Evaluating the activity of the filamentous growth mitogen-activated protein kinase pathway in yeast.
Mitogen-activated protein kinase (MAPK) pathways are evolutionarily conserved signaling pathways that regulate diverse processes in eukaryotes. One such pathway regulates filamentous growth, a nutrient limitation response in budding yeast and other fungal species. This protocol describes three assays used to measure the activity of the filamentous growth pathway. First, western blotting for phosphorylated (activated) MAPKs (P∼MAPKs; Slt2p, Kss1p, Fus3p, and Hog1p) provides a measure of MAPK activity in yeast and other fungal species. Second, the PGU1 gene is a transcriptional target of the filamentous growth pathway. Cells that undergo filamentous growth secrete Pgu1p, an endopolygalacturonase that degrades the plant-specific polysaccharide pectin. We describe an assay that measures secreted pectinase activity, which reflects an active filamentous growth pathway. Finally, in yeast, two mucin-like glycoproteins, Msb2 and Flo11, regulate filamentous growth. Secretion of the processed and shed glycodomain of Msb2 is an indicator of MAPK activity. Flo11, the major adhesion molecule that controls filamentous growth and biofilm/mat formation, is also shed from cells. Detecting shed mucins with epitope-tagged versions of the proteins (secretion profiling) provides information about the regulation of filamentous growth across fungal species.
Department of Biological Sciences, State University of New York at Buffalo, Buffalo, New York 14260.
Paul J
PJ
Cullen
D025301:Hyphae / Q000201:enzymology* / Q000254:growth & development*; D047908:Intracellular Signaling Peptides and Proteins / Q000032:analysis; D008562:Membrane Glycoproteins / Q000032:analysis; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010368:Pectins / Q000378:metabolism; D011096:Polygalacturonase / Q000032:analysis*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis; D015398:Signal Transduction; D015003:Yeasts / Q000201:enzymology* / Q000254:growth & development*
C110690:FLO11 protein, S cerevisiae; D047908:Intracellular Signaling Peptides and Proteins; C076360:MSB2 protein, S cerevisiae; D008562:Membrane Glycoproteins; D029701:Saccharomyces cerevisiae Proteins; D010368:Pectins; D020928:Mitogen-Activated Protein Kinases; C493097:PGU1 protein, S cerevisiae; D011096:Polygalacturonase