Genetic and physical interaction of Ssp1 CaMKK and Rad24 14-3-3 during low pH and osmotic stress in fission yeast

The Ssp1 calmodulin kinase kinase (CaMKK) is necessary for stress-induced re-organization of the actin cytoskeleton and initiation of growth at the new cell end following division in Schizosaccharomyces pombe. In addition, it regulates AMP-activated kinase and functions in low glucose tolerance. ssp1− cells undergo mitotic delay at elevated temperatures and G2 arrest in the presence of additional stressors. Following hyperosmotic stress, Ssp1-GFP forms transient foci which accumulate at the cell membrane and form a band around the cell circumference, but not co-localizing with actin patches. Hyperosmolarity-induced localization to the cell membrane occurs concomitantly with a reduction of its interaction with the 14-3-3 protein Rad24, but not Rad25 which remains bound to Ssp1. The loss of rad24 in ssp1− cells reduces the severity of hyperosmotic stress response and relieves mitotic delay. Conversely, overexpression of rad24 exacerbates stress response and concomitant cell elongation. rad24− does not impair stress-induced localization of Ssp1 to the cell membrane, however this response is almost completely absent in cells overexpressing rad24.

The CaMKK and CaMKI and CaMKV calcium-calmodulin (Ca 2þ /CaM)dependent signalling cascade is highly conserved and involved in a number of important cellular processes including cell cycle and neuronal-and immune- CaMKs as well as protein kinase B and AMP-activated kinase (AMPK) [17]. CaMKK activity is regulated by activating and inhibitory phosphorylation as well as Ca 2þ /CaM binding. CaMKKb retains 50-70% constitutive activity but requires Ca2 þ /CaM binding for full activation [18].
The fission yeast CaMKK orthologue Ssp1 [19] was identified [20] in a screen for suppressors of the cell morphology mutants ppe1 [21] and sts5-7 [22], and independently as a pH and temperature sensitive loss of function cell-cycle mutant [23]. Ssp1 phosphorylates Ssp2, the catalytic subunit of AMPK [24,25] and is required for efficient growth in low glucose conditions [19]. AMPKs regulate energy homoeostasis and respond to glucose [26], playing a role directly or indirectly in coupling nutritional response to cell differentiation in fission yeast [24]. In budding yeast, glucose depletion and environmental stressors lead to the activation of AMPK homologue SNF1 via SAK1, TOS3 or ELM1 kinases [27,28]. AMPK negatively regulates glycerol-3-phosphate dehydrogenases GPD1 and GPD2. GPD1 is inhibited in high glucose by TORC2-dependent kinases and AMPK and activated upon glucose limitation. Cells rapidly adapt to hypertonicity through a rapid increase in GPD1 activity via reduction of TOR2C-YPK1/2-mediated phosphorylation, and transcriptionally also upregulate GDP1 within 60 min. When glucose is restricted, AMPK inhibits GPD2 to limit glycerol production [29]. ssp1 2 has a pleiotropic phenotype and is synthetically lethal with spc1 2 under conditions permissible for either single mutant [23]. At high temperatures, ssp1 mutants grow as monopolar cells with a reduced capacity for transient stress-activated dispersion of actin monomers, suggesting a role for Ssp1 in actin mobilization [20,23]. Loss of ssp1 disturbs growth polarity and increases cell morphology aberrations, for example branching [30]. At high temperatures, in the presence of low pH (3.5) or hyperosmolarity (0.6 M KCl), ssp1 mutants cannot proliferate; instead they complete DNA replication and arrest as highly elongated cells in G 2 [20,23].
Although largely cytoplasmic in localization, several pools of Ssp1 exist in the cell, and following osmotic stress a portion localizes to the cell membrane or cortex. Here, we explore the physical interaction of the CaMKK Ssp1 with the 14-3-3 orthologues Rad24 and Rad25 and their relationship to the rapid movement of a portion of the Ssp1 cytoplasmic pool to the cell cortex following stress.
