Sterol regulatory element-binding protein (SREBP), a highly conserved family of membrane-bound transcription factors, is an essential regulator for cellular cholesterol and lipid homeostasis in mammalian cells. Sre1, the homolog of SREBP in the fission yeast Schizosaccharomyces pombe (S. pombe), regulates genes involved in the transcriptional responses to low sterol as well as low oxygen. Previous study reported that casein kinase 1 family member Hhp2 phosphorylated the Sre1 N-terminal transcriptional factor domain (Sre1N) and accelerated Sre1N degradation, and other kinases might exist for regulating the Sre1 function. To gain insight into the mechanisms underlying the Sre1 activity and to identify additional kinases involved in regulation of Sre1 function, we developed a luciferase reporter system to monitor the Sre1 activity through its binding site called SRE2 in living yeast cells. Here we showed that both ergosterol biosynthesis inhibitors and hypoxia-mimic CoCl2 caused a dose-dependent increase in the Sre1 transcription activity, concurrently, these induced transcription activities were almost abolished in Δsre1 cells. Surprisingly, either AMPKα Subunit Ssp2 deletion or Glycogen Synthase Kinases Gsk3/Gsk31 double deletion significantly suppressed ergosterol biosynthesis inhibitors- or CoCl2-induced Sre1 activity. Notably, the Δssp2Δgsk3Δgsk31 mutant showed further decreased Sre1 activity when compared with their single or double deletion. Consistently, the Δssp2Δgsk3Δgsk31 mutant showed more marked temperature sensitivity than any of their single or double deletion. Moreover, the fluorescence of GFP-Sre1N localized at the nucleus in wild-type cells, but significantly weaker nuclear fluorescence of GFP-Sre1N was observed in Δssp2, Δgsk3Δgsk31, Δssp2Δgsk3, Δssp2Δgsk31 or Δssp2Δgsk3Δgsk31 cells. On the other hand, the immunoblot showed a dramatic decrease in GST-Sre1N levels in the Δgsk3Δgsk31 or the Δssp2Δgsk3Δgsk31 cells but not in the Δssp2 cells. Altogether, our findings suggest that Gsk3/Gsk31 may regulate Sre1N degradation, while Ssp2 may regulate not only the degradation of Sre1N but also its translocation to the nucleus.
Sterol regulatory element-binding protein (SREBP), a highly conserved family of membrane-bound transcription factors, is an essential regulator for cellular cholesterol and lipid homeostasis in mammalian cells. Sre1, the homolog of SREBP in the fission yeastSchizosaccharomyces pombe (S. pombe), regulates genes involved in the transcriptional responses to low sterol as well as low oxygen. Previous study reported that casein kinase 1 family member Hhp2 phosphorylated the Sre1 N-terminal transcriptional factor domain (Sre1N) and accelerated Sre1N degradation, and other kinases might exist for regulating the Sre1 function. To gain insight into the mechanisms underlying the Sre1 activity and to identify additional kinases involved in regulation of Sre1 function, we developed a luciferase reporter system to monitor the Sre1 activity through its binding site called SRE2 in living yeast cells. Here we showed that both ergosterol biosynthesis inhibitors and hypoxia-mimic CoCl2 caused a dose-dependent increase in the Sre1 transcription activity, concurrently, these induced transcription activities were almost abolished in Δsre1 cells. Surprisingly, either AMPKα Subunit Ssp2 deletion or Glycogen Synthase Kinases Gsk3/Gsk31 double deletion significantly suppressed ergosterol biosynthesis inhibitors- or CoCl2-induced Sre1 activity. Notably, the Δssp2Δgsk3Δgsk31 mutant showed further decreased Sre1 activity when compared with their single or double deletion. Consistently, the Δssp2Δgsk3Δgsk31 mutant showed more marked temperature sensitivity than any of their single or double deletion. Moreover, the fluorescence of GFP-Sre1N localized at the nucleus in wild-type cells, but significantly weaker nuclear fluorescence of GFP-Sre1N was observed in Δssp2, Δgsk3Δgsk31, Δssp2Δgsk3, Δssp2Δgsk31 or Δssp2Δgsk3Δgsk31 cells. On the other hand, the immunoblot showed a dramatic decrease in GST-Sre1N levels in the Δgsk3Δgsk31 or the Δssp2Δgsk3Δgsk31 cells but not in the Δssp2 cells. Altogether, our findings suggest that Gsk3/Gsk31 may regulate Sre1N degradation, while Ssp2 may regulate not only the degradation of Sre1N but also its translocation to the nucleus.
