Abstract
Frataxin is a highly conserved protein encoded by the frataxin (FXN) gene. The full-length 210-amino acid form of protein frataxin (1–210; isoform A) expressed in the cytosol of cells rapidly translocates to the mitochondria, where it is converted to the mature form (81–210) by mitochondrial processing peptidase. Mature frataxin (81–210) is a critically important protein because it facilitates the assembly of mitochondrial iron-sulfur cluster protein complexes such as aconitase, lipoate synthase, and succinate dehydrogenases. Decreased expression of frataxin protein is responsible for the devastating rare genetic disease of Friedreich’s ataxia. The mitochondrial form of frataxin has long been thought to be present in erythrocytes even though paradoxically, erythrocytes lack mitochondria. We have discovered that erythrocyte frataxin is in fact a novel isoform of frataxin (isoform E) with 135-amino acids and an N-terminally acetylated methionine residue. There is three times as much isoform E in erythrocytes (20.9 ± 6.4 ng/mL) from the whole blood of healthy volunteers (n = 10) when compared with the mature mitochondrial frataxin present in other blood cells (7.1 ± 1.0 ng/mL). Isoform E lacks a mitochondrial targeting sequence and so is distributed to both cytosol and the nucleus when expressed in cultured cells. When extra-mitochondrial frataxin isoform E is expressed in HEK 293 cells, it is converted to a shorter isoform identical to the mature frataxin found in mitochondria, which raises the possibility that it is involved in disease etiology. The ability to specifically quantify extra-mitochondrial and mitochondrial isoforms of frataxin in whole blood will make it possible to readily follow the natural history of diseases such as Friedreich’s ataxia and monitor the efficacy of therapeutic interventions.
Highlights
Frataxin is a highly conserved protein encoded by the frataxin gene (FXN), which can be found in both prokaryotes and eukaryotes[1,2]
To clearly show that the erythrocyte frataxin and the mature form frataxin are two independent forms, we ran erythrocyte lysate with frataxin SILAC standard that showed only one band (Fig. 1d, lane 2) with higher molecular weight than the one corresponding to the band coming from the HepG2 lysate with frataxin SILAC standard (Fig. 1d, lane 3)
To eliminate the possibility that within the erythrocyte milieu the mature frataxin form could be modified to a different isoform, we mixed the erythrocyte lysate with the HepG2 lysate and added the frataxin SILAC standard (Fig. 1d, lane 4)
Summary
Frataxin is a highly conserved protein encoded by the frataxin gene (FXN), which can be found in both prokaryotes and eukaryotes[1,2]. An in-frame stop codon in exon 5B of FXN-2 mRNA results in it encoding a shorter protein of 171-amino acids known as isoform B or FXN-2 (Q16595-2, Uniprot, MW = 19,095 Da)[9,13] Another minor alternative transcript, FXN-3 mRNA, differs from FXN-1 mRNA through an insertion of 8 bp due to an alternative splice site at the 5′ end of intron 4. The 8 bp insertion generates a frameshift that introduces a stop codon site, so that this transcript encodes for a protein of 196-amino acids known as FXN-3 (Q16595-3, Uniprot, MW = 21,416 Da)[9,13] These two shorter proteins, both contain the N-terminal mitochondrial targeting sequence[1]. We provide evidence that this N-terminally acetylated form of frataxin is a precursor of extra-mitochondrial mature frataxin that is normally found within the mitochondria
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