Yeast cells respond to increases in external osmolarity by activating the stress-activated Hog1 SAPK. A major outcome of the activation of Hog1 is the regulation of gene expression, and several transcription factors have been proposed as being controlled by the SAPK (de Nadal and Posas, 2010
; de Nadal et al., 2011
; Martinez-Montanes et al., 2010
; Weake and Workman, 2010
). However, data suggest that additional transcription factors may be required for the osmostress-induced gene expression controlled by Hog1 (Miller et al., 2011
). In this paper, we show that the Rtg1/3 transcriptional complex is a new target for Hog1, linking the SAPK to mitochondrial activity during adaptation to stress. Deletion of RTG1
results in a deficient osmostress-induced expression of key genes in the trichloroacetic acid (TCA) cycle, such as CIT2
, and PYC1
, known to be activated by the RTG pathway upon respiratory dysfunction (Liao et al., 1991
; Chelstowska et al., 1999
; Epstein et al., 2001
). In addition deletion of RTG1
displays osmosensitivity. Of note, the osmosensitivity observed in rtg1
mutants is stronger than that observed in deletions of other known transcription factors targeted by Hog1, but less sensitive than observed in hog1
Osmostress alters mitochondrial function by reducing the mitochondrial electron transport in plants (Hamilton and Heckathorn, 2001). Recently it was reported that constitutive activation of Hog1 provokes a decrease in respiration levels, which leads to cell death caused by an increase in reactive oxygen species (Vendrell et al., 2011). On the other hand, the Rtg proteins are essential for respiration under limiting respiratory conditions, when several TCA cycle and respiratory chain enzymes are significantly up-regulated (Fendt and Sauer, 2010). Therefore it is likely that RTG-mediated transcription increases to compensate the down-regulation of respiration that rapidly occurs upon stress. Correspondingly, mutants in different mitochondrial components showed hypersensitivity to increases in NaCl, suggesting that mitochondrial biogenesis and function are needed for efficient adaptation to osmostress (Martinez-Pastor et al., 2010). Taken together, the data suggest that cells exposed to osmostress have compromised mitochondrial respiration, and Rtg1 and Rtg3 transcription factors might be important determinants for maintaining or restoring mitochondrial function.
Production of glycerol is critical for adaptation in response to osmostress. Glycerol synthesis requires the action of NADH-dependent enzymes, and mitochondria are designed to oxidize NADH back to NAD+
. Thus it could seem contradictory that osmostress can up-regulate mitochondrial function. However, the pool of NADH/NAD+
in the cytosol and mitochondria are different, although NADH can diffuse from the cytosol into the mitochondria in certain conditions (Rigoulet et al., 2004
). Therefore the function of inducing the retrograde function by osmostress might be related to the ability of the cells to reactivate respiration once cells have started to adapt rather than to maintenance of the balance between of NADH/NAD+
. Alternatively, due to the delay required for the Rtg response when compared with glycerol accumulation, it is possible that an elevation in mitochondrial respiration provides more ATP to improve long-term survival under osmostress.
Gene expression analysis showed that, specifically upon osmostress, the HOG pathway is required for proper induction of Rtg1/3 target genes. The retrograde response is linked to other pathways, such as TOR signaling (Komeili et al., 2000
; Butow and Avadhani, 2004
). However, activation of the retrograde pathway by addition of rapamycin does not require the SAPK, indicating that RTG transcription can be activated by different signaling pathways, depending on the extracellular stimuli. A key regulatory step in the regulation of the RTG pathway is the translocation of Rtg1/3 proteins from the cytoplasm to the nucleus. When the RTG pathway is inactive, Rtg1 and Rtg3 are sequestered together in the cytoplasm. On activation, Rtg3 undergoes changes in its phosphorylation state and translocates into the nucleus together with Rtg1 (Sekito et al., 2000
). Hog1 interacts with the Rtg1/3 complex in vivo, and this interaction seems to be critical for the nuclear accumulation of the complex in response to osmostress. Indeed, localization of Rtg1 and Rtg3 in cells expressing Hog1 with abolished catalytic activity and altered subcellular localization correlates with the localization of Hog1 independently upon its catalytic activity. Thus the sole interaction of the transcription factor with the SAPK is sufficient for Rtg1 and Rtg3 nuclear accumulation. There are other stress-responsive transcription factors for which translocation is the key regulatory event in stress induction of transcription. For instance, Msn2 and Msn4 activators accumulate in the nucleus under stress conditions. However, in this case, the nuclear localization seems to occur independent of Hog1 (Gorner et al.
