Accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggers the so-called unfolded protein response (UPR), a conserved signaling pathway that drives the transcription of genes such as chaperones and folding enzymes. Nevertheless, the activity of the UPR accounts only for a part of the gene expression program activated upon ER stress. Moreover, the mechanism(s) for how cells adapt and survive to this stress are largely unknown. Here, we show that the yeast high osmolarity glycerol (HOG) pathway plays a role in ER stress resistance. Strains lacking the MAPK Hog1p displayed sensitivity to tunicamycin or ␤-mercaptoethanol, whereas hyperactivation of the pathway enhanced their resistance. However, these effects were not due to Hog1p-mediated regulation of the UPR. Northern blot analysis demonstrated that Hog1p controls the tunicamycininduced transcriptional change of GPD1 and that wild-type cells exposed to the drug accumulated glycerol in a Hog1p-dependent manner. Consistent with this, deletion of genes involved in glycerol synthesis caused increased sensitivity to tunicamycin, whereas overexpression of GPD1 provided higher tolerance to both wildtype and hog1⌬ mutant cells. Quite remarkably, these effects were mediated by the basal activity of the MAPK because tunicamycin exposure does not trigger the phosphorylation of Hog1p or its nuclear import. Hence, our results describe new aspects of the yeast response to ER stress and identify additional functions of glycerol and the Hog1p MAPK to provide stress resistance.

All cells are delimited by biological membranes, which are consequently a primary target of stress-induced damage. Cold alters membrane functionality by decreasing lipid fluidity and the activity of membrane proteins. In Saccharomyces cerevisiae, evidence links sphingolipid homeostasis and membrane phospholipid asymmetry to the activity of the Ypk1/2 proteins, the yeast orthologous of the mammalian SGK1-3 kinases. Their regulation is mediated by different protein kinases, including the PDK1 orthologous Pkh1/2p, and requires the function of protein effectors, among them Nce102p, a component of the sphingolipid sensor machinery. Nevertheless, the mechanisms and the actors involved in Pkh/Ypk regulation remain poorly defined. Here, we demonstrate that Sng1, a transmembrane protein, is an effector of the Pkh/Ypk module and identify the phospholipid asymmetry as key for yeast cold adaptation. Overexpression of SNG1 impairs phospholipid flipping, reduces reactive oxygen species (ROS) and improves, in a Pkh-dependent manner, yeast growth in myriocin-treated cells, suggesting that excess Sng1p stimulates the Pkh/Ypk signalling. Furthermore, we link these effects to the association of Sng1p with Nce102p. Indeed, we found that Sng1p interacts with Nce102p both physically and genetically. Moreover, mutant nce102∆ sng1∆ cells show features of impaired Pkh/Ypk signalling, including increased ROS accumulation, reduced life span and defects in Pkh/Ypk-controlled regulatory pathways. Finally, myriocin-induced hyperphosphorylation of Ypk1p and Orm2p, which controls sphingolipid homeostasis, does not occur in nce102∆ sng1∆ cells. Hence, both Nce102p and Sng1p participate in a regulatory circuit that controls the activity of the Pkh/Ypk module and their function is required in response to sphingolipid status.

a b s t r a c tWe examined the physiological significance of the nuclear versus cytosolic localization of the MAPK Hog1p in the ability of yeast cells to cope with osmotic and ER (endoplasmic reticulum) stress. Our results indicate that nuclear import of Hog1p is not critical for osmoadaptation. Plasma membrane-anchored Hog1p is still able to induce increased expression of GPD1 and glycerol accumulation. This is a key osmoregulatory event, although a small production of the osmolyte coupled with the nuclear import of Hog1p is sufficient to provide osmoresistance. On the contrary, the nuclear activity of Hog1p is dispensable for ER stress adaptation.