The minimum domain of Pho81 is not sufficient to control the Pho85–Rim15 effector branch involved in phosphate starvation-induced stress responses
- First Online:
- Cite this article as:
- Swinnen, E., Rosseels, J. & Winderickx, J. Curr Genet (2005) 48: 18. doi:10.1007/s00294-005-0583-3
- 173 Views
The phosphate regulatory mechanism in yeast, known as the PHO pathway, is regulated by inorganic phosphate to control the expression of genes involved in the acquisition of phosphate from the medium. This pathway is also reported to contribute to other nutritional responses and as such it affects several phenotypic characteristics known also to be regulated by protein kinase A, including the transcription of genes involved in the general stress response and trehalose metabolism. We now demonstrate that transcription of post-diauxic shift (PDS)-controlled stress-responsive genes is solely regulated by the Pho85–Pho80 complex, whereas regulation of trehalose metabolism apparently involves several Pho85 cyclins. Interestingly, both read-outs depend on Pho81 but, while the previously described minimum domain of Pho81 is sufficient to sustain phosphate-regulated transcription of PHO genes, full-length Pho81 is required to control trehalose metabolism and the PDS targets. Consistently, neither the expression control of stress-regulated genes nor the trehalose metabolism relies directly on Pho4. Finally, we present data supporting that the PHO pathway functions in parallel to the fermentable growth medium- or Sch9-controlled pathway and that both pathways may share the protein kinase Rim15, which was previously reported to play a central role in the integration of glucose, nitrogen and amino acid availability.
Inorganic phosphate (Pi) is an essential nutrient for all organisms, being required for the biosynthesis of nucleic acids, phospholipids and cellular metabolites as well as for energy metabolism and signal transduction. Microorganisms often encounter limiting concentrations of this essential nutrient and have evolved complex signal transduction networks that enable them to make optimal use of the scarce available phosphate sources. When Pi in the medium becomes limiting, the budding yeast Saccharomyces cerevisiae activates a regulatory mechanism known as the PHO pathway, which results in an increased expression of multiple genes involved in the acquisition, uptake and storage of phosphate (Ogawa et al. 2000; Persson et al. 2003). This transcriptional activation is mediated by the transcription factor Pho4 in cooperation with the coactivator Pho2 (Lenburg and O’Shea 1996). Typical Pho4 target genes include PHO84 and PHO89, encoding the high-affinity phosphate transporters, and PHO5, encoding a secreted acid phosphatase. The activity of Pho4 is regulated through phosphorylation at several serine residues by the cyclin-dependent kinase (CDK) Pho85 when associated with the Pho80 cyclin (Kaffman et al. 1994). Phosphorylation of Pho4 leads to its inactivation via disruption of the Pho4–Pho2 interaction, stimulation of Pho4 nuclear export and inhibition of its nuclear import (O’Neill et al. 1996; Kaffman et al. 1998a, b; Komeili and O’Shea 1999). Regulation of the kinase activity of the Pho85–Pho80 complex in response to phosphate levels occurs through the Pho81 CDK inhibitor (CKI) protein (Schneider et al. 1994; Ogawa et al. 1995). Under low-phosphate conditions, Pho81 inhibits the kinase activity of the Pho85–Pho80 complex, thereby allowing nuclear import and activation of the Pho4 transcription factor. Notably, only one particular domain of Pho81, the so-called Pho81 minimum domain, is essential for inhibition of the Pho85–Pho80 kinase activity towards the Pho4 target (Huang et al. 2001). Since expression of PHO81 is itself controlled by Pho4, it is the target of a positive feedback loop where a low-Pi signal results in enhanced expression of PHO81 (Creasy et al. 1993; Ogawa et al. 1993, 1995). In addition, it was recently shown that the Pho80 and Pho81 proteins are subjects for Pho85-mediated phosphorylation and this phosphorylation appears to be required for the proper in vivo functioning of these components, indicating a multitude of internal feedback loops within the pathway (Knight et al. 2004; Waters et al. 2004).
