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. Author manuscript; available in PMC: 2010 Oct 19.

Published in final edited form as: Methods Enzymol. 2010 Mar 1;471:291–317. doi: 10.1016/S0076-6879(10)71016-8

SLN1 pathway organization and regulation. (a) Schematic showing flow of phosphoryl groups through the SLN1 pathway. Plasma membrane associated Sln1 kinase is autophosphorylated on the H576 residue under normal growth conditions. Hyper-activation occurs under conditions causing weakening of the cell wall (wall stress). Hyperosmolarity and other conditions causing reduced turgor lead to a reduction in Sln1 kinase activity and accumulation of Sln1 in the dephosphorylated form. The phosphoryl group on H576 (H) is transferred to D1144 (D) in the receiver domain of Sln1 and then to H64 of the phosphotransferase, Ypd1, and finally to D554 and D427 in the receiver domains of the Ssk1 and Skn7 response regulators, respectively. When Ssk1 is unphosphorylated it interacts with and stimulates the activity of the Ssk2 and Ssk22 MAPKKKs of the HOG1 pathway. Phosphorylation of Ssk1 renders it inactive with respect to the HOG1 pathway. Phosphorylation of Skn7 leads to activation of SKN7 dependent genes such as the mannosyltransferase, OCH1. (b) Schematic depicting the domain structure of Sln1. Two molecules of a Sln1 dimer are shown associated with the plasma membrane via two transmembrane domains (TMDs). An extensive extracellular domain (ECD) protrudes into the periplasmic space. This protein configuration is conducive to detection of changes in pressure against the membrane (turgor pressure) and changes in composition of the cell wall which may lead to altered rigidity. The stimulus is transduced through several juxtamembrane domains including a coiled-coil domain (CC) which appears to be contiguous with the phosphoaccepting H576 residue. Rotation of the coiled-coil domain in response to a stimulus is thought to regulate access of H576 to the kinase domain (HK) and hence the level of kinase phosphorylation. The structure of the Thermotoga maritima TM0853 HK domain (PDB ID 2C2A) (Marina et al., 2005)is depicted here. Transfer of the H576 phosphoryl group to the receiver domain (Rec) is a prerequisite for subsequent phosphotransfer to Ypd1 and the response regulators Ssk1 and Skn7 (not shown). The structure of the Sln1-Rec domain is shown here (PDB ID 2R25) (Zhao et al., 2008)and the dashed lines represent linker regions of unknown structure. (c) Several types of regulation are embodied in the Sln1 pathway. (1) The Sln1 kinase itself is positively regulated by changes in the cell wall structure, and negatively regulated by hyper-osmolarity and other situations leading to reduced turgor. (2) The HOG1 pathway is positively regulated by an inactive SLN1 pathway. Only the dephosphorylated form of the Ssk1 response regulator stimulates the Ssk2 and Ssk22 MAPKKKs of the HOG1 pathway. Finally, (3) the HOG1 pathway is inactivated and Skn7 is activated by the phosphorylated SLN1 pathway intermediates.

SLN1 pathway organization and regulation. (a) Schematic showing flow of phosphoryl groups through the SLN1 pathway. Plasma membrane associated Sln1 kinase is autophosphorylated on the H576 residue under normal growth conditions. Hyper-activation occurs under conditions causing weakening of the cell wall (wall stress). Hyperosmolarity and other conditions causing reduced turgor lead to a reduction in Sln1 kinase activity and accumulation of Sln1 in the dephosphorylated form. The phosphoryl group on H576 (H) is transferred to D1144 (D) in the receiver domain of Sln1 and then to H64 of the phosphotransferase, Ypd1, and finally to D554 and D427 in the receiver domains of the Ssk1 and Skn7 response regulators, respectively. When Ssk1 is unphosphorylated it interacts with and stimulates the activity of the Ssk2 and Ssk22 MAPKKKs of the HOG1 pathway. Phosphorylation of Ssk1 renders it inactive with respect to the HOG1 pathway. Phosphorylation of Skn7 leads to activation of SKN7 dependent genes such as the mannosyltransferase, OCH1. (b) Schematic depicting the domain structure of Sln1. Two molecules of a Sln1 dimer are shown associated with the plasma membrane via two transmembrane domains (TMDs). An extensive extracellular domain (ECD) protrudes into the periplasmic space. This protein configuration is conducive to detection of changes in pressure against the membrane (turgor pressure) and changes in composition of the cell wall which may lead to altered rigidity. The stimulus is transduced through several juxtamembrane domains including a coiled-coil domain (CC) which appears to be contiguous with the phosphoaccepting H576 residue. Rotation of the coiled-coil domain in response to a stimulus is thought to regulate access of H576 to the kinase domain (HK) and hence the level of kinase phosphorylation. The structure of the Thermotoga maritima TM0853 HK domain (PDB ID 2C2A) (Marina et al., 2005)is depicted here. Transfer of the H576 phosphoryl group to the receiver domain (Rec) is a prerequisite for subsequent phosphotransfer to Ypd1 and the response regulators Ssk1 and Skn7 (not shown). The structure of the Sln1-Rec domain is shown here (PDB ID 2R25) (Zhao et al., 2008)and the dashed lines represent linker regions of unknown structure. (c) Several types of regulation are embodied in the Sln1 pathway. (1) The Sln1 kinase itself is positively regulated by changes in the cell wall structure, and negatively regulated by hyper-osmolarity and other situations leading to reduced turgor. (2) The HOG1 pathway is positively regulated by an inactive SLN1 pathway. Only the dephosphorylated form of the Ssk1 response regulator stimulates the Ssk2 and Ssk22 MAPKKKs of the HOG1 pathway. Finally, (3) the HOG1 pathway is inactivated and Skn7 is activated by the phosphorylated SLN1 pathway intermediates.