Sterol homeostasis is essential for eukaryotic cells to maintain the normal structure and fluidity of cell membrane as well as to regulate the function of membrane proteins and sterol synthesis. Sterol regulatory element binding protein (SREBP), a subfamily of basic helix-loop-helix leucine zipper (bHLH-LZ) transcription factors that are widely conserved from fungi to human, is an important factor that regulates sterol levels in cells [1, 2]. SREBP, as a non-activated precursor protein synthesized in the endoplasmic reticulum (ER), consists of an N-terminal transcription factor domain and a C-terminal domain that forms a complex with a sterol sensing protein, SREBP cleavage activating protein (Scap) [3]. Under sterol replete conditions, Scap binds cholesterol, and the SREBP-Scap complex is retained in the ER [4]. Upon sterol depletion, Scap undergoes a conformational change and SREBP-Scap enters COPII vesicles for transport to the Golgi [5, 6], and then SREBP is cleaved sequentially in the Golgi, resulting in the release of the SREBP transcription factor domain, which then translocate into the nucleus and bind to the specific DNA sequence (sterol regulatory element, SRE) of the target genes involved in sterol synthesis [7].In fission yeast, Sre1, the homologue of mammalianSREBP, is not only a factor for controlling sterol homeostasis but also a principal regulator of low oxygen gene expression [2, 8]. It has been reported that, upon conditions of low oxygen, ergosterol biosynthesis decreases, and Scp1, the homologue of Scap, transports Sre1 from the ER to the Golgi where Sre1 is proteolytically cleaved, releasing the active Sre1 N-terminal transcription factor fragment (Sre1N). Under low oxygen or low sterols conditions, the Sre1-Scp1 transports and cleavages at the Golgi increase dramatically. After released, Sre1N enters the nucleus and promotes transcription of the target genes involved in sterol synthesis as well as oxygen responsive genes. Upon reintroduction of oxygen or sterol, Sre1N degradation is accelerated through a proteasome-dependent pathway, allowing rapid down-regulation of Sre1N [9, 10]. Factors controlling Sre1 cleavage and activation have been largely studied [11, 12], but the mechanism underlying Sre1 degradation remains not fully understand.The initial characterization of the fission yeastSREBP pathway revealed that the activated transcription factor Sre1N could be hyper-phosphorylated, indicating potential regulation of sterol homeostasis by kinases [8]. Recent studies have shown that a highly conserved casein kinase 1 family member Hhp2 phosphorylates Sre1N and accelerates Sre1N degradation, but it seems like that Hhp2 is not the sole Sre1N kinase. Sre1N contains at least 22 phosphorylated serine and threonine residues [13], suggesting that additional kinases might be involved in Sre1 activity regulation.Here, our studies focus on whether additional protein kinases are involved in regulation of Sre1 activity. We monitored the transcriptional activity of Sre1 in living cells by using a luciferase reporter system with three tandem repeats of SRE2 fused to firefly luciferase gene. We found that ergosterol biosynthesis inhibitors- or hypoxia-mimic CoCl2-induced Sre1 transcriptional activity was significantly suppressed in AMPKα Subunit Ssp2 deletion or Glycogen Synthase Kinases Gsk3/Gsk31 double deletion as well as double deletion of Ssp2 and Gsk3 or Gsk31, respectively. In particular, the nuclear fluorescence of GFP-Sre1N were dramatically reduced in these deletion cells. In addition, the immunoblot showed a dramatic decrease in Sre1N levels in the Δgsk3Δgsk31 or Δssp2Δgsk3Δgsk31 cells but not in the Δssp2, Δssp2Δgsk3 or Δssp2Δgsk31 cells. Our findings reveal the involvement of AMPKα Subunit Ssp2 and Glycogen Synthase Kinases Gsk3/31 in regulation of SREBP activity in fission yeast, which may pave a way for further studying similar mechanisms in higher eukaryotes.
Materials and methods
Strains, media, and genetic and molecular biology methods
S. pombe strains used in this study are listed in Table 1. The normal minimal medium EMM (Edinburgh minimal medium), the complete medium yeast extract-peptone-dextrose (YPD) and the rich yeast extract with supplements (YES) have been described previously [14]. Standard genetic and recombinant-DNA methods [15] were used except where noted. Gene disruptions are denoted by lower-case letters representing the disrupted gene followed by two colons and the wild type gene marker used for disruption (for example, sre1::ura4+). Gene disruptions are abbreviated by the gene preceded by Δ (for example, Δsre1). Proteins are denoted by Roman letters and only the first letter is capitalized (for example, Sre1) [16].
A multicopy plasmid (pKB7665) containing the nmt1 promoter without its cis element, three tandem repeats of SRE2-like sequence (ATCACCCCAT) which is the binding core of the Sre1 transcriptional activator identified in the sre1 promoter, and the destabilized luciferase from pGL3 (R2.2) version containing PEST, CL1, and AU-rich repeats was constructed as described previously [17], except that the CDRE oligonucleotides were replaced by the SRE2-like oligonucleotides (sense, 5’-GGC TT; antisense, 5’-TCG AGT GCA T, SRE2-like sequence underlined). Then, an integration 3×SRE2::luc (R2.2) plasmid was constructed by inserting the SRE2-like oligonucleotides and arg1+ into pBC SK(+) (Stratagene) to give pSY291 [18]. The multicopy plasmid and integration plasmid of 3×SRE2::luc (R2.2) were all used for real-time monitoring assay of Sre1-mediated transcriptional activity.The truncated fragment of sre1 gene, encoding the active N-terminal transcription factor domain of Sre1 (Sre1N) was amplified by PCR with the genomic DNA of S. pombe as a template. The sense primer used for PCR was 5’-CGC (BamHI site are underlined), and the antisense primer was 5’-ACG C (SalI site are underlined). The amplified product was digested with BamHI/SalI, and the resulting fragment was subcloned into Bluescript SK (+) (Stratagene, USA). To assess the subcellular localization and the total protein levels of Sre1N, the truncated fragment of sre1+ gene which encoding N-terminal transcriptional factor domain was ligated to the C terminus of the GFP or GST expressing GFP-Sre1N or GST-Sre1N and subcloned into the pREP1 expression vector containing a thiamine-repressible nmt1 promoter [19]. Expression was repressed by the addition of 4 μM thiamine to EMM.
Real-time monitoring assay of Sre1-mediated transcriptional activity
The multicopy 3×SRE2::luc (R2.2) reporter plasmid pKB7665 was transformed into fission yeast wide-type cells and mutations to perform the luciferase reporter assays as described previously [20]. In addition, to obtain the chromosome-borne 3×SRE2::luc (R2.2), the integration reporter plasmid linearized with StuI was integrated into the chromosome at the arg1+ locus of both KP2101 (h
leu1-32 arg1) and CM156 (h
leu1-32 ura4-D18 arg1 sre1::ura4) as described previously [18]. The cells transformed with the multicopy reporter plasmid or the chromosome-borne cells were selected and cultured in EMM or EMM with leucine to midlog phase at 30°C and recovered by centrifugation respectively. Then the cells were resuspended in refresh EMM or EMM with leucine containing different concentrations of drugs. Luciferin was used as a substrate for Firefly luciferase, and yielding luminescence was detected using a luminometer (AB-2350; ATTO Co., Tokyo, Japan) at 1-min intervals and reported as relative light units (RLU).