Aside from its role in the PHO pathway, Pho85 has additional functions in many other pathways (for reviews, see Moffat et al. 2000; Carroll and O’Shea 2002). This is reflected in the multiple phenotypes displayed by a pho85Δ strain. Cells deleted for the PHO85 gene grow slowly on glucose and fail to properly utilize a number of other carbon sources including galactose, sucrose and several non-fermentable carbon sources (Lee et al. 2000). Other phenotypes include hyperaccumulation of glycogen, sporulation defects, morphological abnormalities, hypersensitivity to a large number of stress conditions and aberrant expression profiles during the diauxic shift (Gilliquet and Berben 1993; Timblin et al. 1996; Measday et al. 1997; Huang et al. 2002; Nishizawa et al. 2004). Consistent with the diverse roles of Pho85, ten genes encoding known or putative Pho85 cyclins (Pcl) have been identified (Measday et al. 1997). Hence, the different functions of Pho85 may relate to specific cyclin subunits; and in some cases the molecular targets for particular kinase complexes have indeed been identified (Huang et al. 1996, 1998; Wilson et al. 1999). For most processes involving the Pho85 CDK, however, neither the required cyclin partners nor the molecular targets for the kinase complex are known. Another issue concerns the regulation of the activities of the different Pho85 complexes. Only for the Pho85–Pho80 complex, is regulation via the Pho81 CKI well documented. Other Pho85 complexes have been reported to be regulated by a cell cycle-dependent transcriptional control on cyclin expression (Ogas et al. 1991; Espinoza et al. 1994; Measday et al. 1994, 1997; Aerne et al. 1998; Tennyson et al. 1998; Lee et al. 2000) or by activation of the cyclin in response to nutrient availability (Shemer et al. 2002).
In this paper, we focused on phenotypes known to be dependent on protein kinase A (PKA), including trehalose metabolism and expression of stress-responsive genes (Roosen et al. 2004). We demonstrate that the PHO pathway is required for proper regulation of these read-outs in response to phosphate levels. We provide evidence that different Pho85 complexes may regulate trehalose metabolism, while specifically the Pho85–Pho80 complex seems to be responsible for expression-regulation of several stress-responsive genes, especially those regulated by Gis1, a transcription factor that binds the post-diauxic shift (PDS) DNA element. We also show that Pho81 takes part in this regulation but, in contrast to the regulation of the PHO5 encoded acid phosphatase activity, the Pho81 minimum domain as described by Huang et al. (2001) seems not to be sufficient. Consistently, none of the phenotypic read-outs strictly depends on Pho4. Since expression of stress-responsive genes and trehalose metabolism are known targets of the Sch9-controlled fermentable growth medium (FGM) pathway, which converges downstream of PKA on the protein kinase Rim15 (Reinders et al. 1998; Pedruzzi et al. 2003; Roosen et al. 2005), we analyzed a possible connection between this signalling cascade and the PHO pathway. We demonstrate that, similar to Sch9, Pho85 also appears to regulate Rim15 and the Rim15 effectors. Interestingly, both kinases appear to function in parallel signalling routes, as indicated by the synthetic lethality when the deletions of SCH9 and PHO85 are combined.
Materials and methods
Yeast strains and plasmids
Strains used in this study
BY4741 (wild type)
Matahis3Δ1 leu2Δ0 met15Δ0 ura3Δ0
BY4741 with pho4::KANMX4
BY4741 with pho80::HIS3
BY4741 with pho81::KANMX4
BY4741 with pho85::KANMX4
BY4741 with pho81::KANMX4 pho85::KANMX4
BY4741 with pho81::KANMX4 pho80::HIS3
BY4741 with rim15::KANMX4
BY4741 with pho85::KANMX4 rim15::KANMX4
BY4741 with pho80::HIS3 rim15::KANMX4
BY4741 with sch9::HIS3
Matα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0
BY4742 with pho85::KANMX4
Culture and starvation conditions
Cells were cultured at 30°C into exponential phase (optical density at 600 nm = 1.5–2.0) in YP medium (1% yeast extract, 2% bacto peptone) with 2% glucose. Mid-exponential cells were harvested and transferred to phosphate-starvation medium (5.7 g l−1 yeast nitrogen base without phosphate) with 4% glucose, uracil (50 mg l−1), leucine (250 mg l−1), histidine (100 mg l−1) and methionine (20 mg l−1). Cells were starved for phosphate for 3 days at 30°C under continuous shaking and starvation medium was refreshed daily. The pho81Δ and pho4Δ strains with constitutive overexpression of specific PHO81 constructs were grown in SD-ura medium (synthetic medium without uracil containing 2% glucose) until the exponential phase and transferred to phosphate-starvation medium (without uracil) containing 4% glucose.
Incubation conditions for phosphate-addition assays
The phosphate-starved, glucose-repressed cells were rapidly cooled on ice and harvested by centrifugation. The pellet was washed twice with ice-cold MES buffer, pH 6.0, resuspended in fresh phosphate-starvation medium with 4% glucose and incubated at 30°C with shaking. After 30 min of incubation, 10 mM KH2PO4 was added to the culture and samples were taken at the indicated time-points.
