Two Zinc-Cluster Transcription Factors Control Induction of Alternative Oxidase in Neurospora crassa

Abstract

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.

Mutations in Two Zinc-Cluster Proteins Activate Alternative Respiratory and Gluconeogenic Pathways and Restore Senescence in Long-Lived Respiratory Mutants ofPodospora anserina

“…In N. crassa, electrophoretic mobility shift assays showed that AOD2 and AOD5 act synergistically to bind an alternative oxidase induction motif (AIM) present in the promoter of the aod-1 gene, which encodes the alternative oxidase. These data support the hypothesis that the two proteins interact with each other (Chae et al 2007b). The AIM motif consists of one pair of CGG repeats separated by 7 bp and is essential for the inducible expression of the aod-1 gene.…”

“…The rse2-1 gene was shown to contain a single T-to-G substitution changing a Tyr into an Asp codon at position 300. Sequence comparisons revealed that this protein is homologous to the recently reported AOD2 protein from N. crassa, which acts synergistically with another transcription factor of the zinc-cluster family, AOD5 (Chae et al 2007b). This prompted us to search by BLAST the homolog of this protein in the P. anserina genome.…”

“…We identified three mutations localized in two zinc-cluster transcription factors that control AOX expression both in P. anserina (this work) and in N. crassa (Chae et al 2007b). Interestingly, these two mutations activate the expression of the alternative oxidase and also of gluconeogenic genes.…”

Alternative oxidase: an inter-kingdom perspective on the function and regulation of this broadly distributed 'cyanide-resistant' terminal oxidase

“…It was hypothesised that the motif, which consists of a pair of CGG repeats separated by 7 bp, might be recognised by a member of the zinc-cluster family of transcription factors that might activate aod-1 expression by binding to the sequence (Chae et al 2007a). Indeed, the aod-2 and aod-5 gene products have recently been identified as transcription factors of this family (Chae et al 2007b). It is worth noting that the induction motif present in the aod-1 gene of N. crassa was only conserved in other members of the fungal order Sordariales.…”

Genetic and Functional Investigation of Zn2Cys6Transcription Factors RSE2 and RSE3 in Podospora anserina

“…In plants, the alternative oxidase is present in some tissues or developmental stages and may also be induced in response to a variety of stresses (7)(8)(9). In most fungi, it is undetectable, or present at very low levels, under normal growth conditions but becomes expressed when the cytochrome respiratory chain is restricted by inhibitors such as antimycin, azoxystrobin, and cyanide or by mutations (10)(11)(12)(13). Under these conditions, the AOX bypasses respiratory complexes III and IV.…”