Differential complementation of a Neurospora crassa Gα_i mutation using mammalian Gα protein genes

Abstract

Heterotrimeric (αβγ) G proteins interact with sensory receptors to transduce signals to downstream effectors in eukaryotes. We previously reported that GNA-1 from Neurospora crassa is a microbial member of the Gα_i family found in higher organisms. Deletion of gna-1 leads to female sterility, slower growth rates on normal and hyperosmotic solid medium, and increased resistance to heat and oxidative stress. In this study we compare mammalian genes for proteins of the Gα_i subfamily (Gα_i, Gα_o, Gα_t and Gα_z), and Gα_s (which is not a member of the Gα_i family) with the N. crassa gna-1 gene with respect to their ability to complement Δgna-1 phenotypes. Northern analysis detected full-length transcripts of all these genes, except that for Gα_i, in N. crassa transformants. Measurements of pertussis toxin-catalyzed ADP-ribosylation and Western analysis showed that the GNA-1, Gα_z, Gα_o and Gα_s proteins were present in the respective transformed strains. Strains in which the mammalian Gα protein could be detected were subjected to phenotypic testing. During the vegetative cycle, none of the mammalian Gα genes complemented the thermotolerance phenotype of Δgna-1. However, the three expressed mammalian Gα genes achieved at least partial complementation of the defects in vegetative apical extension rate. cAMP levels did not correlate with restoration of vegetative growth rate by the mammalian genes. During the sexual cycle, Gα_o was the only mammalian Gα gene that rescued the defect in female fertility characteristic of Δgna-1 strains. Alignment of GNA-1, Gα_z, Gα_o and Gα_s protein sequences revealed correlations between the observed complementation pattern and the degree of identity to GNA-1 in various functional motifs. The finding that Gα_z gave the best restoration of vegetative growth but could not restore normal female fertility implies that GNA-1 regulates different pathways that are important for vegetative and sexual growth in N. crassa.