The stationary phase is defined as a metabolically dormant state characterized by turning off genes that are required for the mitotic cell cycle (Werner-Washburne et al., 1993) and turning on genes that are required for survival in stress conditions, including starvation, heat shock and chemical treatment (Bataille et al., 1991; Petko and Lindquist, 1986). The fission yeast enters a stationary phase from either G1 or G2 of the cell cycle, depending on the specific nutrients that are in short supply. Entry into stationary phase occurs from G1 when nitrogen or phosphate is exhausted, while it occurs from G2 in the absence of a carbon source (Bostock, 1970; Costello et al., 1986). Cyclic AMP is a second messenger that detects the availability of a carbon source and plays a critical role in switching between the mitotic cell cycle and the stationary phase (Kawamukai et al., 1991). Disruption of the cAMP pathway causes cell cycle arrest in G1 and subsequent entry into G0 in budding yeast (Iida and Yahara, 1984). This pathway is not essential for cell proliferation in fission yeast, but is involved in the control of differentiation (Maeda et al., 1990). Disruption of the regulatory subunit of protein kinase A (PKA) renders Schizosaccharomyces pombe cells sterile and causes a loss of cell viability upon nitrogen or carbon starvation, due to an inability to enter the stationary phase (DeVoti et al., 1991), suggesting that the activation and function of PKA is required for adaptation of the stationary phase (Hoffman, 2005; Roux et al., 2006). Activation of a MAP kinase (MAPK) pathway is also crucial for responding to external stress (Kyriakis and Avruch, 1996; Ruis and Schuller, 1995). The MAPKK homologue wis1+ gene is essential for cell survival under stress, and the MAPK homologue Spc1/Sty1 is activated by Wis1 in response to osmotic stress and nutrient limitation. The integrity of the Wis1–Sty1 pathway is required for survival under extreme conditions of heat, osmolarity, oxidation or limited nutrition (Kato et al., 1996; Samejima et al., 1997; Shiozaki and Russell, 1995). Sty1 is negatively regulated by tyrosine phosphatases Pyp1 and Pyp2 via the direct dephosphorylation of tyrosine-173 (Degols et al., 1996; Millar et al., 1995; Shiozaki and Russell, 1995). The Wis1–Sty1 cascade is linked to the G2–M cell cycle control. Under optimal growth conditions, sty1– mutants exhibit moderate delay at the onset of mitosis, which is greatly exacerbated upon stress (Shiozaki and Russell, 1995). The transcription factor Atf1, a known substrate for Sty1, is responsible for the Sty1-dependent regulation of gene expression in response to various stresses, although atf1– mutants do not exhibit the mitotic delay phenotype of sty1– cells (Shiozaki and Russell, 1995; Wilkinson et al., 1996). Previously, sds23/psp1/moc1 gene was characterized as one of the Sz. pombe regulators of anaphase-promoting complex and as a phosphoprotein in stationary phase (Ishii et al., 1996; Jang et al., 1997). This gene was also found as to overcome sterility caused by high expression of adenylyl cyclase (Yakura et al., 2006). The phosphorylation of serin-333 by Cdc2-Cdc13 affected the sexual development and exit mitosis and was not involved in maintaining viability in stationary phase (Jang et al., 1997; Yakura et al., 2006). Recently, Hanyu et al. (2009) reported that Sds23/Psp1/Moc1 is a regulator of PP2A-related phosphatase Ppe1 under the limitation of glucose for the cell number increase. In addition to the functions of phosphatases and kinases for adaptation under the stress condition, the degradation of aberrant proteins induced by stress is also an important issue. Ufd2, a multi-ubiquitin chain assembly factor (E4) facilitates the polyubiquitination of the target protein by transferring additional ubiquitin moieties (Koegl et al., 1999). In this study we focused on the multiple phosphorylations in other sites of Sds23/Psp1/Moc1, and their significance in maintaining cell viability in extended culture. Additionally, we show that the multi-phosphorylated form of Sds23/Psp1/Moc1 interacts preferably with Ufd2, suggesting that Sds23/Psp1/Moc1 may be one of the target proteins of the ubiquitin fusion degradation pathway to cope under stressful conditions.
