Identification of Genes Required for Alternative Oxidase Production in the Neurospora crassa Gene Knockout Library

Abstract

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.

Analysis of RNA expression of normal and cancer tissues reveals high correlation of COP9 gene expression with respiratory chain complex components

“…Jab1/COPS5 was previously identified as an apoptosis inducing factor through modulation of mitochondrial BH3-domain containing protein BclGs [27]. A study found that in Neurospora crassa , three subunits of the COP9 were required to recover from deficiency of the alternative oxidase function, which is important for mitochondrial functions [28]. However, no follow up studies have been carried out for mammalian cells.…”

Alternative Oxidase: A Mitochondrial Respiratory Pathway to Maintain Metabolic and Signaling Homeostasis during Abiotic and Biotic Stress in Plants

“…In extant prokaryotes, AOX is limited to some α-proteobacteria (e.g., Novosphingobium aromaticivorans , [43]) and it is likely that AOX entered eukaryotic lineages via the ancient proteobacterial endosymbiont that gave rise to mitochondria [39]. AOX is also found sporadically in protists and fungi, in many cases within pathogenic organisms within these groups [44,45], but also within important non-pathogenic model systems such as the fungi Neurospora crassa [46] and Podospora anserina [47] Surprisingly, AOX is also present in many animal phyla, although clearly absent from vertebrates and arthropods [39,48]. The finding of AOX in the animal kingdom has spawned several recent studies in which the protein has been introduced into different vertebrates, as a means to study aspects of mitochondrial bioenergetics and signaling [49–51].…”

Characterization of Single Gene Deletion Mutants Affecting Alternative Oxidase Production in Neurospora crassa: Role of the yvh1 Gene

“…A number of genes that affect aod-1 expression have been identified [ 23 , 28 , 29 ]. Two zinc cluster transcription factors, encoded by the aod-2 and aod-5 genes [ 23 , 30 ], bind constitutively as a heterodimer in the upstream region of the aod-1 gene and are required for the increased transcription of aod-1 that occurs under inducing conditions [ 19 , 30 , 31 , 32 , 33 ].…”