Abstract
The Saccharomyces cerevisiae Snf1 protein kinase, a member of the Snf1/AMPK (AMP-activated protein kinase) family, has important roles in metabolic control, particularly in response to nutrient stress. Here we have addressed the role of Snf1 in responses to other environmental stresses. Exposure of cells to sodium ion stress, alkaline pH, or oxidative stress caused an increase in Snf1 catalytic activity and phosphorylation of Thr-210 in the activation loop, whereas treatment with sorbitol or heat shock did not. Inhibition of respiratory metabolism by addition of antimycin A to cells also increased Snf1 activity. Analysis of mutants indicated that the kinases Sak1, Tos3, and Elm1, which activate Snf1 in response to glucose limitation, are also required under other stress conditions. Each kinase sufficed for activation in response to stress, but Sak1 had the major role. In sak1Delta tos3Delta elm1Delta cells expressing mammalian Ca(2+)/calmodulin-dependent protein kinase kinase alpha, Snf1 was activated by both sodium ion and alkaline stress, suggesting that stress signals regulate Snf1 activity by a mechanism that is independent of the upstream kinase. Finally, we showed that Snf1 protein kinase is regulated differently during adaptation of cells to NaCl and alkaline pH with respect to both temporal regulation of activation and subcellular localization. Snf1 protein kinase becomes enriched in the nucleus in response to alkaline pH but not salt stress. Such differences could contribute to specificity of the stress responses.
Highlights
Snf1 protein kinase is required for the adaptation of yeast cells to glucose limitation and for growth on carbon sources that are less preferred than glucose, such as sucrose and nonfermentable carbon sources (6)
Exposure of cells to sodium ion stress, alkaline pH, and oxidative stress resulted in phosphorylation of Thr-210 of Snf1 and elevation of Snf1 catalytic activity, whereas treatment with sorbitol and heat shock did not
Inhibition of respiratory metabolism by addition of antimycin A to cells growing on nonfermentable carbon sources caused an increase in Snf1 activity
Summary
To determine whether these kinases activate Snf1 in response to other stresses, we exposed sak1⌬ tos3⌬ elm1⌬ triple mutant cells to 1 M NaCl, pH 8, or 0.3 mM H2O2 for 5 min as described above. These findings indicate that activation of Snf1 protein kinase in response to all of these different stress conditions requires Sak1, Tos3, or Elm1.
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