Materials and methods
Strains and media
The strain ED665 h–ade6-M210 leu1-32 ura4-D18 was used as a normal haploid strain. YE-glucose (YE) medium and Edinburgh minimal medium (EMM) were used as described by Gutz et al. (1974). When necessary, 75 µg/ml of each of adenine and uracil or 250 µg/ml of leucine was added to the growth medium as a supplement. For repression or induction of the nmt1 promoter function, we used the same medium as previously described (Jang et al., 1997; Maundrell, 1993). The sds23/psp1/moc1 deletion mutant (sds23/psp1/moc1::Kan h– ade6-M210 leu1-32 ura4-D18) was purchased from Bioneer Corp. (Daejon, Korea). The ufd2 (the systematic name is SPAC20H4.10), pka1, or sty1 deletion mutant (gene::ura4 h–ade6-M210 leu1-32 ura4-D18), and the pka1 and wis1 double deletion mutant (pka1::ura4 wis1::Kan h–ade6-M210 leu1-32 ura4-D18) were cultured in EMM containing adenine and leucine.
Site-directed mutagenesis using PCR methods
The procedures for PCR mutagenesis used in this study have been extensively described in our previous reports (Jang et al., 1997). To change serine residues of Sds23/Psp1/Moc1, GRQ18S, TRS267S and RRG391S into alanines, we performed recombinant PCR with mutated primer sequences: GRQA (m1), 5′-GCTGGACGTCAGGCCTTTGG-3′; TRSA (m2), 5′-TACACGTTCTGCTTCTG-3′; RRGA (m3), 5′-ACGATGGCTGCTTGTGCTTCCT-3′. The sense strand of the amino terminus and the antisense strand of the carboxyl terminus were used as previously described (Jang et al., 1997). Two or three point mutations at Ser18 and 267 and/or Ser391 were constructed by similar recombinant PCR methods, with combinations of the appropriate primers. The changes in the sequences were confirmed by sequencing the amplified fragments. For the production of mutant proteins, the amplified DNAs were cloned into the GST–fusion vector pGEX 4 T-2 (m2, m3, m12, m13 and m123).
Protein kinase assay
To determine whether Sds23/Psp1/Moc1 is a substrate of protein kinase A in vitro, 1–2 µg of the purified wild-type and mutant Sds23/Psp1/Moc1 proteins were mixed with 10 U protein kinase A catalytic subunit (New England Biolabs) and incubated at 30°C for 20 min in the presence of 200 μm ATP and 25 μCi [γ-32P] ATP (Slice and Taylor, 1989). As a standard substrate of protein kinase A, 1–2 µg/ml histone H1 (Boehringer Mannheim) was used (Pearson and Kemp, 1991).
For the kinase assay of Sty1 MAP kinase, GST–sty1 gene fusion construct was generated using pESP1 plasmid (Stratagene) containing the GST fusion construct under the nmt1 promoter. The resulting pESP–sty1 was introduced into Sz. pombe strain ED665. The expression of Sty1 from the nmt1 promoter was induced by incubating the transformant at 30°C for > 50 h in thiamine-deficient medium. Proteins were purified from Sz. pombe cell extracts using agarose beads. The beads were washed three times in the kinase buffer (50 mm Tris–HCl, pH 7.2, 10 mm MgCl2, 0.1 mm EGTA, 0.1 mm sodium orthovanadate, 1 mm DTT). For the in vitro phosphorylation of Sds23/Psp1/Moc1, the bead-purified Sty1 was added as a source of kinase and the suspension was incubated at 30°C for 20 min in kinase buffer supplemented with 25 μCi [γ-32P] ATP. Sds23/Psp1/Moc1 phosphorylated was analysed by SDS–PAGE and autoradiography.
Preparation of yeast extracts, protein binding assay and immunoprecipitation
Yeast extracts were prepared as described by Stone et al. (1993), with modifications. Cells were grown to 2 × 107 cells/ml and harvested. After washing in appropriate buffers, the cells were frozen at –70°C for further study. Two different buffers were used: PB buffer (20 mm HEPES, pH 7.5, 150 mm NaCl, 2 mm MgCl2, 1 mm PMSF) for the protein binding assay; and IP buffer (50 mm Tris–HCl, pH 7.5, 10 mm EDTA, pH 8.0, 5 mm EGTA, pH 7.5, 400 mm NaCl, 1 mmβ-mercaptoethanol, 1 mm PMSF) for immunoprecipitation. Approximately 2 × 108 cells were broken with glass beads. After centrifugation at 14 000 rpm for 20 min, the supernatants were used as the extracts. For immunoprecipitation, yeast extract in IP buffer, as previously described, was mixed with 2–20 µl antisera of Sds23/Psp1/Moc1 protein, and protein A-agarose (25 µl bed volume, Sigma). This mixture was incubated overnight at 4°C, washed four times with IP buffer and then used for further analysis. In vitro protein binding assay was performed as follows. To prepare the protein products of the sds23/psp1/moc1 and ufd2 genes, the coding region DNAs of these genes were cloned into the GST- and/or MBP-fusion vector. Fusion proteins were expressed in an Escherichia coli system in the presence of 0.3 mm IPTG at 37°C. GST- and MBP-fusion proteins from the crude E. coli extract were purified with glutathione– and maltose–agarose beads, respectively, as described previously (Smith and Johnson, 1988). The purified proteins on the beads were then incubated with yeast extracts in PB buffer for 16 h at 4°C. After incubation, the beads were washed four times with the same buffer, separated on SDS–PAGE and analysed by western blot.
