Analysis of altered G-protein subunit accumulation in Cryphonectria parasitica reveals a third Galpha homologue

Heterotrimeric G-proteins mediate many responses of eukaryotic cells to external stimuli and have been shown to be important for fungal pathogenicity. In this study, we explored the accumulation of G-protein subunits of the chestnut blight fungus, Cryphonectria parasitica, in mutant strains deleted for one or more putative partner subunits. Using a series of extraction buffers and immunoblot end-point dilution analysis, we established a convenient method to assess the relative abundance of these membrane-associated proteins. Disruption of either cpg-1, which encodes the Galpha subunit CPG-1, or cpgb-1, the Gbeta subunit CPGB-1, consistently reduced the level of its presumptive partner protein. This was not observed in the case of a second Galpha subunit, CPG-2, suggesting that CPG-1 and CPGB-1 regulate each other's stability. Further, analysis of transcript levels indicated that the Galpha and Gbeta protein turnover rates were increased in the mutant strains. Additionally, a previously unidentified protein that was cross-reactive with anti-CPG-1 antiserum was found to be enhanced in liquid culture. We describe the sequence of a new Galpha subunit, CPG-3, that is most similar to three other filamentous fungal Galpha proteins that form a phylogenetically distinct grouping.