To compare growth rates, thiamine-repressed OP-GFP-ssp1int and/or OP-rad24-His 6 int cells were washed in EMM before plating diluted aliquots (EMM, EMMþ 1 M KCl and EMM pH 3.5; all +thiamine) (figure 1d), followed by incubation for 5 days (30 or 378C). At both temperatures, cells overexpressing rad24 displayed inhibited growth compared with cells overexpressing ssp1 or both rad24 and ssp1. Overexpression of rad24 negatively affects proliferation, resistance to hyperosmotic and low pH stress, and increases mitotic delay. Growth inhibition in OP-rad24-His 6 int cells was exacerbated by 1 M KCl (30 and 378C) and OP-GFP-ssp1int did not alleviate this effect. Similar growth delay in rad24 overexpressing cells was evident on EMM pH 3.5 media (30 and 378C) and was not ameliorated by ssp1 overexpression. Low pH conditions may affect nutritional status in these cells. Failure of CaMKK to activate AMPK [24] in cells with a drop in energy may consequently inhibit processes such as protein synthesis and growth, and affect downstream regulators.
3.3. Loss of ssp1 reduces the restrictive temperature for cdc25-22 The cdc25-22 ts (Q300) allele at the restrictive temperature (36 -36.58C) arrests at the G 2 /M boundary as very elongated single cells [33,37,38]. The ssp1 2 background exacerbates the cell elongation phenotype of cdc25-22 ts at semi-permissive temperatures (328C) (figure 2a,b). As ssp1 2 cells are sensitive to KCl stress and Cdc25 is exported out of the nucleus within 10 min after KCl stress [39], we investigated the effect of ssp1 2 rsob.royalsocietypublishing.org Open Biol. 4: 130127 on the nuclear localization of Cdc25. We expressed single-copy, native promoter-integrated cdc25-GFP (cdc25-GFPint) [40] in rad24 2 ssp1 2 (Q4113) and ssp1 2 (Q3974) backgrounds at 258C and shifted to 358C for 4 h. The nuclear localization of Cdc25-GFP is independent of ssp1 2 and rad24 2 single and ssp1 2 rad24 2 double gene deletions in both conditions (figure 2c), suggesting that the loss of ssp1 does not interfere with the nuclear localization of Cdc25-GFP.  [23]. rad24 2 alleviates this arrest at high temperatures on YEA and in the presence of KCl. rad24 2 is therefore epistatic with respect to ssp1 2 cell-cycle arrest following 0.6 M KCl stress at 368C. Proliferation at pH 3.5 at 368C is not rescued and small cell size suggests a block to growth (figure 3, also see figure 1). Growth rate in ssp1 2 is markedly reduced at high temperatures as indicated by the small colony size in spot tests (figure 3b). The response of Ssp1 to 0.6 M KCl stress and to low pH probably occurs through different mechanisms.
3.5. At native expression levels Ssp1-GFP localizes to the cell membrane after KCl stress GFP-Ssp1 localization was previously examined following strong overexpression (nmt1) from a multi-copy plasmid ssp1-GFPint rad24-2HA-His 6 (ura4 þ )int leu1-32 ura4-D18 this study and Russell laboratory Q4119 ssp1-GFPint rad25-His 6 ura4-D18 leu1-32 this study Q4120 ssp1-GFPint rad24-2HA-His 6 (ura4 þ )int rad25-His 6 leu1-32 ura4-D18 this study and Russell laboratory Q4121 ssp1-GFPint rad25-His 6 rad24::ura4 þ ura4-D18 leu1-32 this study Q4122 nmt1:GFP-ssp1int ura4-D18 leu1-32 this study Q4123 ssp1-GFP:kanMX6int ura4-D18 leu1-32 this study Q4124 pREP1-rad24-His 6 in ssp1-GFP:kanMX6int ura4-D18 leu1-32 this study rsob.royalsocietypublishing.org Open Biol. 4: 130127 [20,23,41], however variations in cell shape, strength and duration of fluorescence signals in unperturbed and osmotically challenged cells were evident [23]. Cells overexpressing GFP-ssp1 often accumulate fluorescence at the cell membrane even when unperturbed. We expressed the chromosomal integrant ssp1-GFPint (ssp1::ura4 þ ssp1-GFP LEU2) using the ssp1 native promoter. ssp1-GFPint cells grow similarly to wild-type under normal and stress conditions (YEA, YEA þ 0.6 M KCl and EMM pH 3.5; 30 and 368C) (figure 4a,b). To investigate the pattern and timing of re-localization of Ssp1-GFP at native expression levels following KCl stress ssp1-GFPint cells in mid-logarithmic growth were examined in a microfluidic flow chamber (Y2 microfluidic plate, CellASIC), allowing media changes without mechanical perturbation (ONIX Flow Control System, CellASIC). Fluorescence in unperturbed cells expressing ssp1-GFPint is largely cytoplasmic. Vacuoles are faintly discernible, suggesting that Ssp1-GFP levels are lower than those in the cytoplasm (figure 4c). After switching from YEA to YEA þ 0.6 M KCl, cell shapes became more jagged and fission scars became more notable, with a slight