Liquid assay for β-galactosidase activity
For the β-galactosidase activity assay, ortho-Nitrophenyl-β-galactoside (ONPG) was used as a substrate. The amount of the released O-nitrophenol was expressed as units, calculated using the equation (Guarente, 1983):
where t denotes the time of incubation and V the volume (ml) of culture added to the Z buffer.
Survival ratio of Sz. pombe cells
Sz. pombe cells were incubated at 30°C on agar plates or in liquid media. Viable cells were determined by counting colony-forming units (CFU) on thiamine-deficient EMM agar plates and YEA, after incubation for several days.
Sds23/Psp1/Moc1 is phosphorylated by stress-activated MAP kinase Sty1 and cAMP-dependent kinase during prolonged culture
When yeast cells face a stressful situation, such as nutritional deprivation, they undergo a transition from the actively growing stage to the dormant stage. Sds23/Psp1/Moc1 is isolated as a suppressor of a mutant of anaphase promoting complex, and is involved in survival and sexual development in fission yeast (Ishii et al., 1996; Jang et al., 1997; Yakura et al., 2006). In actively growing cells, it is largely present as the unphosphorylated form (Figure 1Aa, lane 1 in α-psp1) (Jang et al., 1997). However, it becomes phosphorylated when Sz. pombe cells enter into stationary phase for 50 h incubation in continuous culture (Figure 1Aa, lane 4 in α-psp1). We initially examined the possibility that Sds23/Psp1/Moc1 in stationary phase might be a target of stress-activated MAP kinase, such as Sty1, since this kinase is activated under nutritionally deficient conditions as well as other stress conditions (Degols et al., 1996; Wilkinson et al., 1996). The hyperphosphorylation of this protein diminished in pka1- and sty1-deletion mutants after 50 h culture (Figure 1Aa, lanes 5 and 6 in α-psp1). When Pyp1, one of the inhibitors of Sty1, was overexpressed under a thiamine-repressible nmt1 promoter, unphosphorylation of Sds23/Psp1/Moc1 was increased in stationary phase (Figure 1Ab, lane 3 in α-psp1). For in vitro kinase assay, Sty1 protein fused to GST was expressed ectopically and then purified from cells in stationary phase (Figure 1Ba, lane 2 in upper panel). Endogenous Sds23/Psp1/Moc1 was co-precipitated with GST–Sty1, as detected by immunoblotting (Figure 1Bb, lane 2 in upper panel). This result indicated that Sty1 kinase physically interacts with Sds23/Psp1/Moc1 in vivo. Moreover, a protein band corresponding to Sds23/Psp1/Moc1 phosphorylated was observed when the purified GST–Sty1 beads were incubated with only [γ-32P] ATP (Figure 1Bb, lane 2 in lower panel). In in vitro kinase assay, GST–Sds23/Psp1/Moc1 was efficiently phosphorylated by Sty1 kinase (Figure 1Bc, lane 2 in 32P) as well as Atf1, a known substrate of Sty1 kinase (Figure 1Bc, lane 3 in 32P). In addition to the Sty1 signalling pathway, cAMP pathway elements, including protein kinase A (PKA), play roles in cells adapting to stationary phase (Hoffman, 2005; Roux et al., 2006). Previously, we found that Pka1 interacted with Sds23/Psp1/Moc1 in a yeast two-hybrid (data not shown). Indeed, endogenous Sds23/Psp1/Moc1 interacted with GST–Pka1 and was detected by immunoblot in a protein pull-down assay (Figure 1Ca, lane 2 in lower panel). When PKA catalytic subunit was incubated with the GST–Sds23/Psp1/Moc1 protein in vitro, Sds23/Psp1/Moc1 was phosphorylated on the same level with histone H1, which is a known substrate of PKA (Pearson and Kemp, 1991) (Figure 1Cb, lanes 2 and 3 in 32P).
