Checkpoints in a Yeast Differentiation Pathway Coordinate Signaling during Hyperosmotic Stress
Figure 5
Constitutively active Hog1 dampens Fus3 activation and induction.
(A) Activation kinetics of Fus3 with constitutively active Hog1; wild-type cells transformed with vector control or plasmid-borne GAL1-SSK2ΔN were grown in SC media with 2% raffinose (Raf). Ssk2ΔN expression was induced by addition of 2% galactose for 60 min followed by addition of 3 µM α factor for 30 min. Cell lysates were resolved by 12.5% SDS-PAGE. P-Fus3 and P-Kss1 were detected with phospho-p44/p42 antibodies. P-Hog1 was detected with phospho-p38 antibodies. Total Fus3 and Hog1 were detected with Fus3 and Hog1 antibodies. G6PDH served as a loading control. All primary antibodies were recognized by fluorescently labeled secondary antibody, detected by fluorescence scanner (Typhoon Trio) and quantified by scanning densitometry (ImageJ). The panels to the right show averaged scanning densitometry of four individual experiments. Error bars represent ± SEM. P-Hog1 reduced P-Fus3 by 49.4%±6.7% at 120 min. (B) Wild-type, hog1Δ, and, hog1K52R cells transformed with GAL1-SSK2ΔN or parent vector control were grown in SC and 2% galactose for 60 min followed by addition of 3 µM α factor or left untreated for 30 min. (C) fus3Δ cells transformed with ADH1-FUS3 and GAL1-SSK2ΔN or vector were grown and stimulated as in B. P-Hog1 (SSK2ΔN) reduced P-Fus3 by 30.7%±3.2%. (D) rck2Δ cells transformed with GAL1-SSK2ΔN or vector were grown and stimulated as in B. P-Hog1 reduced P-Fus3 by 31.0%±6.2%.