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. Author manuscript; available in PMC: 2013 Apr 4.
Published in final edited form as: Cell Rep. 2013 Feb 14;3(2):328–334. doi: 10.1016/j.celrep.2013.01.022

Formation of Sub-Nuclear Foci is a Novel Spatial Behavior of Mating MAPKs during Hyper-Osmotic Stress

Simon E Vidal 1,2,*, David Pincus 1,3,*, Jacob Stewart-Ornstein 1, Hana El-Samad 1,#
PMCID: PMC3616637  NIHMSID: NIHMS438454  PMID: 23416049

SUMMARY

The assembly of signaling components and transcription factors in ordered subcellular structures is increasingly implicated as an important regulatory strategy to modulate the activity of cellular pathways. Here, we document the inducible formation of sub-nuclear foci formed by two mitogen-activated protein kinases (MAPKs) in Saccharomyces cerevisiae upon hyper-osmotic stress. Specifically, we demonstrate that activation of the hyper-osmotic stress response pathway induces the mating pathway MAPK Fus3 and the filamentation pathway MAPK Kss1 to form foci in the nucleus that are organized by their shared downstream transcription factor Ste12. Foci formation of co-localized Ste12, Fus3, and Kss1 requires the kinase activity of the hyper-osmotic response MAPK Hog1 and correlates with attenuated signaling in the mating pathway. Conversely, activation of the mating pathway prevents foci formation upon subsequent hyper-osmotic stress. These results suggest that Hog1-mediated spatial localization of Fus3 and Ste12 into sub-nuclear foci could contribute to uncoupling the pheromone and osmolarity pathways, which share signaling components, under high osmolarity conditions.

INTRODUCTION

Mitogen-activated protein kinases (MAPKs), a family of Ser/Thr protein kinases conserved among eukaryotes, are involved in many cellular programs such as proliferation, differentiation and death (Blenis, 1993; Kim and Choi, 2010; Wagner and Nebreda, 2009). MAPK signaling cascades are organized hierarchically into three-tiered modules. MAPKs are phosphorylated and activated by MAPK-kinases (MAP2Ks), which are phosphorylated and activated by MAP2K-kinases (MAP3Ks) (Chen and Thorner, 2007). MAP3Ks themselves are activated by interaction with members of small GTPase families and other protein kinases, connecting the MAPK module to cell surface receptors or other signaling molecules at the plasma membrane. Three main families of MAPKs exist in mammalian species, grouped by their structures and functions: the extracellular signal-regulated protein kinases (ERKs), the p38 MAPKs, and the c-Jun NH2-terminal kinases (JNKs) (Graves et al., 1995; Seger and Krebs, 1995; Wagner and Nebreda, 2009). Transmission of signals to the nucleus and subsequent modulation of the activity of a number of transcription factors and chromatin remodeling proteins is a particularly important role of MAPKs. This transmission is often mediated by the physical translocation of MAPK proteins to the nucleus (Chen et al., 1992; Cohen-Saidon et al., 2009; Khokhlatchev et al., 1998; Plotnikov et al., 2011).

The budding yeast S. cerevisiae possesses five genes with homology to mammalian MAPKs (HOG1, SLT2, FUS3, KSS1, and SMK1). These kinases, whose active forms have both cytoplasmic and nuclear targets, are important for inducing the activity of condition-specific transcriptional programs via phosphorylation of transcription factors, and in some cases by serving as transcriptional co-activators (Chen and Thorner, 2007). As in mammalian cells, activation-dependent MAPK nuclear translocation is conserved in S. cerevisiae and has been directly observed for Hog1, the MAPK activated in response to hyper-osmotic stress (Ferrigno et al., 1998; Mettetal et al., 2008; Muzzey et al., 2009). Nuclear translocation of Fus3, the pheromone pathway MAPK, and Kss1, the filamentation pathway MAPK, has also been observed (Blackwell et al., 2003; Blackwell et al., 2007; Chen et al., 2010; Maeder et al., 2007; Slaughter et al., 2007).