In addition to its role in the regulation of the Rtg1/3 subcellular localization, Hog1 is further required for Rtg1/3 chromatin binding and transcriptional activity. It is known that Hog1 becomes intimately linked with stress-responsive loci upon osmostress, and this binding is dependent on the presence of stress-mediating transcriptional activators (Alepuz et al., 2001
; Pascual-Ahuir et al., 2006
; Pokholok et al., 2006
; Proft et al., 2006
). Similarly, Hog1 is recruited by Rtg1 to the RTG-dependent promoters in response to osmostress. Thus the Rtg1/3 transcription complex targets the SAPK to specific promoters. On the other hand, our results also showed that recruitment of the Rtg1/3 complex to RTG-dependent promoters during osmostress is strongly reduced in a hog1
Δ strain. These results are in agreement with the subcellular localization experiments indicating that Hog1 regulates the localization of the complex upon stress. However, the association of RTG at promoters not only requires the presence of Hog1 but also its catalytic activity. For instance, in cells containing the catalytic-inactive Hog1KNN
, Rtg1 and Rtg3 are nuclear upon stress but cannot associate to chromatin. Similarly, it has been described that nuclear accumulation of the MAPK or the transcription activator Hot1 are not sufficient for the association of the transcription factor to chromatin, which is also dependent on Hog1 activity (Alepuz et al., 2003
). Overall the interdependence of binding of Hog1 and Rtg1 to promoters of target genes reveals, as in the case of other stress-mediating transcription factors, a functional connection between both proteins for making a stable complex at stress-responsive promoters.
Hog1 also directly phosphorylates Rtg1 and Rtg3 proteins. We have not been able to unravel the role of Rtg1 phosphorylation by Hog1, but Rtg3 phosphorylation seems to be important for proper gene activation. Gene induction is reduced in an Rtg3 nonphosphorylatable mutant, whereas Rtg1/3 recruitment remains unaltered. Moreover, combining Rtg1 and Rtg3 nonphosphorylatable mutants is not synergistic for proper gene activation. Thus Hog1 phosphorylation plays an important role in the regulation of Rtg3 transcriptional activity. A similar scenario has been described for other transcription factors under the control of the SAPK. This is the case of the MEF2-like activator Smp1, the transcriptional activity of which is regulated by Hog1-dependent phosphorylation upon stress (de Nadal et al., 2003
Taken together, our results show that, in contrast with what is known for the regulation of other transcription factors targeted by Hog1, the SAPK regulates the Rtg1/3 heterodimeric transcription complex by multiple mechanisms (see schematic diagram in ), which are: 1) the regulation of the nuclear accumulation of the complex, 2) the stimulation of the association of the complex at RTG-responsive promoters, and 3) the control of Rtg3 transcriptional activity. Notably, whereas the presence of Hog1 is essential for Rtg1/3 nuclear localization upon stress, the activity of the SAPK is required for Rtg1/3 chromatin binding and Rtg3 transcriptional activity regulation. Our data also provide a direct link between Hog1 and the regulation of genes important for mitochondrial activity during stress adaptation.
FIGURE 7: The Hog1 SAPK directly modulates the Rtg1/3 complex upon osmostress by a triple mechanism: (1) regulating the nuclear accumulation of the heterodimeric complex (no catalytic activity of Hog1 is required); (2) stimulating the association of the complex (more ...)