Gene deletion
To delete the sre1 gene, a one-step gene disruption by homologous recombination [21] was performed. The sre1::ura4 disruption was constructed as follows. The cloned open reading frame of the sre1 gene in pBluescript SK (Stratagene) was digested with HindIII, and the resulting fragment containing the sre1 gene was subcloned into the HindIII site of the pBluescript vector. Then a BamHI fragment containing the ura4 gene was inserted into the BamHI site of the previous construct, causing the interruption of the open reading frame. The fragment containing the disrupted sre1 gene was transformed into haploid cells. Stable integrants were selected on medium lacking uracil, and disruption of the gene was checked by Southern blotting.The deletion of either scp1+ or hhp2+ gene with a genetic background of h
leu1-32 ura4-D18 ade6-M210 or M216 and the KanMX cassette was purchased from BioNEER (South Korea) [22]. We constructed scp1 or hhp2 deletion cells that were not auxotrophic for uracil and adenine by the genetic cross between wild-type cells HM123 and the above strains to make CM109 or CM125, respectively (Table 1). We constructed Δssp2Δgsk3Δgsk31 triple deletion by the genetic cross between KP4304 (h
leu1-32 ura4-D48 asn1::loxp ssp2::asn1) and KP5683 (h
his2 leu1-32 ura4-D48 asn1::loxp gsk3::ura4
gsk31::KanMX) to make CM134 (h
leu1-32 ura4-D48 asn1::loxp ssp2::asn1gsk3::ura4
gsk31::KanMX) (Table 1).
Fluorescence microscopy
Cells transformed with pREP1-GFP-Sre1N were grown in EMM medium with 4 μM thiamine to attenuate the expression for 16 h at 30°C, GFP-Sre1N was detected by its own fluorescence expressed in living cells by fluorescence microscope using a Nikon Eclipse Ni-U microscope equipped with a DS-Qi2 camera (Nikon Instruments Inc., Japan). For measurement of fluorescence intensities, images of the cells expressing GFP-Sre1N were taken using an oil-immersion objective lens (UApo 100×, NA 1.3, Nikon) at NA = 0.65; The best-focused image of the eight optical sections was selected for quantification. Quantification of fluorescence intensities was determined as follows. Specify a region including the nucleus, and calculate the mean fluorescence intensity (MeanN) and area values (AreaN) of the nuclear region. Calculate the mean fluorescence intensity in a cytoplasmic region of the same cell as the background mean fluorescence intensity (MeanB). Calculate GFP-Nucleus fluorescence intensity on NE (FNE) according to the following formula: FN = (MeanN—MeanB) × AreaN. Measure the fluorescence intensities of about 50 cells in each strain, and calculate average values and standard deviations. Wide-type cells fluorescence intensity was set to one in calculating relative fluorescence intensities [23, 24].
Cell extract preparation and immunoblot analysis
For the analysis of the total Sre1N protein in various mutants, whole cell extracts were prepared from cultures of wild-type cells or mutants harboring pREP1-GST-Sre1N plasmid grown at 30°C to mid-log phase. Total cell lysates were prepared as follows. Approximately 2 × 107 cells were resuspended in 500 μl of homogenizing buffer (92.5% 2N NaOH, 7.5% β-mercaptoethanol). After cooling on ice for 10 min, the proteins were precipitated by the addition of 500 μl of 50% trichloroacetic acid. Then, cellular debris was removed by centrifugation at 14000 rpm for 5 min. The resulting protein extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [25]. We used separating acrylamide gels (10.0%) with a mono/bis ratio of 29:1 to detect Sre1N. Purified polyclonal anti-GST was used as the primary antibodies, and goat anti-rabbit immunoglobulin G (IgG) Fc fragments was used as the secondary antibodies. Enhanced chemiluminescence was used for immunodetection on the membrane.
Statistical analyses
Quantitative data were expressed as means ± S.D. Multiple comparisons were statistically analyzed by one-way ANOVA followed by Tukey’s test. The difference was considered to be significant, if P value is less than 0.05. All statistical analyses were performed using the SPSS 16.0 software package (SPSS, Inc., Chicago, IL, USA).
Results
Real-time monitoring of Sre1 activity in living cells
Mammalian SREBPs bind a 10-bp DNA sequence in the promoters of target genes, called the sterol regulatory element (SRE) [7]. In fission yeast, two DNA elements (SRE2 and SRE3) in the promoter of sre1+ gene were identified to be necessary and sufficient for oxygen-dependent, Sre1-dependent transcription, but the binding of Sre1 to SRE2 was stronger than to SRE3 [26]. In order to monitor the real-time Sre1 activity for further studying the regulation mechanisms underlying the Sre1 activity, we constructed multicopy reporter plasmid containing three tandem repeats of SRE2 fused to firefly luciferase gene. As shown in Fig 1A–1C, ergosterol biosynthesis inhibitors including clotrimazole (CLZ), terbinafine (TER), and fenpropimorph (FEN), increased the luciferase activity with a peak rise at about 12–13 hours through the SRE2 motif in a dose-dependent manner (Fig 1A–1C). Since it is known that Sre1 can bind to the SRE2 motif for its transcriptional control, we examined the involvement of Sre1 in ergosterol biosynthesis inhibitors-induced activation of SRE2 reporter. The results showed that these ergosterol biosynthesis inhibitors-induced increase in SRE2 reporter activity was completely abolished in Δsre1 cells (Fig 1D–1F), suggesting that the multicopy reporter assay reflected Sre1 activity upon low ergosterol conditions. Given that Scp1 is required for Sre1 cleavage in fission yeast [8], we also examined the effect of Scp1 deletion on ergosterol biosynthesis inhibitors-induced SRE2 reporter activity. As shown in Fig 1D–1F, ergosterol biosynthesis inhibitors-induced increase in SRE2 reporter activity was also abolished in Δscp1 cells (Fig 1D–1F), further strengthening the reliability of our reporter system.
Fig 1
Real-time monitoring of Sre1 activity in living cells.
(A) CLZ (2 μg/ml to 8 μg/ml), (B) TER (2 μg/ml to 8 μg/ml) and (C) FEN (1 μg/ml to 4 μg/ml) induced a marked increase in Sre1 transcriptional activity. Wild-type cells harboring the multicopy 3×SRE2::luc (R2.2) reporter plasmid were cultured and assayed as described in materials and methods. Y-axis values are the relative light units (RLU) of each sample. The data shown are representative of multiple experiments. (D-F) CLZ (2 μg/ml to 8 μg/ml), TER (2 μg/ml to 8 μg/ml) and FEN (1 μg/ml to 4 μg/ml)-induced transcriptional activity was almost completely abolished in Δsre1 and Δscp1 cells. The Δsre1 and Δscp1 cells harboring the multicopy reporter plasmid were cultured and assayed as described in Fig 1A-1C. Y-axis values are the relative light units (RLU) of peak height normalized to cell density (OD660) of each sample at the peak time. The data were averaged from three independent experiments, and each sample was done in triplicate. Error bars indicate means (n = 3) ± S.D. **P<0.01 compared with the vehicle condition for the respective genotype.
