Differential complementation of a Neurospora crassa G alpha(i) mutation using mammalian G alpha protein genes

Heterotrimeric (alpha beta gamma) G proteins interact with sensory receptor s to transduce signals to downstream effecters in enkaryotes. We previously reported that GNA-1 from Neurospora crassa is a microbial member of the G alpha(i) family found in higher organisms. Deletion of gna-1 leads to femal e sterility, slower growth rates on normal and hyperosmotic solid medium. a nd increased resistance to heat and oxidative stress. In this study we comp are mammalian genes for proteins of the G alpha(i) subfamily (G alpha(i), G alpha(o), G alpha(t), and G alpha(z)), and G alpha(s) (which is nota membe r of the G alpha(i) family) with the N. crassa gna-1 gene with respect to t heir ability to complement Delta gna-1 phenotypes. Northern analysis detect ed full-length transcripts of all these genes, except that for G alpha(i). in N. crassa transformants. Measurements of pertussis toxin-catalyzed ADP-r ibosylation and Western analysis showed that the GNA-1, G alpha(z), G alpha (o), and G alpha(s) proteins were present in the respective transformed str ains. Strains in which the mammalian G alpha protein could be detected were subjected to phenotypic testing. During the vegetative cycle, none of the mammalian G alpha genes complemented the thermotolerance phenotype of Delta gna-1. However, the three expressed mammalian G alpha genes achieved at le ast partial complementation of the defects in vegetative apical extension r ate. cAMP levels did not correlate with restoration of vegetative growth ra te by the mammalian genes. During the sexual cycle, G alpha, was the only m ammalian G alpha gene that rescued the defect in female fertility character istic of Delta gna-1 strains. Alignment of GNA-1, G alpha(z), G alpha(o) an d G alpha(s) protein sequences revealed correlations between the observed c omplementation pattern and the degree of identity to GNA-1 in various funct ional motifs. The finding that G alpha(z), gave the best restoration of veg etative growth but could not restore normal female fertility implies that G NA-1 regulates different pathways that are important for vegetative and sex ual growth in N. crassa.