Iron-induced oligomerization of yeast frataxin homologue Yfh1 is dispensable in vivo

Abstract

The neurodegenerative disease Friedreich's ataxia is caused by reduced levels of frataxin, a mitochondrial matrix protein. The in vivo role of frataxin is under debate. Frataxin, as well as its yeast homologue Yfh1, binds multiple iron atoms as an oligomer and has been proposed to function as a crucial iron-storage protein. We identified a mutant Yfh1 defective in iron-induced oligomerization. This mutant protein was able to replace functionally wild-type Yfh1, even when expressed at low levels, when mitochondrial iron levels were high and in mutant strains having deletions of genes that had synthetic growth defects with a YFH1 deletion. The ability of an oligomerization-deficient Yfh1 to function in vivo suggests that oligomerization, and thus oligomerization-induced iron storage, is not a critical function of Yfh1. Rather, the capacity of this oligomerization-deficient mutant to interact with the Isu protein suggests a more direct role of Yfh1 in iron-sulphur cluster biogenesis.

Figure 1

Comparison of Yfh1 and human frataxin. (A) Alignment of sequences corresponding to the α1-helix region of eukaryotic frataxins. Negatively charged residues are in bold. At, Arabidopsis thaliana; Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Hs, Homo sapiens; Mm, Mus musculus; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe. (B) Structural model of Yfh1 generated using SWISS-MODEL program (Schwede et al, 2003). Side chains of solvent-exposed conserved, negatively charged residues in α1-helix are shown.

Figure 2

Oligomerization of wt and 86N/90Q/93Q Yfh1 in the presence of ferrous iron. Purified wt or 86N/90Q/93Q Yfh1 was incubated with Fe(NH₄)₂(SO₄)₂ at indicated Yfh1:iron molar ratios. Samples were subjected to size-exclusion chromatography and fractions analysed. (A) SDS–PAGE stained with SYPRO-Ruby. Positions of thyroglobulin (669 kDa), BSA (67 kDa), ovalbumin (43 kDa) and ribonuclease A (13.7 kDa) are indicated. (B) Iron content measured by atomic absorption spectrometry.

Figure 3

Mutations that alter the oligomerization capacity of Yfh1 do not affect cell growth even at low protein expression or high mitochondrial iron content. (A) Tenfold serial dilutions of cell suspensions of wt, Δyfh1 and Δyfh1 carrying a plasmid expressing either wt or indicated YFH1 mutant plated on rich (YPD) or minimal synthetic (CS) media. (B) Left and centre panels: Tenfold serial dilutions of wt, Δyfh1 and Δyfh1 containing wt or 86/90/93A YFH1 in a plasmid under the control of the tetO promoter. Right panel: Cells grown on YPD plate were harvested, cell extracts prepared and immunoblot analysis using Yfh1specific antibody was carried out. (C) Tenfold serial dilutions of wt, erv1^ts and Δyfh1erv1^ts containing either wt or 86/90/93A YFH1 on a plasmid under the control of the tetO promoter. Cells were grown at 30°C or indicated temperatures for 2 days. YPD− and YPD+ indicate plates lacking (−) or containing (+) 25 μg ml⁻¹ doxycycline.

Figure 4

Genetic interaction between YFH1 and genes involved in ISC biosynthesis. (A) Haploids of indicated genotypes resulting from analysis of tetrads of indicated strains. The genotype of the inviable y/u spore was deduced from the genotype of the three viable spores. (B) Tenfold serial dilutions of indicated double-deletion strains expressing wt or 86/90/93A YFH1 in a plasmid under the control of the tetO promoter. y/u and y/n, 30°C for 2 days; y/q, 34°C for 3 days; y/a1 and y/a2, 30°C for 4 days. y, u, n, q, a1 and a2 indicate deletion of YFH1, ISU1, NFU1, SSQ1, ISA1 and ISA2, respectively. (−) and (+) indicate absence and presence of doxycycline (25 μg ml⁻¹), respectively.

Figure 5

wt and 86/90/93A Yfh1 interact with Isu. His-tagged wt or His-tagged 86/90/93A Yfh1 bound to Ni resin was incubated with wt mitochondrial extracts. Binding of Isu was detected by immunoblot analysis using antibodyspecific for Isu. Binding of Isu in two independent experiments was quantified; binding of wt Yfh1 indicated as 100%. BSA, control for nonspecific binding; In, 5% of total input of mitochondrial extract.