Serine residues are phosphorylated by PKA and/or Sty1 in vivo and in vitro
We performed a phosphoamino acid analysis of the phosphorylation bands in gels from previous kinase assays (Figure 1, lanes 2 in C, D). Both PKA and Sty1 kinase phosphorylated Sds23/Psp1/Moc1 in vitro on serine residue (Figure 2Aa, b). For determination of the phosphorylation site(s), deletion or point mutations at the potential serine sites were introduced in sds23/psp1/moc1 gene, as shown in Figure 2B. Because several putative phosphorylation sites for MAP kinase and PKA are located in the coding region of Sds23/Psp1/Moc1 (RQ18S, RS267S and RG391S) (Kennelly and Krebs, 1991; Pearson and Kemp, 1991), we changed each serine residue at positions 18, 267 and 391 into alanine (m1, m2 and m3), and all mutant constructs were expressed and purified from E. coli system and used in in vitro kinase assays (Figure 2Ca). When the N-terminus (NW) or the C-terminus (CW) of Sds23/Psp1/Moc1 was subjected in Sty1 kinase assay, the C-terminal region was phosphorylated (Figure 2Cb, lane 3). Interestingly, when serine-391 was changed into alanine, the signal phosphorylated by Sty1 totally disappeared (Figure 2Cb, lanes 5 and 7). We then examined the phosphorylation of the mutant constructs of Sds23/Psp1/Moc1 by PKA catalytic subunit in vitro. N- and C-terminal regions and the mutant constructs m2, m3 and m12 were phosphorylated (Figure 2Cc, lanes 2–6). However, Sds23/Psp1/Moc1 mutated at both Ser-18 and Ser-391 (m13) was not phosphorylated, suggested that serine-18 and serine-391 are the phosphorylation sites for PKA (Figure 2Cc, lane 7), and that Ser-391 might be the site phosphorylated by Sty1 and PKA kinases. For evidence of phosphorylation of these sites in vivo, we introduced a mutation on serine-333 in both m13 and wild-type (wt) constructs. Serine-333 was previously reported as the phosphorylation site of Sds23/Psp1/Moc1 by Cdc2 kinase (Jang et al., 1997). Indeed, in vivo phosphor-labelling experiments indicated that both serine-18 and serine-391 are critical for Sds23/Psp1/Moc1 phosphorylation in stationary phase. Whereas Sds23/Psp1/Moc1 containing S333A overexpressed was highly phosphorylated in stationary phase (Figure 2D, lane 4), the Sds23/Psp1/Moc1 mutated at serine-18, serine-391 and serine-333 into alanine was not phosphorylated and showed a similar phosphorylation intensity with endogenous Sds23/Psp1/Moc1 (Figure 2Da, lanes 2 and 3). These data suggest that serine-18 and serine-391 are additional phosphorylation sites of Sds23/Psp1/Moc1 in stationary phase.
Phosphorylation of Sds23/Psp1/Moc1 is required for maintaining cell viability during stationary phase
To examine the physiological role of Sds23/Psp1/Moc1 phosphorylation in stationary phase, we analysed the growth rate and viability of cells expressing various constructs of Sds23/Psp1/Moc1 under control of the nmt1 promoter. The constructs of pnmt1-psp1 (W) and pnmt1-mutant psp1 (m1, m3 and m13) were introduced into a haploid strain, ED665, and the growth rate of cells were measured at indicated time points during culture in thiamine-deficient medium. As cells entered a stationary phase under the thiamine-deficient condition, the growth rate was decreased (Figure 3A). Under this culture condition, cells were harvested at 48, 72 and 100 h after induction (Figure 3Aa–c, Ba–c), washed and cultured continuously in fresh medium. Whereas cell viability began to drop after 60 h in control haploid cells (Figure 3B, filled circle), most of the cells expressing wild-type Sds23/Psp1/Moc1 stayed alive even after 100 h of incubation (Figure 3B, open triangle and point 1). However, cells containing mutant Sds23/Psp1/Moc1 showed a dramatic decrease in viability. More than 70% of cells were defective after 100 h of incubation (Figure 3B, filled triangle and point 3). Generally, when normal haploid cells were grown for ~110 h, they became small and exhibited a resistance to heat-shock (data not shown), which are the characteristics of the G0 resting stage. As cells expressing wild-type Sds23/Psp1/Moc1 became short and round in cellular morphology (Figure 3C, 1). However, cells containing mutant Psp1 showed numerous irregular shaped and dead cells after 110 h or longer of incubation (Figure 3C, 3). These results indicated that as the unphosphorylated form of Sds23/Psp1/Moc1 accumulates during stationary phase, cell viability drops dramatically and overexpression of the mutant Sds23/Psp1/Moc1 might inhibit normal Sds23/Psp1/Moc1 function for keeping cells alive in prolonged culture. Indeed, the viability of deletion mutant of sds23/psp1/moc1 was dramatically decreased after prolonged culture for 70–100 h (Figure 3Da, Δpsp1). When wild-type Psp1 was expressed under the pnmt1 system, the lethality of deletion mutant in stationary phase was recovered (Figure 3Db, –thia, wt). However, expression of phospho-mutant Psp1 (m13) was not particularly effective for recovery (Figure 3Db, –thia, m13), suggested that the phosphorylations are required for Psp1 function to maintain cell viability during stationary phase.