decrease in cell volume. Reversal of this cell 'shrivelling' with the re-establishment of cell turgor depends upon glycerol synthesis and requires gpd1 þ [42]. The nucleus appeared compressed and irregular in shape. Vacuolar areas became more conspicuous, with low fluorescence intensity similar to the nucleus. Within 2-3 min of 0.6 M KCl treatment, small areas of increased Ssp1-GFP fluorescence (foci) in the cytoplasm and near the cell walls and septum appeared. These foci brightened further and by 7 min, additional fluorescent foci along the cell membrane appeared, especially at the cell tips (figure 4c,d). Localization of Ssp1-GFP at the cell membrane started to decline by 20 min, decreased further by 34 min and was similar to cytoplasmic levels by 70 min. Cells accommodated to the increase in extracellular osmolytes, regaining volume and rod-shaped cell morphology at 23-34 min, indicating induction of gpd1 þ and glycerol synthesis [42]. Surface plots of fluorescence intensity of cells at t ¼ 0, t ¼ 7, t ¼ 34 and t ¼ 70 min during 0.6 M KCl stress (figure 4d) highlights the formation of the small foci of fluorescence intensity in the stressed cells. The conspicuous placement of fluorescent foci, often on opposing sides along the cylindrical portion of the cell suggested that Ssp1-GFP localization to this area takes place not only as distinct foci, but instead as a ring. Although overall fluorescence declined, Ssp1-GFP protein levels stayed constant (figure 4e). Expression of multi-copy GFP-ssp1 from the nmt1 promoter (Q4108) facilitated the capture of a multi-image Z-stack. In EMM þ 1.5 M KCl (258C), a band of foci formed around the   3.6. Loss of rad24 or rad25 does not affect localization of Ssp1-GFP after stress To investigate how loss of 14-3-3 proteins affects the subcellular localization of Ssp1-GFP, we expressed ssp1-GFPint in rad24 2 (Q4116) or rad25 2 (Q4115) cells. In YEA (308C), Ssp1-GFP in mid-logarithmic rad24 2 or rad25 2 cells was cytoplasmic and excluded from the nucleus. Ssp1-GFP accumulated along forming and formed septa, and along the cell membrane in a subset of cells. After osmotic stress (YEA, 0.6 M KCl) Ssp1-GFP promptly localized to areas near the cell membrane, forming foci of fluorescence as in rad24 þ rad25 þ cells (figure 6a), indicating that Ssp1-GFP localization to the cell membrane after 0.6 M KCl stress does not require rad24 2 or rad25 2 .
3.7. Ssp1-GFP co-immunoprecipitates with the 14-3-3 proteins Rad24-2HA-His 6 and Rad25-His 6 14-3-3 proteins are abundant in all cells, and in budding yeast interact with at least 271 proteins representing approximately 4.4% of the proteome [44] indicating that only a small amount of the total pool of 14-3-3 associates with any one protein at any given time. Only a portion of the total Ssp1-GFP present in the lysate immunoprecipitated in the Rad24-2HA-His 6 pulldown. To test the relative binding of Rad24 or Rad25 to Ssp1-GFP, protein lysates were prepared from mid-logarithmically growing (YEA, 308C) cells expressing single-copy ssp1-GFPint and either rad24-2HA-His 6 (Q4118) or rad25-His 6 (Q4119) integrated at their native promoters. Ssp1-GFP co-immunoprecipitates with Rad24 and Rad25 (figure 6b,c) and a portion of each of the Ssp1 and 14-3-3 pools co-immunoprecipitate. In budding yeast, the deletion phenotype of the major 14-3-3 isoform BMH1 is complemented by overexpression of the minor isoform BMH2, suggesting that these proteins have similar binding partners [45]. To determine the relative amounts of Rad24 and Rad25 associating with Ssp1, we expressed ssp1-GFP rad24-2HA-His 6 rad25-His 6 (Q4120), where Rad24-2HA-His 6 is distinguishable from Rad25-His 6 by size owing to small differences in molecular weight as well as the presence of the 2HA. Rad24-2HA-His 6 was present at approximately fivefold higher levels (IMAGEJ) compared with Rad25-His 6 protein in the cell lysate (figure 6d) and a similar ratio of Rad24-2HA-His 6 and Rad25-His 6 co-precipitated with Ssp1-GFP.
Ssp1-GFP is a highly phosphorylated protein [19]. We can see doublet formation on gels (see immunoprecipitates; figure 6c) and can separate these bands by running the SDS-PAGE gel for an extended amount of time. The phosphorylation state of Ssp1-GFP will be discussed below.

