THE ROLE OF SCHIZOSACCHAROMYCES POMBE SER/THR KINASE IN GROWTH, STRESS RESPONSE AND NUTRIENT DEPRIVATION
Freitag, Silja I.
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Continuous sensation and reaction to environmental fluctuations is especially critical to the survival of unicellular organisms. Stress response mechanisms are essential for cells during the vegetative and sexual life cycles and quiescence. The Schizosaccharomyces pombe mitotic activator and stress response serine/threonine kinase Ssp1 acts independent of the major fission yeast Spc1 MAP kinase stress response cascade. Ssp1 is required at high temperatures in the presence of other stressors, ensures long-term viability in quiescent cells and allows efficient cell division in low-glucose conditions. Ssp1 is cytoplasmic but briefly localizes to the cell membrane after exposure to extracellular stress. It plays a role in actin depolymerization and is required for the change of growth polarity after cell division. After identifying 14-3-3 proteins Rad24 and Rad25 as putative Ssp1 binding partners, we confirmed the interaction with co-immunoprecipitation. Association of Ssp1 with Rad24 diminishes after 15 minutes of hyperosmotic stress, however Rad25 binding is retained. Loss of the rad24 gene product rescues both ssp1- mitotic delay at elevated temperatures and sensitivity to 0. 6M KCl. Conversely, overexpression of rad24 exacerbates ssp1 stress sensitivity and mitotic delay. Diffuse actin polarity and spheroid morphology in rad24- cells improves in an ssp1- background. Ssp1 localization to the cell membrane is negatively regulated by Rad24. Ssp1 does not co-localize with Arp3C (actin-related protein 3 homologue C) after osmotic stress, but instead appears to form a ring around the cell, suggesting localization to fission scars. Ssp1 is basally phosphorylated and hyperphosphorylated after glucose deprivation. Ssp1 is shuttled in and out of the nucleus and accumulates in the nucleus in an exportin Cmr1 dependent manner. Ssp1-GFP levels are constant in all stages of the vegetative cell cycle and Ssp1-GFP is present in both the sexual life cycle and quiescence. C-terminal and N-terminal truncation of ssp1 alters its subcellular localization. The C-terminal region is the site of hyperphosphorylation following glucose deprivation and is also necessary for membrane localization following osmotic stress.