Real-time monitoring of Sre1 activity in living cells.
(A) CLZ (2 μg/ml to 8 μg/ml), (B) TER (2 μg/ml to 8 μg/ml) and (C) FEN (1 μg/ml to 4 μg/ml) induced a marked increase in Sre1 transcriptional activity. Wild-type cells harboring the multicopy 3×SRE2::luc (R2.2) reporter plasmid were cultured and assayed as described in materials and methods. Y-axis values are the relative light units (RLU) of each sample. The data shown are representative of multiple experiments. (D-F) CLZ (2 μg/ml to 8 μg/ml), TER (2 μg/ml to 8 μg/ml) and FEN (1 μg/ml to 4 μg/ml)-induced transcriptional activity was almost completely abolished in Δsre1 and Δscp1 cells. The Δsre1 and Δscp1 cells harboring the multicopy reporter plasmid were cultured and assayed as described in Fig 1A-1C. Y-axis values are the relative light units (RLU) of peak height normalized to cell density (OD660) of each sample at the peak time. The data were averaged from three independent experiments, and each sample was done in triplicate. Error bars indicate means (n = 3) ± S.D. **P<0.01 compared with the vehicle condition for the respective genotype.
It has been known that Sre1 functions as an important oxygen sensor in fission yeast and Sre1 can be proteolytically cleaved and activated under low oxygen conditions [8]. We then tested whether hypoxia mimic Cobalt chloride (CoCl2) could activate SRE2 reporter. As shown in Fig 2A, CoCl2 also caused a marked dose-dependent increase in the SRE2 reporter response with a peak at about 13 hours in wild-type cells (Fig 2A). Consistently, in Δsre1 cells, the multicopy reporter response was reduced by two orders of magnitude compared to wild-type cells, indicating that CoCl2 could activate Sre1-dependent SRE2 reporter activity (Fig 2B). Likewise, the Δscp1 mutant showed significantly lower SRE2 reporter activity (Fig 2B).
CoCl2 (0.08 mM to 0.32 mM) induced a marked increase in Sre1 transcriptional activity. Wild-type cells harboring the multicopy reporter plasmid were cultured and assayed as described in Fig 1A–1C. (B) Two orders of magnitude reduced from wild type cells were observed in Δsre1 and Δscp1 cells. The Δsre1 and Δscp1 cells harboring the multicopy reporter plasmid were cultured and assayed as described in Fig 1A–1C. Y-axis values are the relative light units (RLU) of peak height normalized to cell density (OD660) of each sample at the peak time. The data were averaged from three independent experiments, and each sample was done in triplicate. Error bars indicate means (n = 3) ± S.D. **P<0.01 compared with the vehicle condition for the respective genotype.
CoCl2 (0.08 mM to 0.32 mM) induced a marked increase in Sre1 transcriptional activity. Wild-type cells harboring the multicopy reporter plasmid were cultured and assayed as described in Fig 1A–1C. (B) Two orders of magnitude reduced from wild type cells were observed in Δsre1 and Δscp1 cells. The Δsre1 and Δscp1 cells harboring the multicopy reporter plasmid were cultured and assayed as described in Fig 1A–1C. Y-axis values are the relative light units (RLU) of peak height normalized to cell density (OD660) of each sample at the peak time. The data were averaged from three independent experiments, and each sample was done in triplicate. Error bars indicate means (n = 3) ± S.D. **P<0.01 compared with the vehicle condition for the respective genotype.Considering that the copy number of the multicopy plasmid stably maintained cells might be affected by various factors, we constructed wild-type and Δsre1 chromosome-borne 3×SRE2::luc (R2.2) cells, respectively (CM150 and CM172 listed in Table 1). Similar to the results of cells harboring the multicopy 3×SRE2::luc (R2.2) reporter, the wild-type chromosome-borne 3×SRE2::luc (R2.2) cells could be activated by various concentrations of CLZ, TER or CoCl2 (Fig 3A–3C) in a dose-dependent manner with a similar peak rise time. Likewise, an extremely low response upon stimulation was observed in Δsre1 chromosome-borne 3×SRE2::luc (R2.2) cells (Fig 3D–3F). These results indicate that both the episomal multicopy and the chromosome integration 3×SRE2::luc (R2.2) reporter could reflect Sre1 activity in living cells.
Fig 3
The luciferase activity expressed from the chromosome-borne 3×SRE2::luc (R2.2) wild-type and Δsre1 cells.
(A) CLZ (1 μg/ml to 4 μg/ml), (B) TER (0.5 μg/ml to 2 μg/ml) and (C) CoCl2 (0.08 mM to 0.32 mM) induced a marked increase in Sre1 transcriptional activity. Wild-type chromosome-borne 3×SRE2::luc (R2.2) cells were cultured and assayed as described in materials and methods. Y-axis values are the relative light units (RLU) of each sample. The data shown are representative of multiple experiments. (D-F) An extremely low response upon stimulation by CLZ (1 μg/ml to 4 μg/ml), TER (0.5 μg/ml to 2 μg/ml) and CoCl2 (0.08 mM to 0.32 mM) was examined in Δsre1 chromosome-borne cells. The Δsre1 chromosome-borne cells were cultured and assayed as described in Fig 3A–3C. Y-axis values are the relative light units (RLU) of peak height normalized to the cell density (OD660) of each sample at the peak time. The data were averaged from three independent experiments, and each sample was done in triplicate. Error bars indicate means (n = 3) ± S.D.
The luciferase activity expressed from the chromosome-borne 3×SRE2::luc (R2.2) wild-type and Δsre1 cells.