Rad25-His 6 and Ssp1-GFP physically interact in the absence of Rad24
For the previous co-immunoprecipitation studies, Rad24-2HA-His 6 or Rad25-His 6 proteins were co-expressed with Ssp1-GFP. We wanted to determine whether Rad25 associates with Ssp1-GFP only as part of a heterodimer with Rad24 or is able to bind Ssp1 as a Rad25-Rad25 homodimer in vivo. Immunoprecipitation of Ssp1-GFP and Rad25-His 6 in rad24 þ and rad24 2 (Q4121) cells showed that Rad25-His 6 co-precipitates with Ssp1-GFP in the absence of Rad24 (figure 6e). We also found that Ssp1-GFP is less stable in a rad24 2 background (figure 6e,f ).

Stress reduces the interaction between Ssp1-GFP and Rad24-2HA-His 6
Harvesting cells by centrifugation at 48C, followed by washes in ice-cold lysis buffer exposes cells to stressors, including increased gravitational forces and hypoxia. This is manifested by transient increased phosphorylation of MAPK Spc1 [46,47] and Atf1 [46]. Cells undergo a brief cell-cycle delay similar to but shorter than the delay after 0.6 M KCl [23,46] and there is a transient depolarization of actin [46]. Hypoxia in pelleted cells causes activation of hypoxia response genes via Sre1 [48,49]. Moderate thermal downshift (28-158C) brings about phosphorylation of Spc1 and induction of the stress response genes ctt1, tps1 and ntp1 [46]. To minimize these stressors, cells were treated with pre-warmed YEA (308C; +KCl to 0.6 M KCl) for 15 min, then rapidly chilled to 08C with frozen, crushed YEA (+0.6 M KCl) preceding centrifugation. Very low temperatures greatly delay Spc1 phosphorylation [46]. Ssp1-GFP localizes to the cell membrane after 15 min of 0.6 M KCl stress and activating phosphorylation of Spc1 MAPK is detected [10]. After KCl treatment approximately 10 times less Rad24-2HA-His 6 co-immunoprecipitated with Ssp1-GFP in the KCl-treated immunoprecipitate than in the untreated controls (figure 7a), however we consistently failed to detect a decrease in the total amount of Rad25-His 6 protein that coimmunoprecipitated with Ssp1-GFP (figure 7b). Ssp1-GFP YEA wild-type  at the membrane in a HOG1-independent manner. Loss of BMH1 increases sensitivity to NaCl, KCl and LiCl without affecting plasma membrane potential [45]. In Schizosaccharomyces pombe, MAPK activation after osmotic stress leads to phosphorylation of Cdc25 by Srk1, binding of 14-3-3 and Cdc25 nuclear export [14]. Loss of rad24 or rad25 does not prevent the stress-dependent localization of Ssp1-GFP to the cell membrane, nor does it increase sensitivity to temperature, low pH or KCl stress ( figure 3a,b). Strong expression of multi-copy pREP1:rad24-His 6 in ssp1-GFPint cells (Q4124) (EMM, 308C) impairs Ssp1-GFP accumulation at the membrane after 1 M KCl treatment (figure 7c). In rad24 þ cells expressing singlecopy integrated GFP-ssp1 under control of the nmt1 promoter (Q4111), the majority accumulate GFP-Ssp1 at the membrane after KCl stress. This localization pattern is suppressed by co-overexpression of single-copy, integrated rad24-His 6 under control of the nmt1 promoter, where this response is only detected in approximately 12% of the cell population (figure 7d).

