Searching for biogenesis factors of the NADH:ubiquinone oxidoreductase using the filamentous fungus Neurospora crassa

The NADH:ubiquinone oxidoreductase is the first and biggest complex of the mitochondrial respiratory chain. Its assembly involves subunits encoded by the mitochondrial as well as the nuclear genome and requires biogenesis factors which are not part of the mature complex I. In a genome wide search 283 genes were found only in yeast forming a respiratory complex I but not in yeast without this complex [1]. In this study we determined the relevance of these genes for the assembly of complex I by characterizing the corresponding knockout mutants of N. crassa obtained by a high-throughput programme [2]. The identification of knockout mutants with complex I defects was accomplished by measuring the NADH:ferricyanide redox activity and Blue Native polyacrylamide gel electrophoresis. Assembly intermediates were detected by western blot analysis using specific polyclonal antibodies against different complex I subunits.
Seven of 290 knockouts analyzed were affected in the assembly of complex I: two conserved hypothetical proteins, two proteins that contain the DUF185, two pentatricopeptide repeat proteins and a methyltransferase that is known to be clinically relevant.