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.
Parsley
Todd B
TB
Center for Biosystems Research (formerly The Center for Agricultural Biotechnology), University of Maryland Biotechnology Institute, 5115 Plant Sciences Building, College Park, MD 20742, USA.
Segers
Gert C
GC
Nuss
Donald L
DL
Dawe
Angus L
AL
D000595:Amino Acid Sequence; D002793:Cholic Acids; D005658:Fungi / Q000235:genetics* / Q000378:metabolism; D044385:GTP-Binding Protein alpha Subunits / Q000302:isolation & purification / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D015151:Immunoblotting; D008969:Molecular Sequence Data
D002793:Cholic Acids; D044385:GTP-Binding Protein alpha Subunits; D019204:GTP-Binding Proteins; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate