To adapt to environmental high osmolarity, the budding yeast Saccharomyces cerevisiae activates the Hog1 mitogen-activated protein kinase, which regulates diverse osmoadaptive responses. Hog1 is activated through the high-osmolarity glycerol (HOG) pathway, which consists of independent upstream signaling routes termed the SLN1 branch and the SHO1 branch. Here, we report that the extracellular cysteine-rich (CR) domain of the transmembrane-anchor protein Opy2 binds to the Hkr1-Msb2 homology (HMH) domain of the putative osmosensor Msb2 and that formation of the Opy2-Msb2 complex is essential for osmotic activation of Hog1 through the MSB2 subbranch of the SHO1 branch. By analyzing the phenotypes of mutants with Opy2 cysteine-to-alanine mutations, we deduced that the CR domain forms four intramolecular disulfide bonds. To probe for the potential induction of conformational changes in the Opy2-Msb2 complex by osmostress, we constructed mutants with a site-specific Cys-to-Ala mutation of the Opy2 CR domain and mutants with a Cys substitution of the Msb2 HMH domain. Each of these mutants had a reduced cysteine. These mutants were then combinatorially cross-linked using chemical cross-linkers of different lengths. Cross-linking between Opy2 Cys48 and Msb2 Cys1023 was sensitive to osmotic changes, suggesting that osmostress induced a conformational change. We therefore propose that the Opy2-Msb2 complex might serve as an osmosensor.
Survival of living organisms depends on their ability to adapt to adverse environmental conditions. A well-known example is the response of the budding yeast Saccharomyces cerevisiae to high external osmolarity. When exposed to high osmolarity, yeast cells initiate a coordinated adaptive response that includes the synthesis and accumulation of the compatible osmolyte glycerol, changes in the global pattern of gene expression and protein synthesis, and a temporary arrest of cell cycle progression (1-3). These responses are controlled by the Hog1 mitogen-activated protein kinase (MAPK), which is activated via the high-osmolarity glycerol (HOG) signal pathway. The HOG pathway employs multiple and redundant routes between the input (external high osmolarity) and the output (Hog1-dependent responses). Specifically, the HOG pathway consists of two independent upstream signaling routes termed the SLN1 branch and the SHO1 branch (Fig. 1A). The osmosensor for the SLN1 branch is the sensor histidine kinase Sln1, which transmits the signal through a two-component phosphorelay mechanism to the redundant MAPK kinase kinases (MAPKKKs) Ssk2 and Ssk22 (4, 5). Ssk2/Ssk22 activates the Pbs2 MAPK kinase (MAPKK), which eventually activates the Hog1 MAPK (5-8).When the SLN1 branch is inactivated by the ssk2⌬ ssk22⌬ double mutation (here abbreviated ssk2/22⌬), yeast can still adapt to high osmolarity using the SHO1 branch. The major osmosensor for the SHO1 branch is the four-transmembrane (TM) protein Sho1 (5, 9). In the SHO1 branch, osmostress activates the Ste11 MAPKKK, which then sequentially activates Pbs2 an...