The phosphorylation state of Ssp1 is not altered after 0.6 M KCl stress
Like all CaMKKs [18], Ssp1 is regulated by phosphorylation. Ssp1 has potential phosphorylation sites at Y58, S59, Y63, T82 and S94, where S59 and S94 were identified in phosphopeptides containing only one phosphorylatable Ser residue [19,50]. Ssp1 is dephosphorylatable in vitro [19]. We investigated whether stress response causes changes in Ssp1 phosphorylation. To preserve the phosphorylation state of Ssp1-GFP in unperturbed and KCl-stressed cells, we collected mid-logarithmically growing cells by gentle filtration [35,51] after 15 min treatment with pre-warmed YEA (+KCl to 0.6 M, 308C). We detected a higher mobility band in extracts treated with Lambda phosphatase (no phosphatase inhibitors; figure 7e, lane 3). The faint upper band and strong lower band represent Ssp1-GFP protein in a partially phosphorylated and dephosphorylated state. The lower band is absent in the mock-treated extract (lane 2), confirming that it is not due to proteolysis. There was no bandshift evident in extracts prepared in the presence of phosphatase inhibitors in either perturbed or KCl-treated cells (lanes 4 and 5). Bands corresponded to the position of the phosphorylated bands produced by the control lysates. Ssp1-GFP thus appears to be phosphorylated in both unperturbed (YEA, collected on filter [52] and stressed with 0.6 M KCl) cells in vivo. We obtained similar results when lysates were extracted from Ssp1-GFP cells after mild temperature stress (368C) (not shown). Our results confirm basal phosphorylation of Ssp1.

Discussion
Regulation of CaMKK occurs through Ca 2þ /CaM binding and inhibitory and activating changes in phosphorylation state. Additional inhibition takes place through 14-3-3 binding [17,32]. Here, we explore the interaction of CaMKK Ssp1 with 14-3-3 and their role in cell-cycle regulation and stress response.

CaMKK Ssp1 does not co-localize with actin patches at the cell membrane
At native expression levels, cytoplasmic Ssp1-GFP accumulates at the cell membrane after perturbation by either 0.6-1.2 M KCl or sorbitol [23]; however ssp1 is required for growth in the presence of KCl but not sorbitol. Similarly, although MAPK Pmk1 is activated by 1.2 M sorbitol, pmk1 2 cells are not sensitive to this hyperosmotic stress [69]. A small pool of Ssp1-GFP accumulates at the cell membrane, while the majority of the protein remains cytoplasmic. This does not support a previous model suggesting that Ssp1 directly localizes actin patches at the cell membrane to support areas of new growth [23]. We show that following hyperosmotic stress GFP-Ssp1 forms a band, while actin patches do not follow this pattern; Arp3C-YFP and Ssp1-CFP do not co-localize. The importance of the compartmentalization of Ssp1 at the membrane is not clear. The S. pombe cell wall is most vulnerable to rupture at the extensile tips and is sturdier in the cylindrical portion of the cell [70]. Ssp1 accumulates in areas corresponding to fission scars, which are less vulnerable to damage than the extensile tips [70]. Accumulation at the cell membrane following hyperosmotic stress is transient; however, Ssp1 is required for long-term cell survival under hyperosmotic conditions.

