Respiratory Complexes III and IV Are Not Essential for the Assembly/Stability of Complex I in Fungi

The functional relevance of respiratory supercomplexes in various eukaryotes including mammals, plants, and fungi is hitherto poorly elucidated. However, substantial evidence indicates as a major role the assembly and/or stabilization of mammalian complex I by supercomplex formation with complexes III and IV. Here, we demonstrate by using native electrophoresis that the long-lived Podospora anserina mutant Cyc1-1, respiring exclusively via the alternative oxidase (AOX), lacks an assembled complex III and possesses complex I partially assembled with complex IV into a supercomplex. This resembles the situation in complex-IV-deficient mutants displaying a corresponding phenotype but possessing I-III supercomplexes instead, suggesting that either complex III or complex TV is in a redundant manner necessary for assembly/stabilization of complex I as previously shown in mammals. To corroborate this notion, we constructed the double mutant Cyc1-1,Cox5::ble. Surprisingly, this mutant lacking both complexes III and IV is viable and essentially a phenocopy of mutant Cyc1-1 including the reversal of the phenotype towards wild-type-like characteristics by the several-fold overexpression of the AOX in mutant Cyc1-1,Cox5::ble (Gpd-Aox). Fungal specific features (not found in mammals) that must be responsible for assembly/stabilization of fungal complex I when complexes III and IV are absent, such as the presence of the AOX and complex I dimerization, are addressed and discussed. These intriguing results unequivocally prove that complexes III and Ware dispensable for assembly/stability of complex I in fungi contrary to the situation in mammals, thus highlighting the imperative to unravel the biogenesis of complex I as well as the true supramolecular organization of the respiratory chain and its functional significance in a variety of model eukaryotes. In summary, we present the first obligatorily aerobic eukaryote with an artificial, simultaneous lack of the respiratory complexes III and IV. (C) 2008 Elsevier Ltd. All rights reserved.