Involvement of a G Protein Regulatory Circuit in Alternative Oxidase Production inNeurospora crassa

Abstract

The Neurospora crassa nuclear aod-1 gene encodes an alternative oxidase that functions in mitochondria. The enzyme provides a branch from the standard electron transport chain by transferring electrons directly from ubiquinol to oxygen. In standard laboratory strains, aod-1 is transcribed at very low levels under normal growth conditions. However, if the standard electron transport chain is disrupted, aod-1 mRNA expression is induced and the AOD1 protein is produced. We previously identified a strain of N. crassa, that produces high levels of aod-1 transcript under non-inducing conditions. Here we have crossed this strain to a standard lab strain and determined the genomic sequences of the parents and several progeny. Analysis of the sequence data and the levels of aod-1 mRNA in uninduced cultures revealed that a frameshift mutation in the flbA gene results in the high uninduced expression of aod-1. The flbA gene encodes a regulator of G protein signaling that decreases the activity of the Gα subunit of heterotrimeric G proteins. Our data suggest that strains with a functional flbA gene prevent uninduced expression of aod-1 by inactivating a G protein signaling pathway, and that this pathway is activated in cells grown under conditions that induce aod-1. Induced cells with a deletion of the gene encoding the Gα protein still have a partial increase in aod-1 mRNA levels, suggesting a second pathway for inducing transcription of the gene in N. crassa. We also present evidence that a translational control mechanism prevents production of AOD1 protein in uninduced cultures.

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

“…The production of the AOD1 protein in N. crassa is known to be controlled at the level of transcription of the aod-1 gene [ 19 , 30 , 31 , 33 , 37 ] and at the level of translation of the transcript [ 19 , 37 ]. To distinguish between the two possible mechanisms preventing expression of the protein in Δ yvh1 cells, we analyzed the levels of aod-1 transcript in the deletion mutant.…”

“…We have also demonstrated that the expression of aod-1 is influenced by a G protein signaling pathway. Strains lacking the FLBA protein have high levels of aod-1 transcript, even when grown in standard medium lacking any inducing drugs [ 37 ]. FLBA is a regulator of G protein signaling.…”

“…However, growth in the presence of typical inducing agents does result in the appearance of the AOD1 protein in flba mutants. These observations were interpreted as evidence for a control system that prevents translation of the aod-1 mRNA under non-inducing conditions [ 37 ].…”

Caracterização Da Enzima Oxidase Alternativa Do Fungo Moniliophthora Perniciosa Com O Auxilio Das Ferramentas De Bioinformática / Characterization of the Enzyme Oxidase Alternative of the Fungus Moniliophthora Perniciosa With the Aid of Bioinformatics Tools

“…Bosnjak et., 2019, carried out a study in which the presence of the AOD-1 gene was verified in the filamentous fungus Neurospora crassa, responsible for encoding the alternative oxidase, thus confirming the presence of AOX in this fungus. In this work, it was observed that the enzyme AOX performs the same function as an alternative pathway seen in basidiomycetes [20].…”

Proteome‐wide analyses reveal diverse functions of acetylation proteins in Neurospora crassa

“…Subsequently, a high‐quality draft sequence of the N. crassa genome has aided in efforts to investigative the molecular biology and genetics of filamentous fungi [8]. Studies of the genes regulating various aspects of Neurospora biology include the cAMP‐dependent and heterotrimeric G‐protein pathways [9, 10], mitogen‐activated protein kinase pathway [11–13], light photobiology and circadian rhythmicity [14–17], secondary metabolism [18–20], cell wall structure and synthesis [21] and so on. With respect to N. crassa DNA, approximately 1.5% of the cytosines are methylated, facilitating genetic studies [22].…”