Alternative oxidase regulatory mutants in Neurospora crassa

Mitochondria are the major sites of ATP generation in eukaryotic cells. While most mitochondrial proteins are encoded by nuclear genes, a few proteins necessary for oxidative phosphorylation are encoded by the mitochondrial genome. Since nuclear gene expression is modulated in response to mitochondrial conditions, an as yet unknown signaling system between mitochondria and the nucleus must exist to allow the coordinate gene expression necessary for proper mitochondrial function. The nuclear gene aod-1, which encodes the alternative oxidase in Neurospora crassa, provides a system to study this signaling. Alternative oxidase shows specific induction: it is produced when the primary oxidative phosphorylation pathway is inhibited, but is absent when the oxidative pathway is not compromised.; Mutants in this unknown signaling pathway were identified through an EMS mutagenesis screen of a strain containing a reporter construct of the aod-1 promoter fused to the tyrosinase structural gene. Fifteen putative regulatory mutants comprising five novel complementation groups were identified in the screen. All five mutant loci fail to induce alternative oxidase under inducing conditions. Three of the mutants produce no aod-1 mRNA or protein under inducing conditions, while the other two mutants produce some protein under inducing conditions. None of the mutations affected global mitochondrial function or growth of the strains, suggesting that the mutations affected aod-1 regulation specifically.; The gene affected in the aod-5 mutant was cloned and found to encode a Zn(II)2Cys6 fungal transcription factor. This protein is predicted to regulate transcription by binding DNA as a dimer. It also has a PAS domain which may be involved in signal transduction.; The assembly of AOD1 protein in N. crassa was also examined. AOD1 is an interfacial integral membrane protein associated with the mitochondrial inner membrane. This protein is specifically localized to mitochondria within the cell. It can be partially extracted from the membrane with sodium carbonate (pH 11.5) treatment and is also released from the membrane upon sonication.