Co-precipitation of Phosphate and Iron Limits Mitochondrial Phosphate Availability in Saccharomyces cerevisiae Lacking the Yeast Frataxin Homologue (YFH1)

Alexandra Seguin,Renata Santos,Debkumar Pain,Andrew Dancis,Jean-Michel Camadro,Emmanuel Lesuisse

doi:10.1074/jbc.m110.163253

Abstract

Saccharomyces cerevisiae cells lacking the yeast frataxin homologue (Δyfh1) accumulate iron in the mitochondria in the form of nanoparticles of ferric phosphate. The phosphate content of Δyfh1 mitochondria was higher than that of wild-type mitochondria, but the proportion of mitochondrial phosphate that was soluble was much lower in Δyfh1 cells. The rates of phosphate and iron uptake in vitro by isolated mitochondria were higher for Δyfh1 than wild-type mitochondria, and a significant proportion of the phosphate and iron rapidly became insoluble in the mitochondrial matrix, suggesting co-precipitation of these species after oxidation of iron by oxygen. Increasing the amount of phosphate in the medium decreased the amount of iron accumulated by Δyfh1 cells and improved their growth in an iron-dependent manner, and this effect was mostly transcriptional. Overexpressing the major mitochondrial phosphate carrier, MIR1, slightly increased the concentration of soluble mitochondrial phosphate and significantly improved various mitochondrial functions (cytochromes, [Fe-S] clusters, and respiration) in Δyfh1 cells. We conclude that in Δyfh1 cells, soluble phosphate is limiting, due to its co-precipitation with iron.

Highlights

Because the phenotypes associated with a lack of frataxin are both very diverse and differ according to the cellular model studied
Yeast mutants affected in [Fe-S] cluster biogenesis accumulate iron in their mitochondria, and the mechanism by which iron mislocalizes in these mutants is unknown [14]. We investigated whether this phenotype of mitochondrial iron accumulation can be detected in vitro; we measured iron uptake by mitochondria isolated from different yeast strains (Fig. 1)
This was true, to a lesser extent for mitochondria isolated from another mutant affected in [Fe-S] cluster biogenesis, ⌬ggc1 (Fig. 1), which lacks the GTP/GDP mitochondrial carrier [35] and that we used as a control throughout this study because it accumulates mitochondrial iron in the same form than the ⌬yfh1 mutant [13]

Summary

EXPERIMENTAL PROCEDURES

Yeast Strains and Growth Conditions—The strains used in this study were YPH499 (wild-type; MATa ura lys801 ade101 trp1-⌬63 his3-⌬200 leu2-⌬1 cyh2), YPH499⌬yfh (⌬yfh1; ⌬yfh1::TRP1), YPH499⌬mrs3⌬mrs (mrs4::kanMX4, mrs3::URA3), YPH499⌬ggc (ggc1::kanMX4)), and ERyfh1 [13]. The cells were grown in defined medium without phosphate (yeast nitrogen base without phosphate), to which various amounts of phosphate was added as K2HPO4/ KH2PO4 (pH 6.5). 100-␮l aliquots were withdrawn and added to ice-cold 0.6 M sorbitol buffered with 50 mM HEPES, pH 7, containing 1 mM unlabeled Fe(II)-ascorbate. The mitochondria were preincubated for 15 min at 30 °C in the same buffer containing 1 mM NADH and 10 ␮M Fe(II)-ascorbate, and 0.5 mM [32P]phosphate (as a mix of 1:1 sodium and potassium orthophosphate, pH 7) was added. Aliquots (100 ␮l) were withdrawn at intervals and added to ice-cold buffer (sorbitol-HEPES) containing 50 mM unlabeled phosphate. Soluble, and insoluble iron (55Fe) contents in whole mitochondria, in the intermembrane space, and in mitochondrial matrices were measured by scintillation counting of the corresponding fractions. Low temperature absorption spectra (Ϫ191 °C) of whole cells were recorded as described previously [33, 34]

RESULTS

Phosphate concentration

DISCUSSION

Camadro and Emmanuel Lesuisse