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.

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 cytochromemediated 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.

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.

Alternative oxidase (AOX) has been found in a large number of filamentous fungi and yeasts with the notable exceptions of Saccharomyces cerevisiae and Schizosaccharomyces pombe. In virtually all of these fungi, AOX is induced by stresses on the cell that compromise the efficiency of the standard mitochondrial electron transport chain. As AOX is encoded in the nucleus and the signals that induce its expression originate in mitochondria, induction of the enzyme provides a classic example of retrograde regulation where signals from mitochondria influence the expression of nuclear genes. We have previously isolated mutants in Neurospora crassa that are incapable of inducing AOX. The genes affected in two of these mutants, aod-2 and aod-5, encode zinc cluster transcription factors that act to control expression of the AOX by binding to an alternative oxidase induction motif (AIM) found in the promoter of the AOX structural gene. We have now used pull-down assays and size-exclusion chromatography to demonstrate that the AOD2 and AOD5 proteins physically interact in vitro. In addition, we have shown that a homolog of the RTG2 protein, which is part of a classic retrograde signaling pathway in S. cerevisiae, is not required for AOX regulation in N. crassa.