Hog1 is instrumental for the specific and faithful activation of the hyper-osmotic stress responsive genes (O’Rourke and Herskowitz, 1998; Saito and Tatebayashi, 2004). Despite the fact that the hyper-osmotic stress pathway shares the same MAP3K (Ste11) with the pheromone response and filamentation pathways, hyper-osmotic stress fails to activate pheromone- or filamentation-responsive genes (O’Rourke and Herskowitz, 1998; Schwartz and Madhani, 2004; Shock et al., 2009). The remarkable ability of hyper-osmotic stress to exclusively activate the hyper-osmotic response requires Hog1 kinase activity and has recently been attributed to Hog1-mediated feedback control, potentially through the adaptor protein Ste50 and the kinase Rck2 (Nagiec and Dohlman, 2012). This model, however, does not fully explain the observation that hyper-osmotic stress activates Fus3 and Kss1, yet the target genes of the pheromone and filamentation pathways remain off (Shock et al., 2009). As a result, some further insulation at the level of the transcription factors downstream of Fus3 and Kss1 might still occur.

In addition to simply binding to regulatory sites to control gene expression, transcription factors have been shown to organize sub-nuclear structures with varied functions (Brickner et al., 2012; McCullagh et al., 2010; Sutherland and Bickmore, 2009). In metazoan cells, many sub-nuclear foci containing transcription factors have been described, such as nuclear stress bodies, histone locus bodies, and polycomb bodies (Mao et al., 2011; Nizami et al., 2010). Rather than being nonfunctional aggregates, these structures within the nucleus implement important layers of regulation.

Here, we show that activation of Hog1 by hyper-osmotic stress induces the formation of sub-nuclear foci containing the transcription factor Ste12 and the MAPKs of the mating and filamentation pathway. Our results are consistent with a model in which these sub-nuclear structures prevent pheromone and filamentation pathway target gene activation in response to hyper-osmotic stress. This work documents a novel spatial behavior of the MAPK proteins, and suggests that transcription factor mediated sequestration of signaling components could serve as a general mechanism to modulate the activity of signaling pathways.

RESULTS

Fus3 and Kss1 are unique among MAPK proteins in their ability to form foci under high osmolarity stress

To gain insight into the regulation of localization of S. cerevisiae MAP kinases, we fused yellow fluorescent protein (YFP) to the C-termini of Fus3, Kss1, Hog1 and Slt2 and imaged these strains under a panel of metabolic and stress conditions. In vegetatively growing cells, Fus3 and Kss1 were enriched in the nucleus. Slt2 had a reduced nuclear signal compared to Fus3 and Kss1, while Hog1 was mostly cytoplasmic (Figure 1A). In cells treated with 1 μM α-factor, none of the four kinases changed their localization until Fus3 partially redistributed from the nucleus to the emerging shmoo tip after 1 hour of treatment (Figure S1A), consistent with previous data (Slaughter et al., 2007).

Figure 1. Ste12, Fus3 and Kss1 co-localize in foci under high osmolarity stress.

Figure 1

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(A) The MAPKs Fus3 and Kss1, but not Slt2 and Hog1, form punctuate and transient foci under high osmolarity stress. Endogenous Fus3, Kss1, Hog1 and Slt2 were fused on the C-termini to YFP and spatial localization was followed by confocal microscopy after addition of 0.5 M sorbitol. (B) Fus3 and Kss1 co-localize under high osmolarity stress. Endogenous Fus3 and Kss1 were fused on the C-termini to YFP and mKate, respectively. Foci formation was determined by confocal microscopy 15 minutes after the addition of 0.5 M sorbitol. (C) Fus3 and Ste12 co-localize under high osmolarity stress. Endogenous Fus3 and Ste12 were fused on the C-termini to mKate and YFP, respectively. Foci formation was determined by confocal microscopy 15 minutes after adding 0.5M sorbitol. (D) Fus3 and Kss1 fail to form foci following osmotic stress in cells lacking Ste12. However, Ste12 continued to form foci following osmotic stress even in a fus3Δkss1Δ strain. Endogenous Ste12 was fused on the C-termini to YFP. Fus3, Kss1 and Ste12 were deleted by homologous insertion. Foci formation was determined by confocal microscopy 15 minutes after addition of 0.5 M sorbitol. (E) Fus3 co-localizes with the nuclear pore under high osmolarity stress. Endogenous Fus3 and nup49 (nuclear pore marker) were tagged with YFP and mKate, respectively. Foci formation was followed by confocal microscopy in cells treated with 0.5 M sorbitol. Images were taken 10–15 minutes after adding sorbitol.