(A) CLZ (1 μg/ml to 4 μg/ml), (B) TER (0.5 μg/ml to 2 μg/ml) and (C) CoCl2 (0.08 mM to 0.32 mM) induced a marked increase in Sre1 transcriptional activity. Wild-type chromosome-borne 3×SRE2::luc (R2.2) cells were cultured and assayed as described in materials and methods. Y-axis values are the relative light units (RLU) of each sample. The data shown are representative of multiple experiments. (D-F) An extremely low response upon stimulation by CLZ (1 μg/ml to 4 μg/ml), TER (0.5 μg/ml to 2 μg/ml) and CoCl2 (0.08 mM to 0.32 mM) was examined in Δsre1 chromosome-borne cells. The Δsre1 chromosome-borne cells were cultured and assayed as described in Fig 3A–3C. Y-axis values are the relative light units (RLU) of peak height normalized to the cell density (OD660) of each sample at the peak time. The data were averaged from three independent experiments, and each sample was done in triplicate. Error bars indicate means (n = 3) ± S.D.
Deletion of Ssp2 or/and Gsk3/Gsk31 markedly suppressed CLZ, TER or CoCl2-induced Sre1 activity
Next, our studies focused on whether additional protein kinases were involved in regulation of Sre1 activity. Consistent with the notion that casein kinase 1 family member Hhp2 accelerates Sre1N degradation [13], we found that deletion of hhp2+ significantly increased SRE2 reporter activity in the presence/absence of CLZ, TER or CoCl2 (Fig 4A–4C), thus further validating our reporter system. Surprisingly, in deletion of ssp2+ gene, encoding the AMP-activated protein kinase (AMPK) α Subunit, CLZ, TER or CoCl2-induced Sre1 activity was significantly suppressed (Fig 5A–5C). It should be noted that, compared with wild-type cells, deletion of Ssp2 significantly delayed the peak rising of the SRE2 reporter (Fig 5D–5F).
Fig 4
Deletion of Hhp2 markedly increased Sre1 activity.
Deletion of hhp2+ gene markedly increased Sre1 activity in the presence/absence of CLZ (2 μg/ml to 8 μg/ml) (A), TER (2 μg/ml to 8 μg/ml) (B) or CoCl2 (0.08 mM to 0.32 mM) (C). Cells harboring the multicopy reporter plasmid were cultured and assayed as described in Fig 1A–1C. Y-axis values are the relative light units (RLU) of peak height normalized to the cell density (OD660) of each sample at the peak time. The data were averaged from three independent experiments, and each sample was done in triplicate. Error bars indicate means (n = 3) ± S.D. ##P<0.01 compared with wild-type cells treated with the same drug concentration.
Fig 5
Deletion of Ssp2 or/and Gsk3/Gsk31 markedly suppressed CLZ, TER or CoCl2-induced Sre1 activity.
The Δssp2, Δgsk3Δgsk31, Δssp2Δgsk3, Δssp2Δgsk31 or Δssp2Δgsk3Δgsk31 cells showed a significant decrease in Sre1 activity and delay in the peak rising of the SRE2 reporter stimulated with 8 μg/ml CLZ (A), 8 μg/ml TER (B) or 0.32 mM CoCl2 (C). Cells harboring the multicopy reporter plasmid were cultured and assayed as described in Fig 1A–1C. Y-axis values are the relative light units (RLU) of peak height normalized to the cell density (OD660) of each sample at the peak time. The data were averaged from three independent experiments, and each sample was done in triplicate. Error bars indicate means (n = 3) ± S.D. *P<0.05 and **P<0.01 compared with wild-type cells. #P<0.05 and ##P<0.01 compared between different genotypes as indicated. (D-F) The data shown are representative of multiple experiments as described in Fig 5A–5C. Y-axis values are the relative light units (RLU) of each sample. (G) The Δssp2Δgsk3Δgsk31 triple deletion cells showed more marked temperature sensitivity than any of their single or double deletion. Wild-type, Δgsk3, Δgsk31, Δssp2, Δgsk3Δgsk31, Δssp2Δgsk3, Δssp2Δgsk31 or Δssp2Δgsk3Δgsk31 cells were spotted onto each plate as indicated, and then incubated at 30°C, 35°C or 36°C for 3 days.
Deletion of Hhp2 markedly increased Sre1 activity.
Deletion of hhp2+ gene markedly increased Sre1 activity in the presence/absence of CLZ (2 μg/ml to 8 μg/ml) (A), TER (2 μg/ml to 8 μg/ml) (B) or CoCl2 (0.08 mM to 0.32 mM) (C). Cells harboring the multicopy reporter plasmid were cultured and assayed as described in Fig 1A–1C. Y-axis values are the relative light units (RLU) of peak height normalized to the cell density (OD660) of each sample at the peak time. The data were averaged from three independent experiments, and each sample was done in triplicate. Error bars indicate means (n = 3) ± S.D. ##P<0.01 compared with wild-type cells treated with the same drug concentration.
Deletion of Ssp2 or/and Gsk3/Gsk31 markedly suppressed CLZ, TER or CoCl2-induced Sre1 activity.
