Functional analysis of a human ATPAF2 gene mutation in yeast S.cerevisiae.

Aims : Mitochondrial ATP synthase or complex V couples the proton gradient, generated by the respiratory chain, to ATP synthesis. Assembly of this enzyme requires the activities of several accessory proteins. Here, we report on the functional effect of a p.W94R mutation, present in the ATPAF2 gene of an infant with a complex V decreased activity. The ATPAF2 gene is the human (hu) orthologue of the yeast (y) ATP12 gene. Methods : Multi and single copy plasmid constructs containing the wild type ATPAF2 or ATP12 gene, as well as the human mutant p.W94R or the yeast mutant counterpart p.W103R were generated. These constructs were introduced in an ATP12 respiratory deficient yeast strain. ATP synthase activity of individual yeast transformants was assayed by growth studies on a non-fermentable carbon source and by Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) stained for ATP synthase catalytic activity. WT plasmid constructs rescue the respiratory defect of a y delatp12 mutant strain. Results : Growth on a non-fermentable carbon source was strongly impaired for the human W94R mutant, while the growth of the yeast W103R mutant strain was normal. These results were confirmed by BN-PAGE studies and measurements of the complex V activity of the plasmid constructs in yeast cells. Conclusions : Our yeast complementation studies showed clearly that the p.W94R mutant of the huATPAF2p does not confer respiratory competence to an delatp12 yeast strain and is the cause of the complex V dysfunction and early death of our 14 months-old patient. Additional studies with delatp12delfmc1 double mutants, and at 37°C, the altered yeast protein was no longer able to rescue the respiratory deficient phenotype of the S.cerevisiae host strain.