By contrast, addition of the osmotic stressor sorbitol (0.5 M) triggered the redistribution of Hog1, Fus3 and Kss1. Hog1 transiently localized to the nucleus, within 15 minutes of treatment, and returned to the cytoplasm within 30 minutes (Figure 1A). Strikingly, following sorbitol treatment, Fus3 and Kss1 appeared in small puncta in approximately half the cells in a given confocal microscope section (Figure 1A). Each cell in which the puncta were visible contained 1–3 discrete foci 15 minutes after sorbitol addition, and these foci dispersed within 30 minutes; Fus3 and Kss1 returned to their pre-stress diffuse localization in all cells 30 minutes after sorbitol addition (Figure 1A).

Fus3 also formed sub-nuclear foci under other hyper-osmotic stress conditions such as 0.5 M NaCl and 0.8 M glucose (Figure S1B). Importantly, these sub-nuclear foci did not form under other stress conditions such as hypo-osmotic shock (50% H2O), heat shock (40°C), oxidative stress (0.5% H202), cell wall stress (100 μg/ml zymolyase), ER stress (5 mM DTT), or Tor inhibition (20 mM caffeine) (Figure S1C). These data strongly suggest that the Fus3 and Kss1 foci are a specific phenotype associated with high osmolarity stress.

Fus3/Kss1 foci formation under hyper-osmotic stress is dependent on the pheromone response transcription factor Ste12

We next investigated whether Fus3 and Kss1-containing foci were the same spatial structure. To do so, we measured the localization of Fus3-YFP and Kss1-mKate2 coexpressed in the same cell upon stimulation with sorbitol. Under these conditions, Fus3-YFP and Kss1-mKate2 co-localized in nuclear foci (Figure 1B), indicating that Fus3 and Kss1 are both contained within the same sub-nuclear structures.

The presence of nuclear foci involving the pheromone pathway transcription factor Ste12 has previously been documented. In strains deleted for DIG1, a gene encoding a negative regulator of Ste12, Ste12 molecules localize into discrete sub-nuclear foci (McCullagh et al., 2010). Because of the propensity of Ste12 to form these structures in dig1Δ cells, we hypothesized that Ste12 molecules might also be components of the Fus3/Kss1 foci. To test this possibility, we monitored the localization of Fus3- mKate2 and Ste12-YFP co-expressed in the same cells. Upon treatment with 0.5 M sorbitol, Ste12 indeed formed nuclear foci. Importantly, Fus3 co-localized with Ste12 in these foci (Figure 1C). Moreover, just as Ste12 is constitutively localized in foci in dig1Δ cells, Fus3 also appeared in constitutive foci in the absence of sorbitol in dig1Δ cells (Figure S1D). Furthermore both Dig1 and its paralog Dig2 were present in sorbitol-induced foci in wild type cells (Figure S1E). In summary, these data indicate that the MAPKs Fus3 and Kss1, along with their shared transcription factor Ste12 and its negative regulators Dig1 and Dig2, are components of hyper-osmotic stress induced sub-nuclear structures.

To determine which components are required for the emergence of these structures, we monitored foci formation upon sorbitol addition in strains lacking FUS3, KSS1 or STE12. Interestingly, neither individual deletions of FUS3 or KSS1, nor the FUS3/KSS1 double deletion impacted the formation of Ste12 foci (Figure 1D). By contrast, Fus3 and Kss1 failed to form foci in ste12Δ cells under osmotic stress conditions (Figure 1D).

