The disease-associated mutation of the mitochondrial thiol oxidase Erv1 impairs cofactor binding during its catalytic reaction

Abstract

Erv1 (essential for respiration and viability 1) is an FADdependent thiol oxidase of the Erv/ALR (augmenter of liver regeneration) sub-family. It is an essential component of the mitochondrial import and assembly (MIA) pathway, playing an important role in the oxidative folding of the mitochondrial intermembrane space (IMS) proteins and linking the MIA pathway to the mitochondrial respiratory chain via cytochrome c (cyt c). The importance of the Erv/ALR enzymes was also demonstrated in a recent study where a single mutation in the human ALR (R194H) leads to autosomal recessive myopathy [Di Fonzo, Ronchi, Lodi, Fassone, Tigano, Lamperti, Corti, Bordoni, Fortunato, Nizzardo et al. (2009) Am. J. Hum. Genet. 84, 594-604]. However, the molecular mechanism of the disease is still unclear. In the present study, we use yeast Erv1 as a model to provide clear evidence for a progressive functional defect in the catalytic activity of the corresponding Erv1 R182H mutant. We show that the FAD cofactor was released from Erv1 R182H during its catalytic cycle, which led to the inactivation of the enzyme. We also characterized the effects of the mutation on the folding and stability of Erv1 and tested our in vitro findings in vivo using a yeast genetic approach. The results of the present study allow us to provide a model for the functional defect in Erv1 R182H, which could potentially be extended to human ALR R194H and provides insights into the molecular basis of autosomal recessive myopathy.

Redox characterisation of Erv1, a key component for protein import and folding in yeast mitochondria

“… in terms of the relative reduction potentials between the proximal disulphide and FAD cofactor, we carried out Erv1 reduction titration using reduced Mia40. The functional C‐terminal domain of Mia40 was reduced (rMia40c) by incubation of the purified protein with 0.5 m m TECP as described previously , and followed by buffer exchange inside an anaerobic glove box to remove TCEP and molecular oxygen. The UV‐visible spectra of Erv1 were measured during rMia40c titration and are shown in Fig.…”

“…For Erv1 (the WT and mutants), the pET‐24a(+) plasmid containing yeast ERV1 gene was expressed in Escherichia coli Rosetta‐gami™ 2 cells (Novagen, Merck KGaA, Darmstadt, Germany) and purified using Ni‐NTA (Ni 2+ ‐nitrilotriacetate) His‐bind beads (Novagen) as described previously . Further purification was done by size‐exclusion chromatography (SEC) using buffer AE (BAE: 50 m m Tris/HCl, 150 m m NaCl and 1 m m EDTA, pH 7.4) on a Superdex 200 or Superdex 200plus 100/300 GL column (GE Healthcare Bio‐sciences, Uppsala, Sweden).…”

“…Further purification was done by size‐exclusion chromatography (SEC) using buffer AE (BAE: 50 m m Tris/HCl, 150 m m NaCl and 1 m m EDTA, pH 7.4) on a Superdex 200 or Superdex 200plus 100/300 GL column (GE Healthcare Bio‐sciences, Uppsala, Sweden). Similarly, Mia40c (C‐domain of Mia40: residues 284–403) was expressed in Escherichia coli Rosetta‐gami™ 2 cells (Novagen) and purified using Ni‐NTA beads . Superdex 75 100/300 GL column was used to further purify and isolate the monomer proteins for this study.…”

“…The catalytic (or FAD‐binding) domain of yeast ( Saccharomyces cerevisiae) Erv1 is at the C terminus. The FAD‐binding domain also contains a redox active‐site CXXC disulphide bond (Cys130‐Cys133), which is also known as the proximal disulphide as it is located proximal to the isoalloxazine ring of FAD, and a CX 16 C structural disulphide (Cys159‐Cys176) . Furthermore, Erv1 has a functionally important shuttle disulphide bond (Cys30‐Cys33) in the nonconserved and unfolded N‐terminal domain .…”

Protein trafficking in the mitochondrial intermembrane space: mechanisms and links to human disease

“…Erv1 itself has no structural similarity to other Mia40 substrates, though it is related to the Erv2 of the endoplasmic reticulum and shares a 30% similarity with its C-terminal domain (though their N-terminal domains are different) [80–82]. Erv1 exists as a head-to-tail homodimer [83], and has a human homolog, ALR (augmenter of liver regeneration) [84]. The N-terminus of Erv1 contains an amphipathic helix with flexible loops on either side [85], and is thought to be required for Erv1 dimerization, which is essential for its function.…”