Cells were collected and glycerol content was analyzed as described in Fig 4B. stress was initiated by addition of sorbitol to 1 1.2M. Cells were collected and glycerol content was analyzed as described in UC-1728 Fig 4B. Error bars show SD.(TIF) pgen.1008115.s002.tif (349K) GUID:?DE368165-7956-45F9-8820-0509AAEC93F9 S3 Fig: Regulation of Cyc8-SUMOylation during adaptation to hyperosmotic stress is not affected by targets of the broad-spectrum kinase inhibitor staurosporine. (A). At 1 M staurosporine, PKA is inhibited, while Hog1 is not. Cells of the indicated genotypes were incubated with the indicated concentrations of staurosporine or an equal volume of DMSO for 1 hour, then treated with 1.2 M sorbitol for 15 min. Cells UC-1728 were collected, lysed, and subjected to Western analysis for the phosphorylated PKA motif, autophosphorylated Hog1, and Pgk1 as a loading control. (B) Cyc8 deSUMOylation kinetics are not affected by kinase activity. Indicated cells were incubated with 1 M staurosporine or an equal volume of DMSO Flrt2 for 1 hour, then treated, collected, and analyzed as described in Fig 1A. Cyc8 was identified by Western analysis using anti-HSV antibodies. Total Cyc8 in the input fraction was used as a loading control, while anti-pP38 was used as a control for Hog1 activation.(TIF) pgen.1008115.s003.tif (1.3M) GUID:?3FA3110C-1817-41EF-ABE3-CB2179CCE7E0 Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract Environmental stressors can severely perturb cellular homeostasis and compromise viability. To cope with environmental stressors, eukaryotes have developed distinct signaling programs that allow for adaptation during different stress conditions. These programs often require a host of post-translational modifications that alter proteins to elicit appropriate cellular responses. One crucial protein modifier during stress is the small ubiquitin-like modifier SUMO. In many cases, however, the functions of stress dependent protein SUMOylation remain unclear. UC-1728 Previously, we showed that the conserved Cyc8-Tup1 transcriptional corepressor complex undergoes transient hyperosmotic stress-induced SUMOylation and inclusion formation, which are important for appropriate regulation of hyperosmotic-stress genes. Here, we show the osmostress-responsive MAP kinase Hog1 UC-1728 regulates Cyc8 SUMOylation and inclusion formation via its role in the transcriptional activation of glycerol biosynthesis genes. Mutations that ablate Cyc8 SUMOylation can partially rescue the osmosensitivity of cells, and this is facilitated by inappropriate derepression of glycerol-biosynthesis genes. Furthermore, cells specifically unable to synthesize the osmolyte glycerol cause transient Cyc8 SUMOylation and inclusions to persist, indicating a regulatory role for glycerol to reestablish the basal state of Cyc8 following adaptation to hyperosmotic stress. These observations unveil a novel intersection between phosphorylation and SUMOylation networks, which are critical for shifting gene expression and metabolic programs during stress adaptation. Author summary The ability to sense and react to diverse environmental cues is a central aspect in the maintenance of cellular homeostasis. In response to harsh conditions, cells must rapidly deploy specific stress responses in order to adapt, survive, and proliferate. To ensure optimal spatial and temporal control over stress responses, many proteins undergo biophysical and biochemical alterations. More specifically, these alterations include conformational changes and post-translational modificationsCsuch as phosphorylation, ubiquitination, and SUMOylationCthat alter the function, localization, and interactome of target proteins. In this study, we show that the Hog1 MAPK regulates SUMOylation and biomolecular condensation of the yeast transcription corepressor complex Cyc8-Tup1 during exposure to hyperosmotic stress. In turn, this signaling relationship functions to effectively rewire yeast metabolism toward the biosynthesis of the compatible osmolyte glycerol, which serves as the ultimate signal to reset this genetic circuit. Introduction Cellular stresses are abiotic perturbations that can severely and irreversibly damage biomolecules UC-1728 and essential cell structures. All organisms experience cellular stress and subsequently must adjust a variety of cellular programs to reestablish homeostasis. Many of these include signal transduction networks, metabolic pathways, gene expression programs, cell-cycle progression, and protein quality control systems. Resiliency in the face of stress is crucial to cellular survival with the deterioration of adaptive measures thought to underlie a variety of age-related human diseases such as cancer, heart disease, and neurodegeneration [1C3]. Post-translational protein modifications are critical for the responses to cellular stress, and often occur rapidly upon the onset of stress. One of the major modifications that have been observed to occur during a wide range of cellular stresses is the addition.