Since Ste12, a transcription factor, is a crucial component for the formation of these foci, we sought to ask whether DNA also associated with these foci. To test this possibility, we performed ChIP-PCR experiments using Ste12-YFP as bait in either untreated, pheromone treated or sorbitol treated cells (Figure S1F). In untreated cells, we found that Ste12 associated with pheromone target gene promoters (AGA1 and FUS1), but not with osmo-stress target gene promoters (HOR2 and GPD1) or metabolic gene promoters (PGK1 and TDH3). We did not observe Ste12 binding to osmo-stress gene promoters or metabolic gene promoters in any condition. Notably, we found an increase in Ste12 binding to pheromone target promoters upon pheromone treatment, but we did not observe any depletion of Ste12 from the pheromone target gene promoters upon sorbitol treatment (Figure S1G). These data indicate that Ste12 remains specifically bound to its target gene promoters while simultaneously localizing to sub-nulcear foci.

Genomic loci have been reported to localize to the nuclear membrane both for activation and repression (Brickner and Walter, 2004; Green et al., 2012). Since the hyper-osmotic stress induced foci are associated with DNA, we next asked if the foci localize to the nuclear periphery. To this end, we monitored Fus3-YFP in cells in which the nuclear pore component Nup49 was fused to mCherry. After addition of sorbitol, we observed that Fus3 foci indeed localized with or near Nup49 (Figure 1E). Taken together, these data demonstrate that the common transcription factor Ste12 is necessary to organize the MAPKs Fus3 and Kss1 into the sub-nuclear foci induced by high-osmolarity stress, and that these foci are associated with DNA and are localized to the nuclear periphery.

Hog1 kinase activity is required for formation of Fus3/Kss1/Ste12 foci in conditions of hyper-osmotic stress

Under high osmolarity conditions, the effector MAPK Hog1 is phosphorylated by the MAP2K Pbs2 and translocates from the cytoplasm to the nucleus where it plays transcriptional and post-translational roles in the hyper-osmotic stress response (Ferrigno et al., 1998; Mettetal et al., 2008). Therefore, Hog1 is a prime candidate for a regulatory role in Fus3/Kss1/Ste12 foci formation. To address this notion, we first investigated whether Hog1 residence in the nucleus correlated with Fus3/Kss1/Ste12 foci formation. Fus3 and Ste12 foci form and dissipate with identical kinetics (Figure S2A, B), so we chose to simultaneously monitor Fus3-YFP and Hog1-mKate2 in the same cell. The kinetics of Fus3 foci formation and dissolution correlated with Hog1 nuclear residence (Figure 2A, B). Hog1 began to translocate to the nucleus within 2 minutes of 0.5 M sorbitol, and Fus3 began to form Foci within 3 minutes. Both Hog1 nuclear enrichment and Fus3 foci formation achieved their peaks between 5–10 minutes after sorbitol addition. Within 15 minutes, Fus3 foci began to dissipate while Hog1 persisted longer in the nucleus, beginning to relocalize to the cytoplasm only after 20 minutes (Figure 2A, B). Due to our imaging technique, we were not able to observe Fus3 foci in all cells, likely due to the foci falling above or below the confocal plane. Nonetheless, the strong correlation between Fus3 foci and Hog1 nuclear residency held across a range of osmotic stress magnitudes that produced different but correlated Hog1 and Fus3 dynamics (Fig S2C). Moreover, Fus3, Kss1 and Hog1 are phosphorylated and dephosphorylated with similar kinetics, matching the kinetics of foci formation and Hog1 nuclear translocation (figure S2D).

Figure 2. Fus3/Kss1/Ste12 Foci formation requires Hog1 kinase activity.

Figure 2

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(A) Hog1 residence in the nucleus correlates with Fus3/Kss1 foci formation. Endogenous Fus3 and Hog1 were fused on the C-termini to YFP and mKate, respectively. Distribution of Fus3 and Hog1 was followed by confocal microscopy at different time points after the addition of 0.5 M sorbitol. (B) Quantification of Hog1 nuclear residence and Fus3 foci formation. The percentage of cells with foci and nuclear Hog1 was calculated using image J. (C) The kinase activity of Hog1 is a prerequisite for Fus3 and Kss1 foci formation under high osmolarity stress. Following hyper-osmotic stress, Fus3-YFP failed to form foci in cells harboring a hog1-as allele pre-treated with the cell-permeable adenine analog 1-NM-PP1 (1 μM). (D) The percentage of cells with foci following hyper-osmotic stress was calculated using image J for cells with wilde-type HOG1, hog1-as allele untreated with 1-NM-PP1 and cells with a hog1-as allele after 1-NM-PP1 treatment. Compromised Hog1 kinase activity generates dramatic reduction in foci formation. Results represent the mean ?SD of three independent experiments.