The Δssp2, Δgsk3Δgsk31, Δssp2Δgsk3, Δssp2Δgsk31 or Δssp2Δgsk3Δgsk31 cells showed a significant decrease in Sre1 activity and delay in the peak rising of the SRE2 reporter stimulated with 8 μg/ml CLZ (A), 8 μg/ml TER (B) or 0.32 mM CoCl2 (C). Cells harboring the multicopy reporter plasmid were cultured and assayed as described in Fig 1A–1C. Y-axis values are the relative light units (RLU) of peak height normalized to the cell density (OD660) of each sample at the peak time. The data were averaged from three independent experiments, and each sample was done in triplicate. Error bars indicate means (n = 3) ± S.D. *P<0.05 and **P<0.01 compared with wild-type cells. #P<0.05 and ##P<0.01 compared between different genotypes as indicated. (D-F) The data shown are representative of multiple experiments as described in Fig 5A–5C. Y-axis values are the relative light units (RLU) of each sample. (G) The Δssp2Δgsk3Δgsk31 triple deletion cells showed more marked temperature sensitivity than any of their single or double deletion. Wild-type, Δgsk3, Δgsk31, Δssp2, Δgsk3Δgsk31, Δssp2Δgsk3, Δssp2Δgsk31 or Δssp2Δgsk3Δgsk31 cells were spotted onto each plate as indicated, and then incubated at 30°C, 35°C or 36°C for 3 days.Previously, we identified two glycogen synthase kinases encoding genes, gsk3+ and gsk31+ as multicopysuppressors of Ssp2 deletion, and revealed a genetic interaction between Ssp2 and Gsk3 or Gsk31 in cell growth and sexual differentiation [27]. Then we sought to investigate whether Gsk3 or/and Gsk31 was required for Sre1 activity upon CLZ, TER or CoCl2 treatment. We examined the Sre1 activity of Δgsk3 or Δgsk31 single deletion as well as Δgsk3Δgsk31 double deletion by using SRE2 reporter. The results showed that the Δgsk3 single deletion or the Δgsk31 single deletion slightly, but the Δgsk3Δgsk31 double deletion extremely suppressed the CLZ, TER or CoCl2-induced Sre1 activity (Fig 5A–5C). Similar to deletion of Ssp2, the Δgsk3Δgsk31 double deletion significantly delayed the peak rising of the SRE2 reporter (Fig 5D–5F).We also tested the Sre1 activity in either Δssp2Δgsk3 or Δssp2Δgsk31 cells by using SRE2 reporter. Upon CLZ or TER treatment, the Sre1 activity of either Δssp2Δgsk3 or Δssp2Δgsk31 cells was slightly lower than that of Δssp2 cells, but significantly lower than that of Δgsk3 or Δgsk31 cells (Fig 5A and 5B). On the other hand, upon treatment with CoCl2, the Sre1 activity of either Δssp2Δgsk3 or Δssp2Δgsk31 was almost equal to that of Δssp2 cells, slightly lower than that of Δgsk3 or Δgsk31 cells respectively (Fig 5C). Additionally, the Δssp2Δgsk3 or Δssp2Δgsk31 cells also significantly delayed the peak rising of the SRE2 reporter, similar to deletion of Ssp2 (Fig 5D–5F).To further investigate the roles of Ssp2 and Gsk3/Gsk31 in the Sre1 activity, we constructed the strains lacking all of these three genes and tested the Sre1 activity in this triple deletion cells. As shown in Fig 5A–5C, the Δssp2Δgsk3Δgsk31 cells showed extremely lower but still measurable Sre1 activity upon treatment with CLZ, TER or CoCl2, compared with any of their single or double deletion cells (Fig 5A–5C). Consistently, the Δssp2Δgsk3Δgsk31 triple deletion showed more marked temperature sensitivity than any of their single or double deletion (Fig 5G).
Deletion of Ssp2 or/and Gsk3/Gsk31 reduced the nuclear fluorescence of GFP-Sre1N as well as GST-Sre1N protein levels
Given that Sre1N exists as a hyperphosphorylated protein that contains at least 22 phosphorylated serine and threonine residues [8], and protein kinase Hhp2 regulates Sre1N degradation, We then wanted to know whether deletion of Ssp2, Gsk3 or Gsk31 affect Sre1N degradation. Sre1N protein tagged with GFP was visualized and the effect of deletion of Ssp2, Gsk3 or Gsk31 was examined. GFP-Sre1N is functional as its expression complemented the CoCl2-sensitive growth defect of the Δsre1 cells (Fig 6A). It is known that Sre1N is released at Golgi and enters the nucleus for further promoting transcription of the target genes [28]. As expected, GFP-Sre1N clearly localized at the nucleus in the wild-type cells (Fig 6B). In Δgsk3 or Δgsk31 cells, GFP-Sre1N also localized at the nucleus, similar to that of wild-type cells. However, in Δssp2, Δgsk3Δgsk31, Δssp2Δgsk3, Δssp2Δgsk31 or Δssp2Δgsk3Δgsk31 deletion cells, significantly weaker fluorescence intensity of GFP-Sre1N was observed at the nucleus compared with that of wild-type cells (Fig 6B and 6C). As these data seem consistent with changes in Sre1N activity as well as Sre1N protein levels, we further performed the immunoblot analysis to detect the total protein levels of Sre1N in wild-type cells and the deletion mutants. GST-Sre1N is also functional as its expression complemented the CoCl2-sensitive growth defect of the Δsre1 cells (Fig 6A). Consistent with their weak fluorescence intensity, the whole amount of GST-Sre1N was reduced in the mutants except that in Δgsk3 or Δgsk31 cells (Fig 6D). However, the total amount of GST-Sre1N was markedly different between Δssp2 and Δgsk3Δgsk31 deletion cells. The immunoblot showed a dramatic decrease in Sre1N levels in the Δgsk3Δgsk31 double mutant cells, while deletion of Ssp2 has just a minor effect on Sre1N levels (Fig 6D). This was different from the nuclear localization, where Δssp2 mutant had a major effect that was similar to the Δgsk3Δgsk31 double mutant (Fig 6B and 6C). In comparing the Δgsk3Δgsk31 double mutant versus the Δssp2Δgsk3Δgsk31 triple mutant, there is no additive defect in Sre1N levels and the two strains look identical (Fig 6D). These results suggest that Gsk3/Gsk31 might play the primary role in regulating Sre1N degradation, whereas Ssp2 might regulate not only Sre1N degradation but also nuclear localization of Sre1N.
Fig 6
Deletion of Ssp2 or/and Gsk3/Gsk31 reduced the nuclear fluorescence of GFP-Sre1N as well as GST-Sre1N protein levels.