The role of 14-3-3 binding to CaMKK
At least some cytoplasmic Ssp1 is bound to Rad24 and Rad25 in unperturbed cells. After applying hyperosmotic stress, Ssp1 is released from 14-3-3 and Ssp1 accumulates at the cell membrane. Our data also show that Rad24 and Rad25 are dispensable for the translocation of Ssp1-GFP to the cell membrane. This response can be repressed in cells overexpressing Rad24 even when there is an excess of Ssp1-GFP. 14-3-3 proteins commonly act as cytoplasmic anchors, providing negative regulation of proteins through sequestration. In Drosophila melanogaster, 14-3-3 binds phosphorylated b-catenin antagonist Chibby (Cby), promoting cytoplasmic sequestration of the b-catenin-14-3-3-Cby complex [71]. In mammalian systems cytoplasmic Bax, a Bcl-2-related protein required for c-Jun NH 2 -terminal kinase-dependent apoptosis, localizes to the mitochondria after stress stimuli, where it induces cytochrome c release. 14-3-3-bound Bax remains anchored in the cytoplasm [72]. Upon activation, JNK phosphorylates 14-3-3, Bax is released and translocates to the mitochondria, and apoptosis commences [73]. The Ras/Raf/MEK/ERK-signalling cascade is also regulated by 14-3-3 proteins, which are thought to inhibit activation of plasma membrane-anchored Ras by sequestering its activator Raf-1 in the cytoplasm. This mechanism prevents cascade activation in resting cells [74]. The majority of Ras superfamily G proteins are kept in inactive (GDP-bound) or active (GTP-bound) forms by guanine nucleotide exchange factors and GTPase-activating proteins, respectively. The atypical Rho GTPases Rnd1/2/3 are cell morphology regulators and constitutively bind GTP [75,76]. After phosphorylation by Rock1 kinase or protein kinase Ca (PKCa) [77,78], Rnd3 binds 14-3-3 and translocates from the plasma membrane to the cytoplasm, its function inhibited via sequestration from its site of action [79]. In fission yeast, 14-3-3 proteins also regulate the localization of many proteins. For example, they associate with the primarily cytoplasmic Byr2, preventing binding to Ras1/GTP at the cell membrane during vegetative growth. Loss of 14-3-3 expedites Byr2 translocation [80]. Our data suggest that in fission yeast, 14-3-3 proteins may play a role in the negative regulation of Ssp1 translocation to the cell membrane. Mammalian 14-3-3 isoforms require only their N-termini to dimerize. Dimerization greatly increases their thermostability and single recombinant isoforms form homodimers even if they function as heterodimers in vivo [81,82]. Particular isoforms in 14-3-3 heterodimers allow the interaction of proteins by bringing the two binding partners closer together ( [82][83][84]; reviewed in [85][86][87]). The minor isoform Rad25, which associates with Ssp1, forms homodimers and binds Ssp1, at least in the absence of Rad24. A reduction in Rad24 binding to Ssp1 after osmotic stress could occur owing to a decrease in Rad24-Rad24 homodimer and/or Rad24-Rad25 heterodimer binding. Conversely, following hyperosmotic stress, Rad25-Rad25 homodimers remain bound to Ssp1-GFP. This suggests an interesting and distinct role for the minor 14-3-3 isoform in CaMKK regulation. Future studies will confirm whether the Rad24-bound pool is preferentially located in any particular part of the cell.

Increased stress sensitivity in cells overexpressing rad24 is alleviated by co-overexpression of CaMKK
Rad24 is a negative regulator of mitosis after DNA damage [88,89]. Overexpression of rad24 increases long-term sensitivity to stressors such as KCl and low pH, but sensitivity to high temperature is relieved to some extent by co-overexpression of ssp1. Association with 14-3-3 proteins inhibits CaMKK activity in mammalian systems [32,90], thus if Rad24 binding inhibits Ssp1 activity then augmenting levels of 14-3-3 would diminish Ssp1-mediated stress response. Ssp1-FLAG binds 14-3-3, but it is unclear whether this association affects CaMKK activity [19]. 14-3-3 proteins are involved in many pathways [91,92] and substantially increasing the Rad24 pool causes a complex response in terms of stress sensitivity. Both deletion and overexpression of 14-3-3 BMH1 increase chronological lifespan in nutrient-stressed budding yeast. Cells overexpressing BMH1 survive longer in the absence of additional stressors, probably because an increase in phosphorylated BMH1 S238 decreases the stress response required for longevity [93].

