Abstract
Complex I deficiency is a common cause of mitochondrial disease, resulting from mutations in genes encoding structural subunits, assembly factors or defects in mitochondrial gene expression. Advances in genetic diagnostics and sequencing have led to identification of several variants in NUBPL (nucleotide binding protein-like), encoding an assembly factor of complex I, which are potentially pathogenic. To help assign pathogenicity and learn more about the function of NUBPL, amino acid substitutions were recreated in the homologous Ind1 protein of the yeast model Yarrowia lipolytica. Leu102Pro destabilized the Ind1 protein, leading to a null-mutant phenotype. Asp103Tyr, Leu191Phe and Gly285Cys affected complex I assembly to varying degrees, whereas Gly136Asp substitution in Ind1 did not impact on complex I levels nor dNADH:ubiquinone activity. Blue-native polyacrylamide gel electrophoresis and immunolabelling of the structural subunits NUBM and NUCM revealed that all Ind1 variants accumulated a Q module intermediate of complex I. In the Ind1 Asp103Tyr variant, the matrix arm intermediate was virtually absent, indicating a dominant effect. Dysfunction of Ind1, but not absence of complex I, rendered Y. lipolytica sensitive to cold. The Ind1 Gly285Cys variant was able to support complex I assembly at 28°C, but not at 10°C. Our results indicate that Ind1 is required for progression of assembly from the Q module to the full matrix arm. Cold sensitivity could be developed as a phenotype assay to demonstrate pathogenicity of NUBPL mutations and other complex I defects.
Highlights
Defects in respiratory chain function underlie a large share of mitochondrial disorders, of which a third are due to complex I deficiency (OMIM 252010) [1,2,3]
We focused on assessing the functional impact of patient mutations resulting in a single amino acid change at a conserved position, such as Leu104Pro (L104P, patients 9, 10, 11 and 12), Asp105Tyr (D105Y, patients 5 and 6), Leu193Phe (L193F, patient 8) and Gly287Cys (G287C, patient 13)
While Asn198 and Val182 are not conserved in Y. lipolytica Ind1, the NUBPL Gly138Asp (G138D) variant with an allele frequency of 6.73 × 10-4 does affect a conserved amino acid, corresponding to Gly136 in Y. lipolytica Ind1, and was included in this study
Summary
Defects in respiratory chain function underlie a large share of mitochondrial disorders, of which a third are due to complex I deficiency (OMIM 252010) [1,2,3]. Clinical presentations such as Leigh syndrome usually occur in infancy or early adulthood. Disease-causing mutations have been identified in 27 out of 44 structural genes This only provides a diagnosis for about 50% of cases, as complex I deficiency may be caused by mutations in assembly factors, tRNAs and other genes for mitochondrial gene expression or mitochondrial
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