The Mitochondrial Disulfide Relay System Protein GFER Is Mutated in Autosomal-Recessive Myopathy with Cataract and Combined Respiratory-Chain Deficiency

Abstract

A disulfide relay system (DRS) was recently identified in the yeast mitochondrial intermembrane space (IMS) that consists of two essential components: the sulfhydryl oxidase Erv1 and the redox-regulated import receptor Mia40. The DRS drives the import of cysteine-rich proteins into the IMS via an oxidative folding mechanism. Erv1p is reoxidized within this system, transferring its electrons to molecular oxygen through interactions with cytochrome c and cytochrome c oxidase (COX), thereby linking the DRS to the respiratory chain. The role of the human Erv1 ortholog, GFER, in the DRS has been poorly explored. Using homozygosity mapping, we discovered that a mutation in the GFER gene causes an infantile mitochondrial disorder. Three children born to healthy consanguineous parents presented with progressive myopathy and partial combined respiratory-chain deficiency, congenital cataract, sensorineural hearing loss, and developmental delay. The consequences of the mutation at the level of the patient's muscle tissue and fibroblasts were 1) a reduction in complex I, II, and IV activity; 2) a lower cysteine-rich protein content; 3) abnormal ultrastructural morphology of the mitochondria, with enlargement of the IMS space; and 4) accelerated time-dependent accumulation of multiple mtDNA deletions. Moreover, the Saccharomyces cerevisiae erv1(R182H) mutant strain reproduced the complex IV activity defect and exhibited genetic instability of the mtDNA and mitochondrial morphological defects. These findings shed light on the mechanisms of mitochondrial biogenesis, establish the role of GFER in the human DRS, and promote an understanding of the pathogenesis of a new mitochondrial disease.

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

“…A S. cerevisiae erv1 knockout strain containing R182H ERV1 on a TRP1 plasmid [24] was transformed with URA3 plasmid pYES2 (Sigma-Aldrich) containing WT or R182H ERV1 under the regulation of a GAL10 promoter. Following selection, the transformants were plated on 5-fluoroanthranilic acid (FAA) solid medium to counter-select against TRP1-containing plasmid by incubating for up to 3 days at 30…”

“…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.…”

“…Di Fonzo et al [24] reported the first disease directly related to ALR, the human homologue of Erv1. The authors identified a single arginine-to-histidine substitution (R194H) in the Cterminus of ALR in children with autosomal recessive myopathy.…”

“…The authors identified a single arginine-to-histidine substitution (R194H) in the Cterminus of ALR in children with autosomal recessive myopathy. At the cellular level, the effects included a lower cysteinerich protein content, respiratory-chain deficiency, abnormal mitochondrial morphology and increased accumulation of mitochondrial DNA (mtDNA) deletions [24]. Because this arginine residue is conserved in other ALR homologues (Figure 1b), the authors also used yeast to show that the corresponding erv1 R182H mutant strain displayed genetic instability of the mtDNA, altered mitochondrial morphology, a temperature-sensitive growth phenotype and reduced cyt c oxidase (complex IV) activity [24].…”

“…At the cellular level, the effects included a lower cysteinerich protein content, respiratory-chain deficiency, abnormal mitochondrial morphology and increased accumulation of mitochondrial DNA (mtDNA) deletions [24]. Because this arginine residue is conserved in other ALR homologues (Figure 1b), the authors also used yeast to show that the corresponding erv1 R182H mutant strain displayed genetic instability of the mtDNA, altered mitochondrial morphology, a temperature-sensitive growth phenotype and reduced cyt c oxidase (complex IV) activity [24]. Protein characterizations of human ALR have since then shown that the R194H mutation affects the thermal stability, FAD-binding and proteolysis The X-ray crystal structure of yeast S. cerevisiae Erv1 core domain residues 84-188 (c) and a zoomed in of the structure showing the hydrogen bond formed between Arg 182 and the 2 OH of the ribose moiety of FAD (d) (PDB code: 4EOH [9]).…”