A role for 14-3-3 in CaMKK turnover
The absence of Rad24 increases Ssp1 turnover, suggesting a role for Rad24 in maintaining Ssp1 protein stability. rad24 2 cells do not display hypersensitivity to conditions where ssp1 2 cells are unable to proliferate, indicating that Ssp1 protein levels are maintained at sufficient levels. 14-3-3 proteins are involved in protein stabilization both directly and indirectly in other systems, for example by blocking access of ubiquitin ligases (reviewed in [85,86]). In mammalian systems, association with 14-3-3 prevents Wee1 degradation by masking a degradation motif required for normal Wee1 turnover [94]. Budding yeast 14-3-3 homologues BMH1, BMH2 and ACM1 form a stable complex with the APC/C activator CDH1/CDC20, keeping it inactived by acting as an APC pseudosubstrate [95,96]. Further studies will determine whether the interaction of 14-3-3 and Ssp1 has a direct or indirect effect on CaMKK catalytic function and whether 14-3-3 proteins play a role in the negative regulation of Ssp1 translocation to the cell membrane after stress.

Plasmid construction and chromosomal integration
All DNA amplification was performed by PCR with Expand High Fidelity Taq Polymerase (Roche). T4 ligase and restriction enzymes used were from Promega. For primers, see table 2.

Protein lysates
Lysates were prepared at 48C unless otherwise indicated. Mid-logarithmic growth phase cells were harvested by centrifugation (5 min, 1876.9g), washed in ice-cold stop buffer [97], collected again by pulse centrifugation (13 051g) and

KCl treatment
Mid-logarithmic growth phase cells (1000 -3000 ml, YEA, 308C) were treated for 15 min with warm (308C) YEA (control) or YEA þ KCl to 0.6 M. Cells were chilled to 08C within 1 min by the addition of frozen, crushed YEA or YEA þ 0.6 M KCl and immediate immersion of flask into an ice-water/ethanol slurry.

Immunoprecipitation
Lysate preparation and immunoprecipitation were performed at 48C. Cell cultures (YEA, 308C) were harvested by centrifugation (9927.3g) and washed once in 10 ml ice-cold HB buffer [97] (pH 7.4; containing 2 mM DTT and Roche complete protease inhibitor (EDTA free), washed again and followed by mechanical disruption in ice-cold HB buffer. Lysates were centrifuged (35 4406g; 40 min) and incubated with 100 ml bedvolume Sepharose G beads (4 Fast Flow, GE Healthcare) for 1 h to remove non-specific binding proteins. Lysates were incubated overnight with 15 ml rabbit anti-GFP polyclonal serum (Invitrogen) or 15 ml mouse anti-His 6 antibody (Roche) on a rotator. Control lysates did not contain antibodies. Lysates were incubated with 100 ml bed-volume Protein G Sepharose beads for 2 h. Beads were washed extensively with HB buffer and protein complexes eluted with 0.2 M glycine (pH 2.2) for 15 min and neutralized by addition of saturated Tris ( pH 10). Supernatants were boiled with Laemmli buffer and analysed by immunoblotting.

Phosphatase treatments
Mid-logarithmic phase cells were treated with 308C YEA or YEA þ 0.6 M KCl for 15 min. To preserve phosphorylation state, cells were collected on microfibre filters (934-AH; Whatman) and washed with 5 ml ice-cold stop buffer [97] lysed in lysis buffer [97] (buffers contained 15 mM pNPP and 60 mM b-glycerophosphate). Cells collected by centrifugation were washed (150 mM NaCl, 1 mM EDTA, 1 mM PMSF) and lysed in phosphatase-inhibitor-free lysis buffer (modified from [97]) or washed and lysed with phosphatase-inhibitorenriched buffers. Phosphatase-inhibitor-free protein (5 mg) was treated with 800 units of Lambda Protein Phosphatase (NEB) for 30 min at 308C. Mock treatments did not contain phosphatase.

Microscopy
Images were captured by a high performance CCD (Cooke SensiCam) camera on a Leitz DMRB fluorescence microscope  or a high performance CCD Hamamatsu Orca-ER camera on a Zeiss AxioImager.Z1 fluorescence microscope. SLIDEBOOK image analysis software (Intelligent Image Innovations) was used to perform cell measurements and to analyse Z-stacks.
Funding statement. This study was financially supported with the