Determination of trehalose levels
Samples were collected by filtration at the indicated time-points, washed once with ice-cold water, weighed and frozen in liquid nitrogen. The pellets were resuspended in 1 ml of 0.25 M Na2CO3 per 40 mg of cells and boiled at 95°C for 60 min. Samples were neutralized and intracellular levels of trehalose were determined using Humicola trehalase (Neves et al. 1994; Colombo et al. 1998). Glucose concentrations were measured using the GOD-PAP kit (Dialab).
Sampling, extraction and determination of trehalase activity
Samples of 50 mg cells ml−1 were taken at the indicated time-points. Cells were rapidly cooled by the addition of ice-cold water, centrifuged and resuspended in 500 μl of 25 mM MES buffer, 50 μM CaCl2, pH 7, for extraction. Crude cell extracts were prepared as described by Giots et al. (2003) and dialyzed against 25 mM MES buffer, pH 7, with 50 μM CaCl2. Trehalase activity in dialyzed cell extracts was determined as described by Giots et al. (2003). The specific activity is expressed as nmol glucose liberated min−1 mg protein−1 . The total amount of protein in the samples was determined using a standard method (Lowry et al. 1951).
RNA extraction and Northern blot analysis
Samples were taken at the indicated time-points and rapidly cooled by the addition of ice-cold water. The cells were pelleted, washed once with ice-cold water and stored at −80°C. Isolation of total RNA was performed as described by Giots et al. (2003). Probes were labelled with α−32 P-dCTP, using the High Prime kit (Boehringer Mannheim, Monza, Italy). Northern blots were made by separation of total RNA on gels containing 1% (w/v) agarose in 50 mM boric acid, 1 mM sodium citrate, 5 mM NaOH, pH 7.5, 1% formaldehyde. Subsequently, RNA was transferred by capillary blotting to a Hybond-N membrane (Amersham), using 10× SCC buffer (1.5 M NaCl, 0.15 M Na-citrate, pH 7.0). The filters were hybridized with the 32 P-labelled probes consisting of fragments of the desired coding region. The blots were analyzed using PhosphorImager technology (FUJIX, BAS-1000). Quantification of Northern blot images was performed with TINA ver. 2.0.
Reproducibility of results
Relative basal level and activation of trehalase activity for the different pho mutants described in Fig. 2a. Trehalase activities are expressed as a percentage of wild-type levels (mean values from three independent experiments, with SD in brackets)
Relative basal level and activation of trehalase activity of the pho81Δ strain transformed with different PHO81 constructs (see Fig. 4b). Trehalase activities are expressed as a percentage of wild-type levels (WT + EB0730; mean values from three independent experiments, with SD in brackets). See Materials and methods for a description of the different plasmids
Pho81Δ + EB0730
pho81Δ + EB1361
pho81Δ + EB1362
pho81Δ + EB1363
pho81Δ + EB1364
Relative basal level and activation of trehalase activity of the pho4Δ strain transformed with different PHO81 constructs (see Fig. 5c). Trehalase activities are expressed as percentage of wild-type levels (WT + EB0730; mean values from three independent experiments, with SD in brackets). See Materials and methods for a description of the different plasmids
pho4Δ + EB0730
pho4Δ + EB1361
pho4Δ + EB1364
Pho81-mediated inhibition of the Pho85–Pho80 complex is required for the correct regulation of stress-related phenotypes
As a control for the experiments described above, the expression of typical Pho4 targets, such as PHO84 and PHO89, was monitored during phosphate starvation (Fig. 1b,c) and after re-supplementation of phosphate to the starved cells (Fig. 3a,b). Consistent with previously reported data (Lenburg and O’Shea 1996), the Pho4 target genes are induced in the wild-type strain upon starvation and repressed when phosphate is again available. Their expression is completely abolished in the pho81Δ mutant, while it is constitutive in the single pho85Δ and pho80Δ mutants and in the double pho81Δpho85Δ and pho81Δpho80Δ mutants.
The Pho81 minimum domain is only sufficient to sustain Pho4-mediated transcription of PHO genes
The defect of a strain lacking a functional Pho4 can be suppressed by overexpression of PHO81
These data led us to conclude that Pho4 was not affecting the targets studied directly, but indirectly via the positive feedback loop for expression of PHO81. In line with this hypothesis, the expression of a constitutively active form of Pho4, containing five serine-to-alanine substitutions at its sites of phosphorylation by Pho85–Pho80 (Kaffman et al. 1998a, b), could not alleviate the defective trehalose accumulation or the retarded induction of stress-responsive genes in the pho81Δ mutant during phosphate starvation, despite the fact that it rescued PHO gene transcription (data not shown).
