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Yeast Osmoregulation Content Introduction Sensing osmotic changing HOG signaling pathways Transcriptional Responses Response To Hyperosmotic Shock Quantitative analyses Introduction The osmoregulatory system in the yeast Saccharomyces cerevisiae is particularly well understood. Key to yeast osmoregulation is the production and accumulation of the compatible solute glycerol, which is partly controlled by the high osmolarity glycerol (HOG) signaling system. Sensing Osmotic Changing Sln1 Sln1p is a protein of 1,220 amino acids. Sln1p is the only sensor histidine kinase in the S. cerevisiae proteome. Fig1 Topology of Sln1p Sho1p Sho1p is a protein of 367 amino acids consisting of four predicted transmembrane domains within the Nterminal part, a linker domain, and an SH3 domain for protein-protein interaction. Fig2 Topology of Sho1p Hkr1 and Msb2: Putative Osmosensors? Recent studies have shown that mucin-like transmembrane proteins Hkr1 and Msb2 are the potential osmosensors and the most upstream elements know so far in the SHO1 branch. Fig 3 Schematic models of Hkr1 and Msb2 proteins Fig4 A schematic model of SHO1 branch HOG Signaling Pathways The HOG pathway is one of the best understood and most intensively studied MAPK systems. Two branches of the HOG pathways: SHO1 branch and the SLN1 branch. Fig 5 The yeast HOG pathway signaling system The SLN1 Branch Under low osmolarity, Sln1p constantly autophosphorylates itself on His576. Then transferred to Asp1144, within the receiver domain of Sln1p. The phosphate group is transferred to His64 on Ypd1p and further to Asp554 on Ssk1p. Dephosphorylated Ssk1p activates the MAPKKKs Ssk2p and Ssk22p. Fig 6 A schematic model of the SLN1 branch of the yeast HOG pathway Fig 7 A possible model of Pbs2 activation by Ssk2 The SHO1 Branch Activation of the Sho1 branch of the HOG pathway involves rapid and transient formation of a protein complex at the cell surface, specifically at places of cell growth. Fig 8 A schematic model of SHO1 branch Fig 9 Schematic model of the sequential docking interactions in the SHO1 pathway. Events downstream of the MAPKKKs Pbs2p is activated by phosphorylation on Ser514 and Thr518 by any of the three MAPKKKs Ssk2p/Ssk22p and Ste11p. Dual phosphorylation on the conserved Thr174 and Tyr176 activates the MAP kinase Hog1p. Under hyperosmotic stress, Hog1p is rapidly phosphorylated and translocated immediately to the nucleus, and transcriptional responses are observed. Both phosphorylation of Hog1p and nuclear localization are transient effects. Fig 10 Hyperosmotic stress induces nuclear accumulation of Hog1 Fig 11 Intracellular distribution of Hog1 kinases Regulation of the HOG Pathway The Hog1 MAPK in the HOG pathway is negatively regulated jointly by the protein tyrosine phosphatases Ptp2/Ptp3 and the type 2 protein phosphatases Ptc1/Ptc2/Ptc3. Specificities of these phosphatases are determined by docking interactions as well as their cellular localizations. Fig 12 Protein phosphatases downregulating the Hog1 Fig 13 Protein phosphatases downregulating the Hog1 Transcriptional Responses Several studies have reported global gene expression analyses following a hyperosmotic shock of different intensity. It appears that about 200 to 400 genes are upregulated and that some 150 to 250 genes are downregulated. Several transcription factors seem to be involved in Hog1-dependent responses: Hot1, Sko1, Msn2/Msn4, Msn1, and Smp1. Table 1 Main transcriptional regulators of the HOG pathway Protein Family Function Sko1/Acr1p bZIP, CREB Repressor; also needed for activation from CREs Msn2p/Msn4 Zinc finger p Activator; binds to STREs (CCCCT) and mediates protein kinase Adependent gene expression Hot1p Novel helix-loophelix Activator; present together with Hog1p on some target promoters; needed for normal expression of some genes Msn1p Novel helix-loophelix Activator; also involved in pseudohyphal growth and many more processes Smp1 MADS box Activator Fig 14 Hog1 and control of gene expression. SKO1 Sko1 binds to cAMP response element sites in targets promoters. Active Hog1 converts Sko1 from a repressor to an activator. It appears that Sko1 controls the expression of several regulators of the osmoresponse systems, such as the Msn2 transcription factor and the Ptp3 protein phosphatase. Msn2 and Msn4 are two redundant proteins mediating a general stress response. Msn2/Msn4 nuclear localization is negatively controlled by protein kinase A. Hot1 is a nuclear protein that seems to control a set of less than 10 genes. Hot1 recruits Hog1 to target promoters. Response To Hyperosmotic Shock Metabolism and Transport of Glycerol Metabolism of Trehalose and Glycogen Transport systems involved in osmoadptation Metabolism and Transport of Glycerol Glycerol metabolic pathway. Control of glycerol production under osmotic stress Transmembrane flux of glycerol Fig 15 pathways for production of glycerol, trehalose, and glycogen • Control of glycerol production under osmotic stress Expression is rapidly and transiently stimulated by an osmotic upshift. The mRNA profile of GPD1 and GPP2 depends greatly on the severity of the stress. The rapid and transient transcriptional response to osmotic stress of GPD1 and GPP2 expression is highly dependent on the HOG pathway. • Transmembrane flux of glycerol Upon osmoshock, expression changes of several genes encoding enzymes in lipid metabolism have been observed by global gene expression analysis. Lower levels of ergosterol could make the membrane more compact and less flexible and hence lead to diminished transmembrane flux of glycerol. Transport systems involved in osmoadptation MIP Channels Ion Transport Osmolyte Possible Uptake Roles of the Vacuole in Osmoadaptation MIP Channels: Fps1 These type of proteins are characterized by six transmembrane domains andtwo loops, B and E, that dip into the membrane from both sides, essentially forming a seventh transmembrane domain. It appears that three regions play a role in Fps1 gating: the B loop, the region of about 40 amino acids proximal to the first transmembrane domain, and the 10 amino acids immediately distal to the sixth transmembrane domain. Fig 16 The yeast HOG pathway signaling system and overview of response mechanisms Thank you!