Frataxin and mitochondrial FeS cluster biogenesis

Abstract

Friedreich ataxia is an inherited neurodegenerative disease caused by frataxin deficiency. Frataxin is a conserved mitochondrial protein that plays a role in FeS cluster assembly in mitochondria. FeS clusters are modular cofactors that perform essential functions throughout the cell. They are synthesized by a multistep and multisubunit mitochondrial machinery that includes the scaffold protein Isu for assembling a protein-bound FeS cluster intermediate. Frataxin interacts with Isu, iron, and the cysteine desulfurase Nfs1, which supplies sulfide, thus placing it at the center of mitochondrial FeS cluster biosynthesis.

FIGURE 1.

Scheme showing role of yeast frataxin (Yfh1) in mitochondrial FeS cluster assembly. The mitochondrion is shown as an oval bounded by a double membrane. Yfh1 is shown as a helix-sheet sandwich (green and tan). A, mitochondrial carriers Mrs3 and Mrs4 (box) play a role in transfer of iron in some form (Fe-X) across the mitochondrial inner membrane. Cysteine (Cys-SH) enters mitochondria and is acted on by the enzyme Nfs1 and its accessory protein Isd11 to provide sulfur for FeS clusters. Electrons or reducing equivalents are provided by the reductase Arh1 in the membrane and the associated ferredoxin Yah1. Yfh1 physically interacts with Nfs1, Isd11, and Isu. B, the FeS cluster intermediate (red diamonds) is assembled on the scaffold Isu, which interacts with Nfs1, Isd11, and Yfh1. Another complex is formed by Isu with Ssq1, Jac1, and Mge1, with the binding site being provided by the PVK tripeptide on Isu1. Grx5 acts at this step. C, the precursor proteins (squiggly black lines) are nuclear encoded, translated on cytoplasmic ribosomes, targeted to mitochondria, and imported in an unfolded state. After proteolytic processing, they are folded and acquire the FeS cluster cofactor (red diamonds) by action of the chaperones and glutaredoxin.

FIGURE 2.

Imported frataxin (Yfh1) restores FeS cluster assembly in isolated yeast mitochondria. Upper panel, wild-type mitochondria (left) synthesize FeS clusters on endogenous apoaconitase and imported apoYah1. Mutant Δyfh1 mitochondria (middle) fail to synthesize clusters. Following Yfh1 precursor import, mutant Δyfh1 mitochondria (right) now synthesize clusters. OM, outer membrane; IMS, intermembrane space; IM, inner membrane. Lower panel, mitochondria were isolated from wild-type or Δyfh1 strains. Full-length yeast frataxin (produced and radiolabeled on methionine and cysteine in the polypeptide) was imported (18 °C, 10 min) (lanes 3, 4, 7, and 8), and mitochondria were recovered by centrifugation. In the second stage, [³⁵S]cysteine and ferredoxin precursor Yah1 were imported (30 °C, 10 min) with or without added iron. The mitochondria were recovered, and the matrix fraction was analyzed on a native gel and by autoradiography (lanes 1–8). The imported Yfh1 labeled on the polypeptide is visible on the gel. The imported ferredoxin labeled on the sulfur of the FeS is visible in Δyfh1 mitochondria only following Yfh1 import (lanes 7 and 8). Similarly, endogenous aconitase labeled on its FeS cluster is visible in Δyfh1 only following Yfh1 import (lanes 7 and 8) (23).

FIGURE 3.

A, solution structure of monomeric apo-Yfh1 (Protein Data Bank code 2GA5) with residues involved in Isu binding identified in blue. Yfh1 amino acid substitutions with effects on Isu interactions are as follows: N122A/K123T/Q124A, diminished Isu interaction, low aconitase and succinate dehydrogenase, and iron accumulation (58); single amino acid changes Q129A, W131A, and R141A, diminished Isu interaction and low aconitase (45); and D101A/E103A, no interaction with Isu and decreased aconitase (50). B, solution structure of monomeric apo-Yfh1 with residues involved in iron binding identified in red (37). Published data on frataxins with substitutions of acidic amino acids are as follows: D86A/E90A/E93A/D101A/E103A, decreased iron-binding affinity, low aconitase, and slow growth (50); D86A/E89A/E101A/E103A, decreased iron-dependent Isu interaction, low aconitase, and iron accumulation (51); D86A/E90A/E93A, no oligomerization in response to iron exposure in vitro, normal Isu interaction, no FeS deficit, and no iron accumulation (53); D79A/D82A, low ferroxidase, oxidant sensitivity, normal aconitase, and no iron accumulation (69); and E93A/D97A/E103A, oxidant sensitivity, normal aconitase, and no iron accumulation (69). C-term and N-term, C and N termini.