The PHO pathway functions in parallel to Sch9-controlled signalling to regulate Rim15 and the Rim15 effectors
When yeast cells encounter nutrient limitation, they respond by activating several stress responses, which include the accumulation of the reserve carbohydrates trehalose and glycogen and the induction of a multitude of stress-responsive genes (Lillie and Pringle 1980; Thevelein and de Winde 1999). These properties are known to be tightly regulated by multiple signalling pathways with a major role being played by PKA (Francois and Parrou 2001; Winderickx et al. 2003; Cameroni et al. 2004; Roosen et al. 2004, 2005). The latter was concluded based on several studies showing that cells induce stress responses and become more stress-resistant under conditions that correspond to low PKA activity, i.e. if glucose or one of the essential nutrients is missing, while they display opposed responses and become stress-sensitive under conditions that are considered equivalent to high PKA activity, i.e. when cells are grown on rich glucose-containing medium where all nutrients essential for growth are abundantly present (Hirimburegama et al. 1992; Durnez et al. 1994; Donaton et al. 2003; Giots et al. 2003). The adaptation to the presence of essential nutrients appeared not to be associated with changes in the cAMP level, therefore it was postulated that another pathway could control the activity of the catalytic subunits of PKA. This pathway was called the FGM pathway, with the protein kinase Sch9 being the major controlling component (Thevelein 1994; Crauwels et al. 1997). Most recently, the concept of the FGM pathway was demonstrated to reflect convergence of the Sch9-dependent pathway on Rim15, a protein kinase immediately downstream and under negative control of PKA (Reinders et al. 1998; Pedruzzi et al. 2003; Roosen et al. 2005).
In this paper, we demonstrated the involvement of the yeast PHO pathway for the phosphate-dependent regulation of the abovementioned stress responses. Similar to the control of Pho4-mediated transcription of several PHO genes, Pho81-dependent regulation of Pho85 kinase activity is required for proper trehalose metabolism in response to phosphate availability. Unregulated high activity of Pho85 due to the absence of Pho81 impairs trehalose accumulation during phosphate starvation, whereas the absence of Pho85 activity results in the loss of a quick trehalose mobilization after the re-addition of phosphate to the starved cells. However, in contrast to the known PHO pathway, regulation of trehalose levels apparently requires other Pho85-associating cyclins in addition to Pho80. This could be concluded from the observations that the pho81Δpho80Δ mutant still displays lower levels of trehalose than a wild-type strain, in contrast to the pho81Δpho85Δ mutant and that the increased basal activity of trehalase in the pho81Δ mutant can only be restored by the additional deletion of PHO85 but not by the additional deletion of PHO80. Concerning the rapid phosphate-induced trehalase activation, our results indicate that proper functioning of the PHO pathway is important for maximal signalling. However, other signalling systems are involved, as trehalase activation is not abolished in all the pho mutants tested. For instance, it has been shown that PKA plays an important role in the phosphate-induced activation of trehalase (Giots et al. 2003). Several Pho85 cyclins are already implicated in the regulation of the reserve carbohydrate glycogen and one may argue that they may also be required for correct regulation of trehalose levels. These cyclins include Pcl6, Pcl7, Pcl8 and Pcl10 (Huang et al. 1996, 1998; Wilson et al. 1999, 2005; Lee et al. 2000). Of them, Pcl7 might be a very interesting candidate, since this cyclin has been shown to directly interact with Pho81 in vivo, to regulate the kinase activity of the Pho85–Pcl7 complex in response to phosphate levels (Lee et al. 2000). However, this does not exclude that other Pho85 cyclins are involved in Pho81-dependent processes. For instance, the Pho85–Pcl8 and Pho85–Pcl10 complexes phosphorylate the glycogen synthase enzyme Gsy2 (Huang et al. 1996, 1998; Wilson et al. 1999), but so far it is not known how these kinase activities are regulated. Although Pho81 localizes to the nucleus under phosphate-limiting conditions, where it inhibits the activity of the nuclear Pho85–Pho80 complex, additional localization of Pho81 to the cytosol and plasmamembrane observed under the same conditions may be important for the additional function Pho81 plays in the regulation of trehalose metabolism (Huang et al. 2001). The role of the Pho4 transcription factor in the regulation of trehalose levels is limited to its positive action on Pho81 function via transcriptional induction of the PHO81 gene. This is an interesting result, as it shows that the Pho4-mediated feedback on PHO81 expression (Ogawa et al. 1995) is not only required for continued activation of Pho4 but is also important to allow Pho81 to regulate other Pho85-mediated processes in order to ensure a proper response to phosphate starvation.
