Analysis of altered G-protein subunit accumulation in Cryphonectria parasitica reveals a third Gα homologue
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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.
KeywordsG-protein alpha subunit Protein accumulation Protein turnover
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.
- Choi GH, Larson TG, Nuss DL (1992) Molecular analysis of the laccase gene from the chestnut blight fungus and selective suppression of its expression in an isogenic hypovirulent strain. Mol Plant Microbe Interact 5:119–128Google Scholar
- Churchill ACL, Ciufetti LM, Hansen DR, Van Etten HD, Van Alfen NK (1990) Transformation of the fungal pathogen Cryphonectria parasitica with a variety of heterologous plasmids. Curr Genet 17:25–31Google Scholar
- Dohlman HG, Song J, Ma D, Courchesne WE, Thorner J (1996) Sst2, a negative regulator of pheromone signaling in the yeast Saccharomyces cerevisiae: expression, localization, and genetic interaction and physical association with Gpa1 (the G-protein a subunit). Mol Cell Biol 16:5194–5209PubMedGoogle Scholar
- Puhalla JE, Anagnostakis SL (1971) Genetics and nutritional requirements of Endothia parasitica. Phytopathology 61:169–173Google Scholar
- Sankaram MB, Marsh D (1993) Protein–lipid interactions with peripheral membrane proteins. In: Watts AD (ed) Protein and lipid interactions. Elsevier, Amsterdam, pp 127–162Google Scholar
- Segers GC, Nuss DL (2002) Constitutively activated Ga negatively regulates virulence, reproduction and hydrophobin gene expression in the chestnut blight fungus Cryphonectria parasitica. Fungal Genet Biol (in press)Google Scholar