Identification of genes required for alternative oxidase production in the Neurospora crassa gene knockout library.

The alternative oxidase (AOX) of Neurospora crassa transfers electrons from ubiquinol to oxygen. The enzyme is not expressed under normal conditions. However, when the function of the standard electron transport chain is compromised, AOX is induced, providing cells with a means to continue respiration and growth. Induction of the enzyme represents a form of retrograde regulation because AOX is encoded by a nuclear gene that responds to signals produced from inefficiently functioning mitochondria. To identify genes required for AOX expression, we have screened the N. crassa gene knockout library for strains that are unable to grow in the presence of antimycin A, an inhibitor of complex III of the standard electron transport chain. From the 7800 strains containing knockouts of different genes, we identified 62 strains that have reduced levels of AOX when grown under conditions known to induce the enzyme. Some strains have virtually no AOX, whereas others have only a slight reduction of the protein. A broad range of seemingly unrelated functions are represented in the knockouts. For example, we identified transcription factors, kinases, the mitochondrial import receptor Tom70, three subunits of the COP9 signalosome, a monothiol glutaredoxin, and several hypothetical proteins as being required for wild-type levels of AOX production. Our results suggest that defects in many signaling or metabolic pathways have a negative effect on AOX expression and imply that complex systems control production of the enzyme.

Two zinc-cluster transcription factors control induction of alternative oxidase in Neurospora crassa.

The alternative oxidase transfers electrons from ubiquinol to molecular oxygen, providing a mechanism for bypassing the later steps of the standard cytochrome-mediated electron transport chain. The enzyme is found in an array of organisms and in many cases is known to be produced in response to perturbations of the standard chain. Alternative oxidase is encoded in the nucleus but functions in the inner mitochondrial membrane. This implies the existence of a retrograde regulation pathway for communicating from the mitochondrion to the nucleus to induce alternative oxidase expression. Previous studies on alternative oxidase in fungi and plants have shown that a number of genes are required for expression of the enzyme, but the identity of these genes has remained elusive. By gene rescue we have now shown that the aod-2 and aod-5 genes of Neurospora crassa encode transcription factors of the zinc-cluster family. Electrophoretic mobility shift assays show that the DNA-binding domains of the AOD2 and AOD5 proteins act in tandem to bind a sequence element in the alternative oxidase gene promoter that is required for expression. Both proteins contain potential PAS domains near their C terminus, which are found primarily in proteins involved in signal transduction.

Identification of an alternative oxidase induction motif in the promoter region of the aod-1 gene in Neurospora crassa.

The nuclear aod-1 gene of Neurospora crassa encodes the alternative oxidase and is induced when the standard cytochrome-mediated respiratory chain of mitochondria is inhibited. To study elements of the pathway responsible for alternative oxidase induction, we generated a series of mutations in the region upstream from the aod-1 structural gene and transformed the constructs into an aod-1 mutant strain. Transformed conidia were plated on media containing antimycin A, which inhibits the cytochrome-mediated electron transport chain so that only cells expressing alternative oxidase will grow. Using this functional in vivo assay, we identified an alternative oxidase induction motif (AIM) that is required for efficient expression of aod-1. The AIM sequence consists of two CGG repeats separated by 7 bp and is similar to sequences known to be bound by members of the Zn(II)2Cys6 binuclear cluster family of transcription factors. The AIM motif appears to be conserved in other species found in the order Sordariales.

Alternative oxidase expression in Neurospora crassa.

When electron flow through the cytochrome-mediated electron transport chain is blocked by inhibitors or mutations, the mitochondria of Neurospora crassa contain a KCN-insensitive alternative oxidase, encoded by the aod-1 gene, that transfers electrons directly from the ubiquinone pool to oxygen. The mechanism by which the enzyme is induced is unknown. Comparison of the sequence upstream of the N. crassa aod-1 gene with the corresponding region of Gelasinospora spp. and Aspergillus nidulans revealed a cyclic AMP responsive element (CRE) about 700-800 bp upstream of the start codon in each species. Electrophoretic mobility shift assays showed that a factor from N. crassa cell extracts binds specifically to the CRE sequence. However, transformation of an aod-1 mutant strain with constructs lacking the CRE gave strains that regulate alternative oxidase in a normal fashion. Nuclear run-on assays indicated that uninduced cells transcribe the aod-1 gene at a low constitutive rate and that the transcription rate is increased in cells induced by antimycin A. Non-induced wild-type cultures occasionally contained significant amounts of aod-1 mRNA, but Western blots revealed no detectable AOD1 protein in mitochondria of these cells. This suggests that post-transcriptional events also play a role in alternative oxidase expression. A BLAST search of the Neurospora genome sequence revealed a second gene with the potential to encode an alternative oxidase, which we have named aod-3. Northern blot analysis using probes specific for the aod-1 and aod-3 genes revealed no evidence of expression of aod-3.