(A) Both GFP-Sre1N and GST-Sre1N complemented the CoCl2-sensitive growth defect of the Δsre1 cells. Cells transformed with the empty vector, the pREP1-GFP-Sre1N plasmid or pREP1-GST-Sre1N plasmid were spotted onto each plate as indicated, and then incubated at 30°C for 4 days. (B) Significantly weaker fluorescence of GFP-Sre1N at the nucleus was observed in Δssp2, Δgsk3Δgsk31, Δssp2Δgsk3, Δssp2Δgsk31 or Δssp2Δgsk3Δgsk31 cells. Wild-type, Δgsk3, Δgsk31, Δssp2, Δgsk3Δgsk31, Δssp2Δgsk3, Δssp2Δgsk31 or Δssp2Δgsk3Δgsk31 cells expressing GFP-Sre1N were grown in EMM medium with 4 μM thiamine at 30°C for 16 h, and then observed by fluorescence microscopy for the same 3 seconds exposure. Bar, 10 μm. (C) Fluorescence intensity quantification of GFP-tagged nucleus Sre1N. Fluorescence intensities of about 50 cells from each strain cultured at 30°C were measured. Average values after background subtraction are shown in the bar graph. Error bars represent standard deviations. (D) The whole amount of Sre1N was markedly reduced in the Δgsk3Δgsk31 or Δssp2Δgsk3Δgsk31 cells but just a little reduced in the Δssp2, Δssp2Δgsk3 or Δssp2Δgsk31 cells as assessed by immunoblot analysis. Wild-type, Δgsk3, Δgsk31, Δssp2, Δgsk3Δgsk31, Δssp2Δgsk3, Δssp2Δgsk31 or Δssp2Δgsk3Δgsk31 cells were transformed with pREP1-GST-Sre1N and were cultured in liquid EMM to mid-log phase. Total cell lysates were prepared as described in materials and methods, and then the protein extracts were subjected to SDS-PAGE, and immunoblotted using anti-GST antibodies. β-actin was used as a loading control.
Deletion of Ssp2 or/and Gsk3/Gsk31 reduced the nuclear fluorescence of GFP-Sre1N as well as GST-Sre1N protein levels.
(A) Both GFP-Sre1N and GST-Sre1N complemented the CoCl2-sensitive growth defect of the Δsre1 cells. Cells transformed with the empty vector, the pREP1-GFP-Sre1N plasmid or pREP1-GST-Sre1N plasmid were spotted onto each plate as indicated, and then incubated at 30°C for 4 days. (B) Significantly weaker fluorescence of GFP-Sre1N at the nucleus was observed in Δssp2, Δgsk3Δgsk31, Δssp2Δgsk3, Δssp2Δgsk31 or Δssp2Δgsk3Δgsk31 cells. Wild-type, Δgsk3, Δgsk31, Δssp2, Δgsk3Δgsk31, Δssp2Δgsk3, Δssp2Δgsk31 or Δssp2Δgsk3Δgsk31 cells expressing GFP-Sre1N were grown in EMM medium with 4 μM thiamine at 30°C for 16 h, and then observed by fluorescence microscopy for the same 3 seconds exposure. Bar, 10 μm. (C) Fluorescence intensity quantification of GFP-tagged nucleus Sre1N. Fluorescence intensities of about 50 cells from each strain cultured at 30°C were measured. Average values after background subtraction are shown in the bar graph. Error bars represent standard deviations. (D) The whole amount of Sre1N was markedly reduced in the Δgsk3Δgsk31 or Δssp2Δgsk3Δgsk31 cells but just a little reduced in the Δssp2, Δssp2Δgsk3 or Δssp2Δgsk31 cells as assessed by immunoblot analysis. Wild-type, Δgsk3, Δgsk31, Δssp2, Δgsk3Δgsk31, Δssp2Δgsk3, Δssp2Δgsk31 or Δssp2Δgsk3Δgsk31 cells were transformed with pREP1-GST-Sre1N and were cultured in liquid EMM to mid-log phase. Total cell lysates were prepared as described in materials and methods, and then the protein extracts were subjected to SDS-PAGE, and immunoblotted using anti-GST antibodies. β-actin was used as a loading control.
Discussion
In fission yeast, Sre1, the homologue of mammalianSREBP, regulates sterol homeostasis and hypoxia adaptation [29]. It was known that, as a negative regulator of Sre1, casein kinase 1 family member Hhp2 accelerates Sre1N degradation. However, studies on additional kinases involved in Sre1 activity regulation are still limited. Here, we identified AMPKα Subunit Ssp2 and Glycogen Synthase Kinases Gsk3/Gsk31 as positive regulators of Sre1, which are involved in regulation of Sre1 activity via inhibiting degradation and accelerating translocation of Sre1N into the nucleus. To our knowledge, this is the first report to reveal a novel requirement for protein kinases Ssp2 and Gsk3/Gsk31 in regulation of Sre1 activity in fission yeast.Three evidences support that our luciferase reporter system could reflect Sre1 activity. First, ergosterol biosynthesis inhibitors, namely CLZ, TER and FEN could induce a marked increase in transcriptional activity of Sre1 in a dose-dependent manner. Second, ergosterol biosynthesis inhibitors-induced Sre1 activity was abolished in Δsre1 or Δscp1 cells. Third, loss of Hhp2, a negative regulator of Sre1, significantly increased transcriptional activity of Sre1 in the presence/absence of ergosterol biosynthesis inhibitors, such as CLZ or TER.Our previous studies found that Gsk3 and Gsk31 function redundantly in cell growth at restrictive temperatures and sexual differentiation [27]. In present study, several lines of evidence support the hypothesis that Gsk3 and Gsk31 function redundantly in regulation of Sre1 activity, as well as Ssp2 and Gsk3/31 act on parallel in regulation of Sre1 activity. First, CLZ, TER or CoCl2-induced Sre1 activity in Δgsk3Δgsk31 cells was significantly reduced compared to wild-type cells, but slightly reduced in Δgsk3 or Δgsk31 cells. Second, CLZ, TER or CoCl2-induced Sre1 activity in Δssp2Δgsk3 or Δssp2Δgsk31 was only slightly lower than or almost equal to Δssp2 cells. Third, the deletion of ssp2, gsk3 and gsk31, ssp2 and gsk3, or ssp2 and gsk31 significantly delayed the peak rising of the SRE2 reporter, but the deletion of gsk3 or gsk31 did not. Forth, the Δssp2Δgsk3Δgsk31 cells showed the lowest Sre1 activity compared to any of their single or double deletions. These results suggested that there is a genetic interaction between Ssp2 and Gsk3/Gsk31, and Ssp2 and Gsk3/Gsk31 may act on parallel pathway in regulation of Sre1 activity.Furthermore, we found that the fluorescence of GFP-Sre1N at the nucleus observed in Δssp2, Δgsk3Δgsk31, Δssp2Δgsk3, Δssp2Δgsk31 or Δssp2Δgsk3Δgsk31 cells was significantly weakened compared with that in wild-type cells. To our surprises, while nuclear accumulation of GFP-Sre1N appeared to be diminished to a similar extent in the Δssp2 mutant and the Δgsk3Δgsk31 double mutant, the total amount of GST-Sre1N was markedly different in these two mutants. Thus, it seems possible that Ssp2 regulates not only the degradation of Sre1N but also its translocation to the nucleus, whereas Gsk3/Gsk31 regulate mainly its degradation. Since casein kinase 1 family member Hhp2 accelerates Sre1N degradation, our results suggested that Ssp2/Gsk3/Gsk31 might act as inhibitors of Hhp2, or alternatively act on Sre1N activity independently of Hhp2. Previous studies suggested that Sre1 cleavage, Sre1N stability and Sre1N DNA binding are involved in the regulation of Sre1 activity [30-32]. Based on our present results, we propose that Ssp2 and Gsk3/Gsk31 might affect Sre1N stability to regulate Sre1 activity. However, it is currently undetermined whether Ssp2 and Gsk3/31 are involved in Sre1 cleavage or Sre1N DNA binding for regulation of Sre1 activity.In conclusion, our findings establish new functional link between Sre1 and three protein kinases, namely Ssp2, Gsk3 and Gsk31. The present data strongly suggest that Ssp2 and Gsk3/Gsk31 play cooperative but distinct roles in the regulation of Sre1 activity in fission yeast. Understanding whether Ssp2 or Gsk3/Gsk31 directly phosphorylates Sre1N and inhibits its degradation is important questions to be addressed in the future.