Both Pka1 and Sty1 are required for cell survival when phosphorylation-defective mutant of Sds23/Psp1/Moc1 is overexpressed in prolonged stationary phase
To find the role of Pka1 and Sty1 kinases in Sds23/Psp1/Moc1 function, we examined the functional complementation effect of Sds23/Psp1/Moc1 in pka1- or sty1-deleted cells and analysed the cell viability of these cells in stationary phase. When wild-type or mutant Sds23/Psp1/Moc1 was expressed in the haploid ED665 strain, growth rates in log phase were similar in both cells, and cell numbers reached OD600 = 7.0 in 40 h induction (Figure 4Aa, triangle and rectangle) as shown in Figure 3. In pka1-deleted cells, we did not observe a significant change among their growth rates in comparison with those of normal haploid cells (Figure 4Ab). However, in the sty1– mutant and the pka1– wis1– double-deletion mutant, cells grew slowly and cell number reached only < OD600 = 5 in 35 h induction (Figure 4Ac, d, circle). When mutant Sds23/Psp1/Moc1 was overexpressed in the sty1– and the pka1– wis1– cells, the growth inhibition effect became more severe than that in control cells, and cell numbers reached OD600 = 3.1 and OD600 = 1.6, respectively (Figure 4Ac, d, rectangle). For investigation of viabilities, cells were collected after 40 h incubation under thiamine-deficient induction, and their morphologies (Figure 4Ba) and survival ratio (Figure 4Bb, c) were examined. More than 80% of normal haploid cells containing wild-type and mutant Sds23/Psp1/Moc1 harvested after 40 h incubation survived as expected (Figures 3B and 4Ba, ED665). However, in pka1-deleted cells, < 30% of cells expressing the mutant survived (Figure 4B and C, panel 5 and bar graph 3 in Δpka1), whereas cells containing wild-type protein were less defective (Figure 4B, panel 4 in b and bar graph 2 in c in Δpka1). In, about 50% of the sty1-deleted cells expressing the mutant were survived under the same condition (Figure 4B, panel 5 in b and bar graph 3 in c in Δsty1). 42% and 15% of the pka1– wis1– cells expressing wild-type and mutant Sds23/Psp1/Moc1 survived, respectively (Figure 4B, bar graphs 2 and 3 in c in Δpka1/Δwis1) and were highly elongated and branched (Figure 4B, panel 3 in a in Δpka1/Δwis1). In all cases, overexpression of wild-type Sds23/Psp1/Moc1 was not affected at the basal level of survival of each strain in stationary phase (Figure 4B, bar graphs 1 and 2 in c).
Ufd2, multi-ubiquitin chain assembly factor E4, is identified as an interacting protein with Sds23/Psp1/Moc1 by yeast two-hybrid screening
To identify cellular factors that may function along with Sds23/Psp1/Moc1 for cell survival under the nutrient-deficiency stress condition, we performed yeast two-hybrid screening using the sds23/psp1/moc1 constructs fused to the GAL4 DNA-binding domain gene and a Sz. pombe cDNA library fused to the GAL4-activating domain gene. One of the positive clones with strong binding affinity with Sds23/Psp1/Moc1, as indicated in Figure 5A, was a homologue of the ubiquitin-fusion degradation protein-2 (UFD2) of S. cerevisiae (Johnson et al., 1995). Using this fragment, we screened a Sz. pombe cDNA library and obtained a full-length cDNA encoding a protein having sequence homology with UFD2 homologues in many species, human (Hatakeyama et al., 2001), Caenorhabditis elegans and NOSA of D. discoideum (Pukatzki et al., 1998) (Figure 5B). The full-length DNA of the 2793 base pairs was originally registered in GenBank as ufd2 (Accession No. AF059906). A highly conserved motif, U-box (UFD2-homology domain) resided in the C-terminal region (amino acids 848–918; Figure 5B). The original yeast two hybrid clone, Ufd2C (amino acids 704–931 of full-length Ufd2), contained this conserved U-box. The UFD2 homologues in many species have been grouped as a family of ubiquitin–protein ligases, which are involved in the assembly of the multi-ubiquitin chain (Hatakeyama et al., 2001; Koegl et al., 1999). We performed an in vitro binding experiment using GST–Sds23/Psp1/Moc1 and MBP-Ufd2C protein prepared in the E. coli system. In this pull-down experiment, GST–Sds23/Psp1/Moc1 strongly interacted with the C-terminus of Ufd2 (amino acids 704–931; Figure 5C, lane 2 in lower panel). The MBP-Ufd2C protein also bound to GST–Sds23/Psp1/Moc1 (Figure 5C, lane 4 in lower panel). To determine whether Sds23/Psp1/Moc1 interacted with Ufd2 in vivo, GST fusion constructs of the C-terminus and full-length