To directly test if Hog1 activity is required for foci formation, we constructed an analog sensitive allele (hog1-as) whose kinase activity is susceptible to inhibition by the cell-permeable ATP analog 1-NM-PP1 (Westfall and Thorner, 2006). In this mutant, and by adding 1-NM-PP1, pheromone genes are expressed under high osmolarity stress (Westfall and Thorner, 2006). The hog1-as allele was inserted into the genome of a hog1Δ strain under the control of the native HOG1 promoter. In hog1-as cells, 1-NM-PP1 alone had no effect on Fus3 localization. Moreover, in hog1- as cells, Fus3 formed nuclear foci at a frequency comparable to that of wild type cells when treated with 0.5M sorbitol alone (Figure 2C, D). By contrast, less than 10% of cells pre-treated with 1 μM 1-NM-PP1 formed foci upon addition of 0.5M sorbitol (Figure 2C, D). These data demonstrate that the kinase activity of Hog1 is required for Fus3 foci formation under high osmolarity stress.

Pre-activation of the pheromone pathway inhibits the formation of Fus3 foci

The experiments described above show that Ste12 and Fus3, components of the pheromone response pathway, are recruited into foci in high osmolarity conditions and that Hog1 kinase activity is required for this process to proceed. Despite the fact that the hyper-osmotic stress pathway shares the same MAP3K (Ste11) with the pheromone pathway, hyper-osmotic stress fails to activate pheromone-responsive genes (O’Rourke and Herskowitz, 1998; Schwartz and Madhani, 2004; Shock et al., 2009). Therefore, foci formation could constitute a spatial mechanism for attenuating signaling in the pheromone pathway when cells are exposed to high osmolarity. In this case, the presence of foci should diminish the induction of pheromone target genes. Conversely, conditions where these foci are absent should coincide with un-attenuated pheromone signaling output.

To test the notion that the Fus3/Kss1/Ste12 foci represent a spatial mechanism for attenuating pheromone pathway output during hyper-osmotic stress, we performed a series of experiments in which we treated cells with both α-factor and sorbitol. We hypothesized that if cells treated simultaneously with sorbitol and pheromone prioritize the hyper-osmotic stress response, then these cells would form foci that attenuate signaling in the pheromone pathway. On the other hand, we reasoned that pre-treatment of cells with α-factor would establish active signaling through the pheromone MAPK cascade that would preclude the formation of foci. The absence of foci in this situation should then correlate with un-attenuated signaling and induction of pheromone responsive gene expression.

To test these hypotheses, we first used confocal microscopy to monitor Fus3-YFP cells treated with both 1 μM α-factor and 0.5 M sorbitol either simultaneously or by pre-treating the cells with α-factor for 10 minutes before sorbitol addition. When cells were exposed to α-factor and sorbitol simultaneously, Fus3 formed foci to the same level as in cells treated with sorbitol alone (Figure 3A). However, when cells were pre-incubated in 1 μM α-factor for 10 minutes, addition of 0.5 M sorbitol failed to induce Fus3 foci (Figure 3A). Deletion of Ste7, the MAP2K upstream of Fus3, rescued Fus3 foci formation in cells pre-treated with pheromone and subsequently treated with sorbitol, showing that signaling through the pheromone MAPK cascade is required to prevent foci formation (Figure 3A).

Figure 3. Pre-activation of the pheromone pathway inhibits the formation of Fus3 foci under high osmolarity stress.

