Hyperosmotic stress delays mating differentiation.

<p>(A) The mating pathway (blue) and the HOG pathway (yellow) share components (green). Overlapping lines indicate an interaction and activation of the downstream component, otherwise indicated by an arrow. The mating pathway <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002437#pgen.1002437-Dohlman1" target="_blank">[4]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002437#pgen.1002437-Bardwell1" target="_blank">[57]</a> is activated when mating pheromone binds the receptor (Ste2) and activates a G protein. The Gβγ subunit dimer (Ste4/18) recruits the MAPK complex comprised of the scaffold (Ste5), MAPK (Fus3), MAPKK (Ste7), MAPKKK (Ste11) and the Ste11 adaptor protein Ste50 to the plasma membrane <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002437#pgen.1002437-Elion1" target="_blank">[6]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002437#pgen.1002437-Pryciak1" target="_blank">[7]</a>. At the plasma membrane the small G-protein Cdc42 and the protein kinase Ste20 activate the assembled MAPK complex <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002437#pgen.1002437-Lamson1" target="_blank">[58]</a>. Activated Fus3 induces gene transcription, cell cycle arrest, and cytoskeletal rearrangement <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002437#pgen.1002437-Chang1" target="_blank">[8]</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002437#pgen.1002437-Yu1" target="_blank">[10]</a>. Mating pheromone also activates a second MAPK, Kss1. Kss1 primarily activates the haploid filamentous growth response, but also contributes to a full mating response <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002437#pgen.1002437-Bardwell1" target="_blank">[57]</a>. The HOG pathway <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002437#pgen.1002437-Hohmann2" target="_blank">[59]</a> is activated by hyperosmotic conditions. Two branches, <i>SHO1</i> and <i>SLN1</i>, detect hyperosmotic conditions. Hrk1 and Msb2 activate the <i>SHO1</i> branch <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002437#pgen.1002437-Tatebayashi2" target="_blank">[60]</a>, which shares components with the mating pathway <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002437#pgen.1002437-Tatebayashi1" target="_blank">[11]</a>. The <i>SLN1</i> branch activates the protein kinase Ssk1 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002437#pgen.1002437-Maeda2" target="_blank">[61]</a>, which activates the partially redundant MAPKKKs Ssk2 and Ssk22 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002437#pgen.1002437-Posas1" target="_blank">[62]</a>. The <i>SHO1</i> and <i>SLN1</i> branches converge on the MAPKK, Pbs2, which serves as a scaffold for Ste11, Ssk2, Ssk22 and the MAPK, Hog1. Activated Hog1 induces glycerol production, gene transcription, and cell cycle arrest <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002437#pgen.1002437-Clotet1" target="_blank">[12]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002437#pgen.1002437-Escote1" target="_blank">[13]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002437#pgen.1002437-Rep1" target="_blank">[33]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002437#pgen.1002437-Dihazi1" target="_blank">[63]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002437#pgen.1002437-ORourke2" target="_blank">[64]</a>. (B) Shmoo formation was visualized by microscopy after incubation of cells treated with 100 µM α factor, 100 µM α factor+0.5 M KCl, or 100 µM α factor+0.75 M KCl. The percentages of cells with shmoos or buds are shown. (C) Induction kinetics of Far1; wild-type cells were stimulated with 10 µM α factor and co-stimulated with 10 µM α factor+0.75 M KCl. Cell lysates were resolved by 7.5% SDS-PAGE and Far1-HA detected by immunoblotting with anti-HA antibodies. Glucose-6-phosphate dehydrogenase (G6PDH) served as a loading control.