Hypertonic shock of Saccharomyces cerevisiae activates the Hog1p MAP kinase cascade. In contrast, protein kinase C (Pkc1p) and the "cell integrity" MAP kinase cascade are critical for the response to hypotonic shock. We observed that hypertonic shock transiently relocated many, but not all, nuclear and nucleolar proteins to the cytoplasm. We hypothesized that the relocation of nuclear proteins was due to activation of the Hog1p kinase cascade, yet, surprisingly, Hog1p was not required for these effects. In contrast, Pkc1p kinase activity was required, although the Pkc1p MAP kinase cascade and several factors known to lie upstream and downstream of Pkc1p were not. Moreover, sudden induction of a hyperactive form of Pkc1p was sufficient to relocate nuclear proteins. Taken together, these observations show that the scope of involvement of Pkc1p in the organization of the nucleus considerably exceeds what has been characterized previously. The relocation of nuclear proteins is likely to account for the profound inhibition of RNA synthesis that was observed during hypertonic shock.
INTRODUCTIONThe cellular effects of changes in tonicity have been investigated extensively (Robbins et al., 1970;Banuett, 1998;Gustin et al., 1998;Lang et al., 1998). In particular, analysis of the yeast Saccharomyces cerevisiae has elucidated major roles for two MAP kinase signaling pathways: the Pkc1p "cell integrity" pathway, which is stimulated by hypotonic shock (Watanabe et al., 1994;Davenport et al., 1995), and the Hog1p pathway, which is stimulated by hypertonic shock (Brewster et al., 1993;Posas and Saito, 1997). Each pathway is required for long-term survival in the corresponding medium. Hypertonic shock profoundly affects the actin cytoskeleton (Chowdhury et al., 1992;Mulholland et al., 1994;Delley and Hall, 1999).The Pkc1p pathway can receive input from plasma membrane glycoproteins of the Wsc family and from the Rho GTPases and their regulatory factors, for example in the context of control of cell polarization and cell wall biosynthesis (Gustin et al., 1998). Interestingly, some signaling events that require Pkc1p appear not to require the corresponding downstream MAP kinase cascade, which is linked to activation of the transcription factors Rlm1p and Swi4p/ Swi6p (Lee and Levin, 1992;Gustin et al., 1998;Delley and Hall, 1999; Li et al., 2000). In contrast, the Hog1p pathway is initiated by a two-component phosphorelay involving the cell surface transmembrane protein Sln1p and the cytosolic protein Ypd1p (Posas et al., 1996). Stimulation of this pathway causes phosphorylation and transient nuclear entry of the terminal kinase, Hog1p, as well as of the transcription factor Msn2p (Ferrigno et al., 1998;Gö rner et al., 1998;Reiser et al., 1999). The downstream substrate(s) of Hog1p kinase is not known.We have observed recently that arrest of the secretory pathway in yeast inhibits nuclear import and causes many nucleolar and nucleoplasmic proteins to relocate to the cytoplasm. These events (the "arrest of secretion response") a...