Figure 3

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(A) Cells harboring Fus3-YFP were treated with either 0.5 M sorbitol, 0.5 M sorbitol and 1 μM pheromone at the same time or 1 μM pheromone for 10 minutes followed by 0.5 M sorbitol. While the simultaneous stimulation by pheromone and sorbitol had little qualitative effect on foci formation, foci were absent when pheromone preceded sorbitol addition. Foci formation resumed under this scenario in a Ste7Δ strain. (B &C) Pre-activation of cells harboring a pheromone transcriptional reporter (PAGA1-YFP) exhibits full induction under different concentrations of sorbitol. The time-resolved expression of a pheromone transcriptional reporter (PAGA1-YFP) was recorded for cells treated at the same time (B) with 1 μM pheromone and different concentrations of sorbitol (0.1–0.8 M), or (C) with 1 μM pheromone for 10 minutes followed by different concentrations of sorbitol (0.1–0.8 M). YFP expression was measured by flow cytometry. While increasing amounts of sorbitol attenuate signaling in the mating pathway if cells experience sorbitol and salt simultaneously, this effect disappears is cells are pre-treated with pheromone.

We next monitored PAGA1-YFP, a transcriptional reporter of mating pathway activity, by flow cytometry to measure the output of the pheromone pathway in cells treated with both 1 μM α-factor and 9 different doses of sorbitol, either simultaneously or with pheromone pre-treatment. When cells were simultaneously treated with 1 μM pheromone and sorbitol – conditions in which Fus3 foci form – mating pathway output was diminished in proportion to the dose of sorbitol, consistent with previous data (Nagiec and Dohlman, 2012) (Figure 3B). However, in cells pretreated with 1 μM α-factor for 10 minutes before sorbitol addition – conditions that abolished Fus3 foci formation – PAGA1-YFP exhibited full induction under all sorbitol concentrations (Figure 3C). A control promoter for a metabolic gene that is not pheromone regulated (PTDH3-mCherry) remained constant over time in all conditions, demonstrating that the effects of sorbitol are specific to the pheromone pathway reporter (Figure S3). These data indicate that pre-activation of Ste12 by pheromone signaling inhibits the formation of Fus3/Kss1/Ste12 foci upon subsequent hyper-osmotic stress and abrogates the hyper-osmotic stress inhibition of the pheromone pathway output. These results are consistent with the hypothesis that spatial localization of Fus3, Kss1 and Ste12 into foci could constitute a strategy to isolate the pheromone pathway under high osmolarity conditions.

DISCUSSION

The activities of many signaling proteins are regulated by their physical proximity and access to substrates. A common form of this regulation in eukaryotes is controlling access of transcription factors to DNA by regulating import into the nucleus (Cyert, 2001). An extension to this emerging regulatory model is the control of sub-nuclear localization of transcription factors and genes to the nuclear periphery, nucleoli, or foci (Brickner et al., 2012; Mao et al., 2011; Nizami et al., 2010). Such foci of co-localized transcription factors and coordinately regulated DNA have been observed in metazoans and fungi, and have been suggested to represent transcriptional factories where common transcriptional machinery can be marshaled and therefore modulate gene expression of the localized genes (Sutherland and Bickmore, 2009).

Recent work on the Ste12 transcription factor in yeast suggests that cells prevent the co-localization of genes regulated by this transcription factor through the use of a dedicated repressor, Dig1. The absence of Dig1 results in clustering of Ste12 and its target gene promoters, and a concomitant increase in gene expression noise (McCullagh et al., 2010). Since Ste12 clustering is not observed in wild type cells upon activation by pheromone stimulation, this clustering is likely to play a role other than activation of gene expression.

