Ste11 dimethylation impacts the osmotic stress response.
(A) Depiction of Ste11 pathway. (B) Schematic representation of Ssa1-Ste11 inter protein cross-links detected on NBD of Ssa1 and regulatory domain of Ste11. (C) Halo assay analyzing the phenotype of the Ste11 wild-type and methylation mutants in response to alpha factor. (D) FUS1-lacZ activity of Ste11 mutants in response to pheromone. Indicated yeast were grown to mid-log phase and then were processed as in . (E) Western blot analysis of the effect of Ste11 wild type and mutants in response to pheromone signaling. Indicated cells were grown to mid-log phase, treated with the indicated quantity of alpha factor. Phosphorylation of Fus3 was measured by analysis of lysates via western blotting with indicated antisera. (F) Growth assay of Ste11 methylation mutants in response to hyperosmotic stress. Cells were grown to mid-log phase, were 10-fold serially diluted and then plated onto appropriate media using a 48-pin replica-plating tool. Images of plates were taken after 3 days at 30°C. (G) 8xCRE-lacZ activity of Ste11 mutants in response to osmotic stress. Yeast transformed with the 8xCRE lacZ reporter were grown to mid-log phase and then treated with the indicated stressors. Cell lysate was extracted and beta-galactosidase activity was measured as in . Data shown are the mean and standard deviation of at least 5 biological replicates. (H) Halo assay showing that none of the Ste11 variants permit pheromone response in the absence of Ste20. (I) Impact of Ste11 methylation on chaperone interactions. Yeast transformed with indicated HA-Ste11 constructs were grown to mid-log phase and Ste11 complexes were isolated using HA-magnetic beads. The purified Ste11 complexes were analyzed by SDS-PAGE/western blot analysis using indicated antisera. (J) Model of methylation-dependent regulation of Ste11 activity regulation. Di-methylation of Ste11 selectively alters osmotic signaling but not mating pathway activity. R405 di-methylation may alter Ste11 association with key osmotic signaling proteins such as Ste50. Created with BioRender.com. The data underlying the graphs shown in the figure can be found in S1 Data. CTD, C-terminal domain; NBD, nucleotide-binding domain; SBD, substrate-binding domain.