Abstract
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 Gα subunit CPG-1, or cpgb-1, the Gβ subunit CPGB-1, consistently reduced the level of its presumptive partner protein. This was not observed in the case of a second Gα subunit, CPG-2, suggesting that CPG-1 and CPGB-1 regulate each other's stability. Further, analysis of transcript levels indicated that the Gα and Gβ 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 Gα subunit, CPG-3, that is most similar to three other filamentous fungal Gα proteins that form a phylogenetically distinct grouping.
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Acknowledgements
The authors wish to thank Lynn Geletka for critical evaluation of the manuscript. This work was supported in part by Public Health Service grant number GM55981 to D.L.N. T.B.P. was a postdoctoral trainee on Public Health Service grant number AI07510-3.
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Communicated by S. Hohmann
T.B. Parsley and G.C. Segers contributed equally to this work
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Parsley, T.B., Segers, G.C., Nuss, D.L. et al. Analysis of altered G-protein subunit accumulation in Cryphonectria parasitica reveals a third Gα homologue. Curr Genet 43, 24–33 (2003). https://doi.org/10.1007/s00294-003-0367-6
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DOI: https://doi.org/10.1007/s00294-003-0367-6