Involvement of a G Protein Regulatory Circuit in Alternative Oxidase Production in Neurospora 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 Ga 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 Ga 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.