Abstract
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 ...Continue Reading
References
Mar 20, 1992·Cell·C J GimenoG R Fink
Oct 1, 1994·Yeast·A GainvorsA Belarbi
Nov 1, 1996·Genetics·H LiuG R Fink
Jul 31, 1998·FEMS Microbiology Letters·P BlancoT G Villa
Feb 24, 1999·Yeast·S GogniesA Belarbi
Oct 27, 1999·Proceedings of the National Academy of Sciences of the United States of America·H D MadhaniG R Fink
Jul 17, 2004·Genes & Development·Paul J CullenGeorge F Sprague
Nov 17, 2005·Molecular and Cellular Biology·Elvira RománJesús Pla
Feb 12, 2008·Current Biology : CB·Michael J Lee, Henrik G Dohlman
Jul 2, 2008·The Journal of Cell Biology·Nadia VadaiePaul J Cullen
Mar 25, 2010·PLoS Genetics·Colin A ChavelPaul J Cullen
Jul 14, 2010·Current Biology : CB·Sheelarani KarunanithiPaul J Cullen
Mar 29, 2011·The Plant Cell·Elena Pérez-Nadales, Antonio Di Pietro
Feb 10, 2012·PLoS Pathogens·Eva Szafranski-SchneiderJoachim F Ernst
Aug 30, 2012·Nature Methods·Caroline A SchneiderKevin W Eliceiri
Nov 10, 2012·PloS One·Sumant PuriMira Edgerton
Citations
May 6, 2019·Genetics·Jacky ChowPaul J Cullen
Apr 30, 2017·Genetics·Elyse A HopeMaitreya J Dunham
Mar 4, 2015·Cold Spring Harbor Protocols·Paul J Cullen