Ufd2 were expressed in Sz. pombe cells under thiamine-deficient conditions, and were purified from cells grown in log or stationary phase. The purified proteins, GST–Ufd2C and GST–Ufd2, indicated 55 and 130 kDa proteins, respectively (Figure 5D, upper panel, lanes 3–6). Endogenous Sds23/Psp1/Moc1 interacted with both C-terminus and full-length Ufd2 proteins in stationary phase (Figure 5D, lower panel, lanes 4 and 6). However, they did not interact in actively growing cells of log phase (Figure 5D, lower panel, lanes 3 and 5). When the N-terminus of Ufd2 was overexpressed in cells, endogenous Psp1 bound were not detected even in stationary phase (data not shown). Interestingly, endogenous Sds23/Psp1/Moc1 interacted with full-length Ufd2 proteins in stationary phase was detected as a degraded form (Figure 5D, lower panel, lane 6). These data indicated that the C-terminal region (U-box) of Ufd2 is necessary for binding with Sds23/Psp1/Moc1, that the binding most likely occurred during stationary phase when the phosphorylated form of Psp1 became dominant, and that the interaction with Ufd2 may induce the degradation of phospho-Psp1.
Phosphorylation of Psp1 may recruit Ufd2 to form a complex in prolonged stationary phase
Since the association between Sds23/Psp1/Moc1 and Ufd2 did not occur in log phase (Figure 5), we examined whether the interaction between endogenous Sds23/Psp1/Moc1 and Ufd2 in stationary phase is induced by the phosphorylation of Sds23/Psp1/Moc1. Interaction between phosphorylation-deficient mutant Sds23/Psp1/Moc1 and Ufd2 were weaker than that between wild-type Sds23/Psp1/Moc1 and Ufd2 in yeast two-hybrid screening (Figure 6A, pGAD-Ufd2/pGBT-wt and pGAD-Ufd2/pGBT-m13). In addition to the yeast two-hybrid assay, cell extracts from cells expressing wild-type or mutant Sds23/Psp1/Moc1 in log or stationary phase were subjected to in vitro binding experiment with MBP-Ufd2 protein. Wild-type or mutant Sds23/Psp1/Moc1 in cell extracts from log phase did not bind to Ufd2 (Figure 6B, lanes 1 and 2). However, wild-type Sds23/Psp1/Moc1 in stationary cells strongly interacted with Ufd2 (Figure 6B, lane 3), whereas the mutant Sds23/Psp1/Moc1 did not interact with Ufd2 (Figure 6B, lane 4). These data indicate that the phosphorylated form of Sds23/Psp1/Moc1 prefers the interaction with Ufd2 in stationary phase.
Absence of Ufd2 in Sz. pombe maintains cell survival in prolonged stationary phase
To understand the functional relation between Sds23/Psp1/Moc1 and Ufd2, we first investigated the phenotype of ufd2 deletion on cell growth and viability. For deletion, the ufd2 gene in haploid cells was replaced with the ura4 gene by the one-step gene disruption method. The ufd2-deleted cells showed no detectable defects in growth rate and reached stationary phase within 50 h of incubation (Figure 7A, closed circle). However, their survival ratio increased in prolonged stationary phase more than that of normal haploid cells (Figure 7B, filled and open circles). Moreover, most of the cells survived even after 120 h of incubation, whereas only 50% of normal haploid cells survived (Figure 7B, open circle). When C-terminus and full-length Ufd2 were overexpressed in this ufd2-deleted cells, this increment of viability in the ufd2-deleted cells diminished (Figure 7B, open and filled triangles). The viability was maintained in the ufd2-deleted cells overexpressing wild-type Sds23/Psp1/Moc1, and the severe defect was relieved in overexpression of mutant Sds23/Psp1/Moc1 (Figure 7B, open and filled squares), indicating that the Ufd2-related pathway induces normal cell death under stress conditions and the amount of phospho-Sds23/Psp1/Moc1 in stationary phase is important for maintaining cell survival. In addition to the ufd2-deletion, we investigated the survival effect under the overexpression of these genes in prolonged stationary phase. Cells overexpressing wild-type and phosphorylation-defective mutant Sds23/Psp1/Moc1 survived in 52% and 24% of total cells after 120 h, respectively (Figures 3 and 7D, filled and open triangles). Although there was an observational error, cells overexpressing Ufd2 tended to be more defective than normal haploid cells (Figure 7D, open circles). The defective effect on cells overexpressing Ufd2 was recovered by co-expression of wild-type Sds23/Psp1/Moc1 (Figure 7D, filled rectangles), but not by co-expression of mutant Sds23/Psp1/Moc1 (Figure 7D, open rectangles).
