by Student's t test. B, Fe-S cluster synthesis assays were performed anaerobically in the presence of Fe²⁺ with purified recombinant proteins as described (15). Assays included equal concentrations of NFS1-ISD11, ISCU, and WT- or I154F-FXN^81–210 and 10 eq of Fe²⁺. Each plot shows the mean ± S.D. of at least two independent experiments with different FXN protein preparations.

FIGURE 4.

I154F alters FXN^42–210 solubility in S. cerevisiae. The wild type (WT) or mutant (I154F) FXN^1–210 precursors were expressed in yfh1Δ yeast lacking endogenous yeast frataxin to give the yfh1Δ[WT-FXN] and yfh1Δ[I154F-FXN] strains. A, growth phenotype of the indicated strains at different temperatures on rich medium supplemented with a fermentable (YP-Dextrose) or non-fermentable (YP-Ethanol) carbon source. Liquid cultures were started from freshly streaked frozen glycerol stocks, synchronized to late logarithmic phase in synthetic minimal medium supplemented with dextrose at 30 °C, diluted with fresh medium to A₆₀₀ = 0.1, and grown for 24 h. 10-Fold serial dilutions were spotted onto YP-Dextrose or YP-Ethanol plates that were subsequently incubated at 30 or 37 °C. The red pigmentation is due to the ade2-101^ochre mutation in the genome of the parental YPH501 strain and is indicative of mitochondrial respiratory function. B, mitochondria were isolated from the strains shown in A upon growth in rich medium supplemented with galactose, a fermentable carbon source that does not repress mitochondrial biogenesis as described (29). Total mitochondrial protein was measured, and aliquots were analyzed by Western blotting with specific antibodies. St, shown is a mixture of purified FXN isoforms used as standards. As a recombinant FXN^1–210 protein standard was not available, the precursor band was identified from its apparent molecular mass of ∼30 kDa (5, 7) and from the fact that this is the form of the protein that accumulates in cells when mitochondrial import and/or processing is impaired (7) as seen in Fig. 6A, lane 4. C, total (T), soluble (S), and insoluble (P) mitochondrial fractions were prepared from isolated mitochondria and analyzed by Western blotting. D, yeast cultures were started from freshly streaked frozen glycerol stocks, synchronized to late logarithmic phase in minimal medium supplemented with dextrose at 30 °C for ∼12 h, diluted into fresh medium in the absence or presence of 100 μm FeCl₃, and shifted to 37 °C for another 24 h. Aliquots of the cultures were then plated on YP-Dextrose or YP-Glycerol plates, and colonies were counted after 5 days at 30 °C. The black and gray columns show the total number of viable cells, and the red columns show the total number of non-respiring colonies in each culture (expressed per A₆₀₀ = 1). Shown are the means ± S.D. of three cultures; the asterisk denotes p = 0.002 as determined by Student's t test.

FIGURE 5.

I154F alters FXN^42–210 solubility in mammalian cells. A, WT- and I154F-FXN^1–210 precursors were expressed in COS-7 cells. 24 h post-transfection, cells were harvested and solubilized in 1% Triton X-100 in the presence of protease inhibitors, as described under “Experimental Procedures.” Total cell extracts were centrifuged at 20,800 × g for 15 min, and equivalent amounts of the soluble and insoluble fractions were analyzed by Western blotting with specific antibodies. NT, non-transfected.

FIGURE 6.

W155R blocks FXN^1–210 import and processing in yeast resulting in a virtual frataxin knock-out phenotype. The WT- and W155R-FXN^1–210 precursors were expressed in YFH1 or yfh1Δ yeast that, respectively, contained or lacked endogenous yeast frataxin to give the indicated strains. A, total protein extracts were prepared from the four strains by rapid trichloroacetic acid treatment, and FXN isoforms and other proteins were analyzed by Western blotting. B, growth phenotype of the indicated strains on rich medium supplemented with a fermentable (YP-Dextrose) or non-fermentable (YP-Ethanol) carbon source at 30 °C is shown. Experimental procedures were as described in the legend of Fig. 4B. C, lymphoblastoid cells from one control (designated Control; GM07521, female, 19 years old, normal GAA repeats) and one FRDA patient (designated FRDA; GM16197, male, 14 years old, 760 and 830 GAA repeats) were analyzed by Western blotting before or 48 h after transfection with pcDNA4 vectors containing the W155R-FXN^1–210 or W155R-FXN^1–210_FLAG precursor. Detergent-soluble extracts were prepared as described under “Experimental Procedures”; 20 μg of total protein was analyzed in lanes 1 and 2 and lanes 5 show 6 and 5 and 10 μg in lanes 3 and 4, respectively. Virtually identical results were obtained in two independent experiments, one of which is shown. NT, non-transfected.

FIGURE 7.

W155R weakens FXN^81–210 contacts with NFS1-ISD11. A, COS-7 cells transfected with pcDNA4-WT-FXN^1–210 or pcDNA4-W155R-FXN^1–210 were lysed after 24 h, and the extracts were analyzed by size-exclusion chromatography and Western blotting as described (15). Only the data for cells expressing the mutant precursor are shown. B, cell extracts were prepared as above, and pulldown assays were performed with purified _HisNFS1-ISD11 complex bound to Ni-NTA-agarose beads as described under “Experimental Procedures.” Aliquots of each cell extract (Input) and the total protein bound to Ni-NTA-agarose beads (+_HisNFS1-ISD11) were analyzed by Western blotting. + Beads denotes negative controls carried out with agarose beads without the _HisNFS1-ISD11 complex. C, wild type COS-7 cells were grown for 24 h, and total cell lysate was prepared. This lysate was used to perform pulldown assays with purified oligomeric _HisFXN^42–210 (wild type or bearing the W155R mutation) bound to Ni-NTA Agarose beads as described above.

FIGURE 8.

W155R has dramatic effects on the ability of FXN^81–210 and FXN^42–210 to activate NFS1 and catalyze Fe-S cluster synthesis in vitro. Cysteine desulfurase activity (A) and Fe-S cluster synthesis (B) were measured as described in the legend of Fig. 3 with the indicated proteins. In A, each bar shows the mean ± S.D. of six independent measurements with at least two different FXN protein preparations. The asterisk denotes p = 3.4 × 10⁻⁷ as determined by Student's t test. In B, each plot shows the mean ± S.D. of two independent measurements with two different protein preparations except for W155R-FXN^42–210, which was analyzed only one time, as the amounts of this protein were very limited due to its susceptibility to proteolytic degradation during purification from E. coli.