To cope with life‐threatening high osmolarity, yeast activates the high‐osmolarity glycerol (HOG) signaling pathway, whose core element is the Hog1 MAP kinase cascade. Activated Hog1 regulates the cell cycle, protein translation, and gene expression. Upstream of the HOG pathway are functionally redundant SLN1 and SHO1 signaling branches. However, neither the osmosensor nor the signal generator of the SHO1 branch has been clearly defined. Here, we show that the mucin‐like transmembrane proteins Hkr1 and Msb2 are the potential osmosensors for the SHO1 branch. Hyperactive forms of Hkr1 and Msb2 can activate the HOG pathway only in the presence of Sho1, whereas a hyperactive Sho1 mutant activates the HOG pathway in the absence of both Hkr1 and Msb2, indicating that Hkr1 and Msb2 are the most upstream elements known so far in the SHO1 branch. Hkr1 and Msb2 individually form a complex with Sho1, and, upon high external osmolarity stress, appear to induce Sho1 to generate an intracellular signal. Furthermore, Msb2, but not Hkr1, can also generate an intracellular signal in a Sho1‐independent manner.
The yeast filamentous growth (FG) MAP kinase (MAPK) pathway is activated under poor nutritional conditions. We found that the FG‐specific Kss1 MAPK is activated by a combination of an O‐glycosylation defect caused by disruption of the gene encoding the protein O‐mannosyltransferase Pmt4, and an N‐glycosylation defect induced by tunicamycin. The O‐glycosylated membrane proteins Msb2 and Opy2 are both essential for activating the FG MAPK pathway, but only defective glycosylation of Msb2 activates the FG MAPK pathway. Although the osmoregulatory HOG (high osmolarity glycerol) MAPK pathway and the FG MAPK pathway share almost the entire upstream signalling machinery, osmostress activates only the HOG‐specific Hog1 MAPK. Conversely, we now show that glycosylation defects activate only Kss1, while activated Kss1 and the Ptp2 tyrosine phosphatase inhibit Hog1. In the absence of Kss1 or Ptp2, however, glycosylation defects activate Hog1. When Hog1 is activated by glycosylation defects in ptp2 mutant, Kss1 activation is suppressed by Hog1. Thus, the reciprocal inhibitory loop between Kss1 and Hog1 allows only one or the other of these MAPKs to be stably activated under various stress conditions.
To cope with environmental high osmolarity, the budding yeast Saccharomyces cerevisiae activates the mitogen-activated protein kinase (MAPK) Hog1, which controls an array of osmoadaptive responses. Two independent, but functionally redundant, osmosensing systems involving the transmembrane sensor histidine kinase Sln1 or the tetraspanning membrane protein Sho1 stimulate the Hog1 MAPK cascade. Furthermore, the Sho1 signaling branch itself also involves the two functionally redundant osmosensors Hkr1 and Msb2. However, any single osmosensor (Sln1, Hkr1, or Msb2) is sufficient for osmoadaptation. We found that the signaling mechanism by which Hkr1 or Msb2 stimulated the Hog1 cascade was specific to each osmosensor. Specifically, activation of Hog1 by Msb2 required the scaffold protein Bem1 and the actin cytoskeleton. Bem1 bound to the cytoplasmic domain of Msb2 and thus recruited the kinases Ste20 and Cla4 to the membrane, where either of them can activate the kinase Ste11. The cytoplasmic domain of Hkr1 also contributed to the activation of Ste11 by Ste20, but through a mechanism that involved neither Bem1 nor the actin cytoskeleton. Furthermore, we found a PXXP motif in Ste20 that specifically bound to the Sho1 SH3 (Src homology 3) domain. This interaction between Ste20 and Sho1 contributed to the activation of Hog1 by Hkr1, but not by Msb2. These differences between Hkr1 and Msb2 may enable differential regulation of these two proteins and provide a mechanism through Msb2 to connect regulation of the cytoskeleton with the response to osmotic stress.
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