The viability of cells under stress conditions such as heat, osmotic shock or nutritional deprivation depends on how rapidly the cells move their systems to cope with these situations. The ‘decision’ to undergo continuous proliferation through cell cycles or to enter the dormant stage will rely on the activation of the elements responsible for sensing the signals. Several cell cycle regulators or stress responsive elements are modified by a cascade of kinase/phosphatases or by ubiquitin-activating complex enzymes to exert the appropriate functions during this process. When a stress signal is sensed, protein modification by phosphorylation/dephosphorylation or ubiquitination play critical roles in determining the fate of cells, as to whether to remain in the active proliferate cell cycle, to enter the dormant stage where the cell proliferation stops or to prepare for proper death (Laney and Hochstrasser, 1999; Nash et al., 2001).
The sds23/psp1/moc1 gene of the fission yeast encodes a protein which is multi-phosphorylated during stationary phase (Jang et al., 1997). This protein is also required for destruction of mitotic cyclin and sister chromatid separation (Ishii et al., 1996), and is involved in sexual development (Yakura et al., 2006). In this report, we showed that both stress-activated Sty1 MAP kinase and PKA phosphorylate Sds23/Psp1/Moc1 in vitro and in vivo. The phosphorylation of Sds23/Psp1/Moc1 by Sty1 kinase suggested that Psp1 is a target in stress conditions and/or plays a role in coping with stress conditions such as nutrient starvation in prolonged stationary phase. Generally, when Sz. pombe cells are under stress conditions, such as DNA damage, heat or osmotic shock or nutrient deprivation, the Wis1–Sty1 MAP kinase pathway is activated to alter the ongoing cell cycle and prevent cells from progressing further (Warbrick and Fantes, 1991). Activation of this pathway is essential for establishing a G0-like resting state and for maintaining the dormant state under nutritional starvation. Therefore Sds23/Psp1/Moc1 is likely to be a direct downstream target of Sty1 kinase in altering cell cycle progression under the nutritionally depleted stationary phase. Interestingly, the overexpression of the phosphorylation-deficient mutant Sds23/Psp1/Moc1 and the deletion of Sds23/Psp1/Moc1 showed the similar phenotype; cell viability decreased rapidly during stationary phase (Figure 3) (Yakura et al., 2006). These data indicated that a proper function of Sds23/Psp1/Moc1 is required for maintaining cell viability during the dormant stage. These data reflected the fact that cells lacking the downstream component of the Sty1–MAP kinase pathway quickly lose viability after entering stationary phase (Takeda et al., 1995; Warbrick and Fantes, 1991). Therefore, it is possible that when a nutritional deficient signal is sensed by cells, Sds23/Psp1/Moc1 is phosphorylated by proper protein kinases to enter the stationary phase, thereby protecting cells against catastrophic cell death for some time. Several different branches of the regulatory circuit, such as cAMP-dependent protein kinase and the Wis1–Sty1 kinase pathway, are likely to be involved in the phosphorylation of Sds23/Psp1/Moc1. For proper death, systematized pathways, such as ubiquitination and proteolysis, are used. In Figures 5 and 6, Sds23/Psp1/Moc1 interacted with Ufd2, a novel polyubiquitination factor E4 of Sz. pombe, in a phosphorylation-specific manner, indicating that Sds23/Psp1/Moc1 may be implicated in the ubiquitin-dependent protein degradation pathway in stress conditions. The deletion of Ufd2 increased cell viability in prolonged stationary phase in comparison with that of normal haploid cells, and suppressed a defective phenotype of overexpression of mutant Sds23/Psp1/Moc1 (Figure 7B). However, some similarity in the decrement of cell viability was in between overexpression of Ufd2 and normal cells during stationary phase, and overexpression of Udf2 did not seem to have an effect on overexpression of Sds23/Psp1/Moc1s (Figure 7D). Although it is very difficult to draw conclusions about direct functional relationships in these indirect genetic experiments, we suggest that the Ufd2-related ubiquitination pathway may be essential for proper cell death under prolonged nutrient deprivation, and that Sds23/Psp1/Moc1 can be a negative regulator of Udf2 to keep cell viability for maintaining the dormant state. However, on the other hand, we cannot exclude the possibility that each protein has many independent functions for regulating viability in prolonged culture, and that the overexpression of one binding partner sequesters the other binding partner from interfering with its normal functions, regardless