Steroid-resistant nephrotic syndrome (SRNS) is a frequent cause of end-stage renal failure. Identification of single-gene causes of SRNS has generated some insights into its pathogenesis; however, additional genes and disease mechanisms remain obscure, and SRNS continues to be treatment refractory. Here we have identified 6 different mutations in coenzyme Q₁₀ biosynthesis monooxygenase 6 (COQ6) in 13 individuals from 7 families by homozygosity mapping. Each mutation was linked to early-onset SRNS with sensorineural deafness. The deleterious effects of these human COQ6 mutations were validated by their lack of complementation in coq6-deficient yeast. Furthermore, knockdown of Coq6 in podocyte cell lines and coq6 in zebrafish embryos caused apoptosis that was partially reversed by coenzyme Q₁₀ treatment. In rats, COQ6 was located within cell processes and the Golgi apparatus of renal glomerular podocytes and in stria vascularis cells of the inner ear, consistent with an oto-renal disease phenotype. These data suggest that coenzyme Q₁₀–related forms of SRNS and hearing loss can be molecularly identified and potentially treated.

Coenzyme Q₁₀ (CoQ₁₀) deficiency has been associated with an increasing number of clinical phenotypes that respond to CoQ₁₀ supplementation. In two siblings with encephalomyopathy, nephropathy and severe CoQ₁₀ deficiency, a homozygous mutation was identified in the CoQ₁₀ biosynthesis gene COQ2, encoding polyprenyl-pHB transferase. To confirm the pathogenicity of this mutation, we have demonstrated that human wild-type, but not mutant COQ2, functionally complements COQ2 defective yeast. In addition, an equivalent mutation introduced in the yeast COQ2 gene also decreases both CoQ₆ concentration and growth in respiratory-chain dependent medium. Polyprenyl-pHB transferase activity was 33–45% of controls in COQ2 mutant fibroblasts. CoQ-dependent mitochondrial complexes activities were restored in deficient fibroblasts by CoQ₁₀ supplementation, and growth rate was restored in these cells by either CoQ₁₀ or uridine supplementation. This work is the first direct demonstration of the pathogenicity of a COQ2 mutation involved in human disease, and establishes yeast as a useful model to study human CoQ₁₀ deficiency. Moreover, we demonstrate that CoQ₁₀ deficiency in addition to the bioenergetics defect also impairs de novo pyrimidine synthesis, which may contribute to the pathogenesis of the disease.

Hypocotyls of kidney beans (Phaseolus vulgaris L.) accumulated ascorbate after preincubation with a number of possible precursors, mainlyl-galactono-γ-lactone (l-GL) andl-gulono-γ-lactone. The increase in the intracellular ascorbate concentration was parallel to the high stimulation of thel-GL dehydrogenase (l-GLD) activity measured in vitro using l-GL as a substrate and cytochromec as an electron acceptor. Cell fractionation using a continuous linear Percoll gradient demonstrated that l-GLD is associated with mitochondria; therefore, pure mitochondria were isolated and subjected to detergent treatment to separate soluble from membrane-linked proteins. l-GLD activity was mainly associated with the detergent phase, suggesting that a membrane-intrinsic protein is responsible for the ascorbic acid biosynthetic activity. Subfractionation of mitochondria demonstrated that l-GLD is located at the inner membrane.

Background: COQ4 encodes a protein that organises the multienzyme complex for the synthesis of coenzyme Q₁₀ (CoQ₁₀). A 3.9 Mb deletion of chromosome 9q34.13 was identified in a 3-year-old boy with mental retardation, encephalomyopathy and dysmorphic features. Because the deletion encompassed COQ4, the patient was screened for CoQ₁₀ deficiency. Methods: A complete molecular and biochemical characterisation of the patient's fibroblasts and of a yeast model were performed. Results: The study found reduced COQ4 expression (48% of controls), CoQ₁₀ content and biosynthetic rate (44% and 43% of controls), and activities of respiratory chain complex II+III. Cells displayed a growth defect that was corrected by the addition of CoQ₁₀ to the culture medium. Knockdown of COQ4 in HeLa cells also resulted in a reduction of CoQ_10. Diploid yeast haploinsufficient for COQ4 displayed similar CoQ deficiency. Haploinsufficency of other genes involved in CoQ₁₀ biosynthesis does not cause CoQ deficiency, underscoring the critical role of COQ4. Oral CoQ₁₀ supplementation resulted in a significant improvement of neuromuscular symptoms, which reappeared after supplementation was temporarily discontinued. Conclusion: Mutations of COQ4 should be searched for in patients with CoQ₁₀ deficiency and encephalomyopathy; patients with genomic rearrangements involving COQ4 should be screened for CoQ₁₀ deficiency, as they could benefit from supplementation.

Coenzyme Q (Q) is an essential component of the mitochondrial respiratory chain in eukaryotic cells but also is present in other cellular membranes where it acts as an antioxidant. Because Q synthesis machinery inSaccharomyces cerevisiae is located in the mitochondria, the intracellular distribution of Q indicates the existence of intracellular Q transport. In this study, the uptake of exogenous Q₆ by yeast and its transport from the plasma membrane to mitochondria was assessed in both wild-type and in Q-lesscoq7 mutants derived from four distinct laboratory yeast strains. Q₆ supplementation of medium containing ethanol, a non-fermentable carbon source, rescued growth in only two of the fourcoq7 mutant strains. Following culture in medium containing dextrose, the added Q₆ was detected in the plasma membrane of each of four coq7 mutants tested. This detection of Q₆ in the plasma membrane was corroborated by measuring ascorbate stabilization activity, as catalyzed by NADH-ascorbate free radical reductase, a transmembrane redox activity that provides a functional assay of plasma membrane Q₆. These assays indicate that each of the four coq7 mutant strains assimilate exogenous Q₆ into the plasma membrane. The two coq7 mutant strains rescued by Q₆ supplementation for growth on ethanol contained mitochondrial Q₆ levels similar to wild type. However, the content of Q₆ in mitochondria from the non-rescued strains was only 35 and 8%, respectively, of that present in the corresponding wild-type parental strains. In yeast strains rescued by exogenous Q₆, succinate-cytochrome creductase activity was partially restored, whereas non-rescued strains contained very low levels of activity. There was a strong correlation between mitochondrial Q₆ content, succinate-cytochromec reductase activity, and steady state levels of the cytochrome c ₁ polypeptide. These studies show that transport of extracellular Q₆ to the mitochondria operates in yeast but is strain-dependent. When Q biosynthesis is disrupted in yeast strains with defects in the intracellular transport of exogenous Q, the bc ₁complex is unstable. These results indicate that delivery of exogenous Q₆ to mitochondria is required fore activity and stability of the bc ₁ complex in yeast coqmutants.