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      Heat-stress triggers MAPK crosstalk to turn on the hyperosmotic response pathway

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          Abstract

          Cells make decisions based on a combination of external and internal signals. In yeast, the high osmolarity response (HOG) is a mitogen-activated protein kinase (MAPK) pathway that responds to a variety of stimuli, and it is central to the general stress response. Here we studied the effect of heat-stress (HS) on HOG. Using live-cell reporters and genetics, we show that HS promotes Hog1 phosphorylation and Hog1-dependent gene expression, exclusively via the Sln1 phosphorelay branch, and that the strength of the activation is larger in yeast adapted to high external osmolarity. HS stimulation of HOG is indirect. First, we show that HS causes glycerol loss, necessary for HOG activation. Preventing glycerol efflux by deleting the glyceroporin FPS1 or its regulators RGC1 and ASK10/ RGC2, or by increasing external glycerol, greatly reduced HOG activation. Second, we found that HOG stimulation by HS depended on the operation of a second MAPK pathway, the cell-wall integrity (CWI), a well-known mediator of HS, since inactivating Pkc1 or deleting the MAPK SLT2 greatly reduced HOG activation. Our data suggest that the main role of the CWI in this process is to stimulate glycerol loss. We found that in yeast expressing the constitutively open channel mutant (Fps1-Δ11), HOG activity was independent of Slt2. In summary, we suggest that HS causes a reduction in turgor due to the loss of glycerol and the accompanying water, and that this is what actually stimulates HOG. Thus, taken together, our findings highlight a central role for Fps1, and the metabolism of glycerol, in the communication between the yeast MAPK pathways, essential for survival and reproduction in changing environments.

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          Most cited references45

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          A chemical switch for inhibitor-sensitive alleles of any protein kinase.

          Protein kinases have proved to be largely resistant to the design of highly specific inhibitors, even with the aid of combinatorial chemistry. The lack of these reagents has complicated efforts to assign specific signalling roles to individual kinases. Here we describe a chemical genetic strategy for sensitizing protein kinases to cell-permeable molecules that do not inhibit wild-type kinases. From two inhibitor scaffolds, we have identified potent and selective inhibitors for sensitized kinases from five distinct subfamilies. Tyrosine and serine/threonine kinases are equally amenable to this approach. We have analysed a budding yeast strain carrying an inhibitor-sensitive form of the cyclin-dependent kinase Cdc28 (CDK1) in place of the wild-type protein. Specific inhibition of Cdc28 in vivo caused a pre-mitotic cell-cycle arrest that is distinct from the G1 arrest typically observed in temperature-sensitive cdc28 mutants. The mutation that confers inhibitor-sensitivity is easily identifiable from primary sequence alignments. Thus, this approach can be used to systematically generate conditional alleles of protein kinases, allowing for rapid functional characterization of members of this important gene family.
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            The response to heat shock and oxidative stress in Saccharomyces cerevisiae.

            A common need for microbial cells is the ability to respond to potentially toxic environmental insults. Here we review the progress in understanding the response of the yeast Saccharomyces cerevisiae to two important environmental stresses: heat shock and oxidative stress. Both of these stresses are fundamental challenges that microbes of all types will experience. The study of these environmental stress responses in S. cerevisiae has illuminated many of the features now viewed as central to our understanding of eukaryotic cell biology. Transcriptional activation plays an important role in driving the multifaceted reaction to elevated temperature and levels of reactive oxygen species. Advances provided by the development of whole genome analyses have led to an appreciation of the global reorganization of gene expression and its integration between different stress regimens. While the precise nature of the signal eliciting the heat shock response remains elusive, recent progress in the understanding of induction of the oxidative stress response is summarized here. Although these stress conditions represent ancient challenges to S. cerevisiae and other microbes, much remains to be learned about the mechanisms dedicated to dealing with these environmental parameters.
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              Systematic exploration of essential yeast gene function with temperature-sensitive mutants.

              Conditional temperature-sensitive (ts) mutations are valuable reagents for studying essential genes in the yeast Saccharomyces cerevisiae. We constructed 787 ts strains, covering 497 (∼45%) of the 1,101 essential yeast genes, with ∼30% of the genes represented by multiple alleles. All of the alleles are integrated into their native genomic locus in the S288C common reference strain and are linked to a kanMX selectable marker, allowing further genetic manipulation by synthetic genetic array (SGA)-based, high-throughput methods. We show two such manipulations: barcoding of 440 strains, which enables chemical-genetic suppression analysis, and the construction of arrays of strains carrying different fluorescent markers of subcellular structure, which enables quantitative analysis of phenotypes using high-content screening. Quantitative analysis of a GFP-tubulin marker identified roles for cohesin and condensin genes in spindle disassembly. This mutant collection should facilitate a wide range of systematic studies aimed at understanding the functions of essential genes.
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                Author and article information

                Contributors
                colman-lerner@fbmc.fcen.uba.ar
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                11 October 2018
                11 October 2018
                2018
                : 8
                : 15168
                Affiliations
                [1 ]ISNI 0000 0001 0056 1981, GRID grid.7345.5, Departamento de Fisiología, Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales (FCEN), , Universidad de Buenos Aires (UBA), ; Buenos Aires, Argentina
                [2 ]ISNI 0000 0001 0056 1981, GRID grid.7345.5, Instituto de Fisiología, Biología Molecular y Neurociencias (IFIBYNE), CONICET-UBA, ; Buenos Aires, Argentina
                [3 ]ISNI 0000 0001 0056 1981, GRID grid.7345.5, CIHIDECAR-Departamento de Química Orgánica, FCEN, UBA, ; Buenos Aires, Argentina
                Author information
                http://orcid.org/0000-0002-2557-8883
                Article
                33203
                10.1038/s41598-018-33203-6
                6181916
                30310096
                a0fca9f1-89db-4303-aa0e-92567db32942
                © The Author(s) 2018

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 8 March 2018
                : 21 September 2018
                Funding
                Funded by: FundRef https://doi.org/10.13039/501100003074, Ministry of Science, Technology and Productive Innovation, Argentina | Agencia Nacional de Promoción Científica y Tecnológica (National Agency for Science and Technology, Argentina);
                Award ID: PICT2010-2248
                Award ID: PICT2013-2210
                Award ID: PICT2015-3824
                Award Recipient :
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