No abstract

Yeast cells respond to hyperosmotic stress by activating the high-osmolarity glycerol (HOG) pathway, which consists of two branches, Hkr1/Msb2-Sho1 and Sln1, which trigger phosphorylation and nuclear internalization of the Hog1 mitogen-activated protein kinase. In the nucleus, Hog1 regulates gene transcription and cell cycle progression, which allows the cell to respond and adapt to hyperosmotic conditions. This study demonstrates that the uncoupling of the known sensors of both branches of the pathway at the level of Ssk1 and Ste11 impairs cell growth in hyperosmotic medium. However, under these conditions, Hog1 was still phosphorylated and internalized into the nucleus, suggesting the existence of an alternative Hog1 activation mechanism. In the ssk1ste11 mutant, phosphorylated Hog1 failed to associate with chromatin and to activate transcription of canonical hyperosmolarity-responsive genes. Accordingly, Hog1 also failed to induce glycerol production at the levels of a wild-type strain. Inactivation of the Ptp2 phosphatase moderately rescued growth impairment of the ssk1ste11 mutant under hyperosmotic conditions, indicating that downregulation of the HOG pathway only partially explains the phenotypes displayed by the ssk1ste11 mutant. Cell cycle defects were also observed in response to stress when Hog1 was phosphorylated in the ssk1ste11 mutant. Taken together, these observations indicate that Hog1 phosphorylation by noncanonical upstream mechanisms is not sufficient to trigger a protective response to hyperosmotic stress.

The Kluyveromyces lactis SLN1 phosphorelay system includes the osmosensor histidine kinase Sln1, the phosphotransfer protein Ypd1 and the response regulator Ssk1. Here we show that K. lactis has a functional phosphorelay system. In vitro assays, using a heterologous histidine kinase, show that the phosphate group is accepted by KlYpd1 and transferred to KlSsk1. Upon hyperosmotic stress the phosphorelay is inactivated, KlYpd1 is dephosphorylated in a KlSln1 dependent manner, and only the version of KlSsk1 that lacks the phosphate group interacts with the MAPKKK KlSsk2. Interestingly, inactivation of the KlPtp2 phosphatase in a ΔKlsln1 mutant did not lead to KlHog1 constitutive phosphorylation. KlHog1 can replace ScHog1p and activate the hyperosmotic response in Saccharomyces cerevisiae, and when ScSln1 is inactivated, KlHog1 becomes phosphorylated and induces cell lethality. All these observations indicate that the phosphorelay negatively regulates KlHog1. Nevertheless, in the absence of KlSln1 or KlYpd1, no constitutive phosphorylation is detected and cells are viable, suggesting that a strong negative feedback that is independent of KlPtp2 operates in K. lactis. Compared with S. cerevisiae, K. lactis has only a moderate accumulation of glycerol and fails to produce trehalose under hyperosmotic stress, indicating that regulation of osmolyte production is different in K. lactis.