Our present work reveals that Ste12 also forms sub-nuclear foci physiologically in wild type cells in response to hyper-osmotic stress. In these foci, Ste12 co-localizes with the upstream MAPKs Kss1 and Fus3 and the negative regulators Dig1 and Dig2. Whether and to what extent the formation of Ste12 foci in the two conditions, dig1Δ cells and cells experiencing hyper-osmotic stress, reflects the same underlying mechanism remains to be determined. The most parsimonious model is that Ste12 clustering is a controlled process during hyper-osmotic stress, but gets unspecifically triggered in the absence of Dig1. The fact that foci formation of colocalized Ste12, Fus3, and Kss1 was only observed under hyper-osmotic stress and required the kinase activity of the osmotic stress MAPK Hog1 supports the hypothesis that these foci represent a specific response to osmotic shock rather than a general response to cellular damage. Hyper-osmotic stress poses a unique challenge to the pheromone response and filamentation pathways which share a common MAP3K, Ste11. Both the MAP2K Ste7 and the MAPKs Fus3 and Kss1 become phosphorylated during high osmolarity stress (Shock et al., 2009). Yet, downstream target genes of the pheromone and filamentation pathways remain inactive during osmotic stress. Hog1 kinase activity-mediated clustering of Ste12, Fus3 and Kss1 into sub-nuclear foci may represent a novel spatial mechanism that prevents the inappropriate activation of pheromone and filamentation genes despite the activation of their MAPKs by osmotic stress. Repression of signaling by foci formation has recently been shown to occur in the TOR pathway in yeast upon heat shock, suggesting that attenuation by spatial sequestration might be a general regulatory mechanism (Takahara and Maeda, 2012).

Despite the tight correlation of Ste12/Fus3/Kss1 foci formation with Hog1 activation and the inhibition of pheromone target gene expression, much work is still needed to elucidate the mechanisms of Ste12 foci formation and demonstrate a causal relationship between these foci and crosstalk inhibition between the hyper osmolarity and pheromone response pathways. Nonetheless, a tantalizing model suggested by our data is one in which Fus3 and Kss1 are activated by crosstalk upon osmotic stress, while the activation of downstream target genes is suppressed by sequestration of these kinases into foci. This method of inhibition would be complementary to Hog1 directed repression of upstream pheromone pathway elements such as Ste50. Both mechanisms would collaborate to prevent activation of the pheromone inducible target genes by hyper-osmotic stress, which is crucial given the toxic effects of co-stimulation of pheromone and osmotic pathways (Patterson et al., 2010). This physical sequestration of kinases into sub-nuclear structures organized by their downstream transcription factors provides the overlapping pheromone, filamentation and osmotic stress signaling pathways with a rapid and reversible way to prevent inappropriate signaling.

EXPERIMENTAL PROCEDURES

A detailed description of the experiments is included in the Extended Experimental Procedures in the supplementary materials. Briefly, standard procedure for growth, maintenance, and transformation of yeast and bacteria and for the manipulation of DNA were used throughout the study (Table S1 and Table S2). Subcellular localization of fluorescent fusion proteins was observed by spinning disc confocal microscopy. Reporter fluorescence was measured by flow cytometry. Western blots were visualized via infrared fluorescence using the Li-Cor imaging system. ChIP samples were assayed for enrichment by PCR (Table S3).

Supplementary Material

01
02

  • Yeast mating pathway MAPKs Fus3 and Kss1 form sub-nuclear foci under hyperosmotic stress.

  • The transcription factor Ste12 is present in the foci and required for foci formation.

  • Foci formation requires the kinase activity of the hyper-osmotic stress MAPK Hog1.

  • Foci may prevent mating pathway activation in response to hyper-osmotic stress.

Acknowledgments

We would like to thank Noah Helman and Wendell Lim for the pNH604 and pNH605 vectors, and Chao Zhang and Kevan Shokat for the ATP analog 1-NM-PP1. We are grateful to Hiten Madhani and Ignacio Zuleta for valuable scientific discussions and to members of the El-Samad laboratory for help and advice during the course of this work. The confocal microscope was provided by the UCSF-based Consortium for Frontotemporal Dementia Research housed at the J. David Gladstone Institutes. This work was funded by the NIGMS System Biology Center (P50 GM081879). SEV, DP, JS-O and HES designed the experiments, analyzed the data and wrote the manuscript. SEV, DEP and JSO performed the experiments.

Footnotes

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Associated Data

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Supplementary Materials

01
NIHMS438454-supplement-01.pdf (10.8MB, pdf)
02
NIHMS438454-supplement-02.pdf (8.2MB, pdf)

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