of whether these proteins have a regulatory interaction. Additionally, in Figure 5D, endogenous Sds23/Psp1/Moc1 was destructive by increasing the amount of full-length Ufd2, indicating that Sds23/Psp1/Moc1 may be one of the target proteins of the ubiquitination pathway. In this situation, wild-type Sds23/Psp1/Moc1 is a factor for maintaining cell survival for some time under the stress condition, and the phosphorylations of Sds23/Psp1/Moc1 are important for this function. In spite of all these speculations, it is certain that the association between Ufd2 and Sds23/Psp1/Moc1 occurred only in stationary phase, not in the actively growing stage, and the phosphorylation of Sds23/Psp1/Moc1 was essential for this interaction. Sty1 and PKA activity in stationary phase is essential for interaction between Sds23/Psp1/Moc1 and Ufd2. Since UFD2 is implicated in cell survival under stress conditions in budding yeast and is associated with CDC48, an AAA-type ATPase that is thought to possess protein-folding activity (Koegl et al., 1999), it is likely that selective proteolysis induces cell death or protects other cells against the progression to energy-consuming continuous proliferation, when nutrition is depleted. Recently, Paul et al. (2009) reported that UFD2 interacts with the proteins known as inducers of sexual differentiation, such as Sds23/Psp1/Moc1, Moc3 and Moc4, indicating that Ufd2 might be involved in the sexual differentiation pathway in a large complex mediated by Moc proteins, as well as in survival for G0 cells. Although highly conserved UFD2 homologues in other organisms are known, their functions and targets under stress condition have not been extensively characterized. Again, in the absence of Ufd2, the ‘decision’ to live or die during stationary phase is not regulated properly, and cells may alive and well abnormally even in prolonged deprivation of nutrients (Figure 7B). The phosphorylation of Sds23/Psp1/Moc1 at the transition point between the actively growing and stationary phases seems to be important for targeting the ubiquitin-fusion degradation (UFD) pathway for proper death under nutrient-deficient conditions or for maintaining cell survival during the dormant stage. Although the functional relationship among Sds23/Psp1/Moc1, Ufd2 and other components of the anaphase promoting complex (APC) should be further studied, we propose the following model for Sds23/Psp1/Moc1 and Ufd2 functions under the nutritionally stressed condition, based on previous reports and our data (Figure 8). During cell cycle progression in the actively growing stage, Sds23/Psp1/Moc1 acts as a regulator of the anaphase-promoting complex and Dis2/Sds22 phosphatase (Ishii et al., 1996; Yanagida et al., 2011). When Sz. pombe cells sense nutritional deficiency, Cdc2/Cyclin B kinase, one of the key cell cycle regulators in the starvation response, acts to appropriately change the cell size and shape (Sajiki et al., 2009; Yanagida et al., 2011), phosphorylates Sds23/Psp1/Moc1 at first (Jang et al., 1997) and then Sds23/Psp1/Moc1 is further phosphorylated by PKA and/or Sty1 kinases, which are stress-responsive kinases (Figure 1) (Sajiki et al., 2009; Yanagida et al., 2011) to enter into stationary phase. The fully modified Sds23/Psp1/Moc1 interacts with Ufd2 and is possibly destroyed through the ubiquitin-fusion degradation pathway, which is a proper pathway to remove aberrant and unnecessary proteins in stationary phase, and then cells are finally defective in prolonged culture. If Sds23/Psp1/Moc1 remains or Ufd2 pathway is blocked in stationary phase, the cells can survive and enter a dormant stage until the nutrient supply is restored. In spite of these speculations, under the conditions we used in this study, cells contained both wild-type and mutant Sds23/Psp1/Moc1, so that we only observed a competitive situation of wild-type and mutant. For a clearer explanation of the function of hyperphosphorylation, it will be necessary to create a strain that only expresses mutant psp1. A deletion of the UFD2 human homologue causes neuroblastoma tumours, suggesting that it acts in the transition phase between cell proliferation and differentiation (Krona et al., 2003). Homologues of Sds23/Psp1/Moc1 in budding yeast are known as SDS23 and SDS24 (Goldar et al., 2005), but a mammalian homologue is not yet known. To identify a functional homologue of Sds23/Psp1/Moc1 and further downstream target proteins, it will be necessary to understand the UFD pathway in the cell proliferation–differentiation junction under specific conditions.
We thank M. Yanagida at Kyoto University in Japan for discussion at the beginning of this study. This study was supported by a Dankook Research Grant (2012).