A second common response of yeast cells to nutrient-starvation conditions is the transcriptional induction of a large set of stress-responsive genes and our results confirm that this also applies to phosphate starvation (Crauwels et al. 1997; Thevelein and de Winde 1999; Donaton et al. 2003; Giots et al. 2003). Interestingly, only for the expression-regulation of the PDS-driven genes SSA3 and GRE1 did our data clearly demonstrate the importance of the CKI function of Pho81. For the regulation of the STRE-driven gene HSP26, the involvement of the PHO pathway was much less obvious as its induction during phosphate starvation seemed to be largely independent of Pho81 while its repression upon phosphate re-supplementation to the starved cells was delayed in strains lacking Pho85 or Pho80. Note that also the expression of the genes encoding trehalase and the different subunits of the trehalose synthase complex is controlled in part via STRE elements, though they do not seem to act as other typical STRE-controlled genes (Winderickx et al. 1996; Zahringer et al. 1998, 2000). We also observed this phenomenon as repression of NTH1, TPS1 and/or TPS2 after the re-addition of phosphate to the starved cells was delayed for strains lacking either PHO85 or PHO80 as well as PHO81. Hence, it is possible that the uncoupling between trehalase activation and trehalose mobilization that we and others (Giots et al. 2003) observed in mutant strains affected in phosphate signalling may due to an overshoot of trehalose synthesis as compared to trehalose degradation during the first hours after phosphate re-supplementation to starved cells.
The effects on expression of the PDS genes appeared to be largely dependent on the Pho85–Pho80 complex. Although Pho4 is to date the best known physiological effector of the Pho85–Pho80 kinase, our data show that this transcription factor is not directly involved in transcriptional control of the PDS-driven genes but exerts its effect via the positive feedback loop towards Pho81 (Ogawa et al. 1995). In consequence, another effector must link the Pho85–Pho80 kinase activity to phosphate-dependent transcription regulation of the PDS-driven genes. The existence of an additional Pho85–Pho80 effector has already been suggested by Flick and Thorner, who noticed that the temperature-sensitive growth defect of a plc1Δ strain (Flick and Thorner 1993) could be suppressed either by overexpression of PHO81, by disruption of the PHO80 gene or by growing the cells in a low-phosphate environment (Flick and Thorner 1998). Since these suppressive effects were independent of the Pho4 transcription factor, it was concluded that the Pho85–Pho80 kinase inhibits a target which is also downstream of Plc1 and which is required for the proper response to nutrient levels and is involved in growth at elevated temperatures (Flick and Thorner 1998). Further support for the involvement of an additional Pho85–Pho80 effector comes from the data obtained with expression of truncated PHO81 alleles. Regulation of the activity of the Pho4 transcription factor in response to phosphate levels is known to be accomplished through Pho81-mediated regulation of Pho85–Pho80 kinase activity, for which a region of Pho81 containing only 80 amino acids was shown to be both necessary and sufficient (Schneider et al. 1994; Ogawa et al. 1995; Huang et al. 2001). In our studies, however, this Pho81 minimum domain is not sufficient for proper regulation of the PDS-driven genes in response to phosphate availability and thus other domains of Pho81 must be involved. Intriguingly, these domains must then be able to discriminate between the different effectors of the Pho85–Pho80 complex.
Finally, it should be mentioned that induction of the PDS genes in a pho81Δ strain is not totally abolished, but only severely impaired in response to phosphate depletion. Therefore, these genes are subject to additional control allowing the bypass of the defect in Pho81-dependent inhibition of the Pho85–Pho80 kinase. Since this control becomes apparent only after prolonged starvation of the cells, it may be mediated by the lack of metabolic signals when cells are severely starved for phosphate, for instance depletion of ATP. Thus, yeast cells may have a biphasic response to phosphate starvation. In a first phase, the cells rapidly adapt to the lack of phosphate through activation of the PDS-driven genes via the PHO pathway. In a second, slower phase, the lack of metabolic signals generated after prolonged phosphate starvation triggers a stress signal, leading to induction of the PDS genes independent of the PHO pathway. Possibly, the second phase may depend on the stress-activated transcription factors Msn2 and/or Msn4, which are known to mediate part of the Rim15-dependent transcriptional responses redundantly to Gis1 (Cameroni et al. 2004).
This work was supported by a fellowship from the Fund of Scientific Research in Flanders (FWO-Vlaanderen) to E.S. and by grants from FWO-Vlaanderen and the research fund of K.U. Leuven