The original uncropped and unadjusted western blotting images and all individual data points within curve and column graphs.
(ZIP)Click here for additional data file.20 Nov 2019PONE-D-19-30640AMPKα Subunit Ssp2 and Glycogen Synthase Kinases Gsk3/Gsk31 are involved in regulation of sterol regulatory element-binding protein (SREBP) activity in fission yeastPLOS ONEDear Dr Fang,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.==============================Your manuscript was evaluated by two experts in the field and their reports were returned. As you see, both referees gave favorable reviews. However, they also raised several points, which I agree with. Both referees pointed out caveats of the episomal reporter assay. Please reply satisfactorily to their comments possibly by adding new data. They also raised another point with regards to the levels of nuclear and cytoplasmic GFP-Sre1N. Please address this important point. In addition, Referee 2 raised a few concerns. I hope that you could revise the manuscript in response to these comments. Then, I would be happy to consider acceptance of this manuscript.==============================We would appreciate receiving your revised manuscript by Dr Fang. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocolsPlease include the following items when submitting your revised manuscript:A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.We look forward to receiving your revised manuscript.Kind regards,Takashi Toda PhDAcademic EditorPLOS ONEJournal Requirements:When submitting your revision, we need you to address these additional requirements.Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found athttp://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdfReviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.Reviewer #1: PartlyReviewer #2: Partly**********2. Has the statistical analysis been performed appropriately and rigorously?Reviewer #1: YesReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)Reviewer #1: SREBP (Sre1 in fission yeast) is an evolutionally conserved transcriptional factor regulating the expression of genes involved in sterol biogenesis. In this manuscript, Yue Fang et al develop the reporter system to measure the level of SREBP-dependent transcription in fission yeast. The authors show that the expression of the reporter is upregulated in cells treated with inhibitors of sterol biosynthesis or CoCl2, and that deletion of genes encoding AMPK (Ssp2) and/or GSK3s (Gsk3 and Gsk31) greatly diminishes the expression of the reporter. The authors find the intensity of the GFP-Sre1N fluorescence in the nucleus is reduced in mutant cells lacking ssp2 and/or gsk3/gsk31 genes, and speculate that AMPK and GSK may synergistically inhibit degradation of the N-terminal part of Sre1, which is released by protein cleavage on the Golgi, translocated to the nucleus and promotes the transcription of target genes.While I think that study is well-conducted and the presented results are largely clear, I have two major concerns described below:1) In Figure 4, they measure the luciferase activity expressed form the episomally-introduced multicopy plasmid. Although the authors conclude that the reduction of the luciferase activity in the mutant cells lacking ssp2 and/or gsk3/gsk31 is caused by deficiency of Sre1 (SREBP) function, it may be possibly caused by the reduction of the plasmid copy number. Considering that the copy number of the plasmid stably maintained cells is greatly affected by various factors, the authors cannot exclude the possibility that deletion of these genes may somehow reduce the plasmid copy number as long as the episomal plasmid is used. I strongly suggest the authors to perform the experiment using cells in which the reporter construct is integrated to the chromosome. At the minimum, they should confirm that the cells used in the experiment harbor the same number of the reporter plasmid by measuring the amount of the plasmid DNA in the cells.2) In Figure 5, the authors clams that the reduction of GFP-Sre1N fluorescence in the nucleus in the mutant cells is caused by degradation of the GFP-Sre1N protein. However, I believe that it is equally possible that deletion of ssp2 and/or gsk3/gsk31 genes may somehow perturb translocation of the GFP-Sre1N protein from the cytoplasm to the nucleus and the protein may be dispersed throughout the cells. The authors should perform the immunoblotting experiment to show more directly that the whole amount of GFP-Sre1N is reduced in the mutants. If the accumulation of degraded product of GFP-Sre1N in the mutant could be detected in immunoblot, their conclusion would be further strengthened. If phosphorylation of Sre1N causes its band-shift, the author may be also able to see how much the Sre1N phosphorylation is affected by deletion of ssp2 and/or gsk3/gsk31 genes.Reviewer #2: This paper by Miao et al. investigates protein kinases that regulate the SREBP response in fission yeast. Past work has shown that CK1 kinase Hhp2 phosphorylates Sre1N (the cleaved and activated region of SREBP) to accelerate its degradation. Here, the authors generated a luciferase-based reporter for Sre1N activity, and identified a role for AMPK/Ssp2 and GSK-3 orthologs Gsk3 and Gsk31 in regulating Sre1N activity. The authors’ data support a model where Ssp2, Gsk3, and Gsk31 promote Sre1N activity and/or levels. The underlying mechanisms remain undefined in the current work, but identifying these new regulators would represent progress on this pathway and therefore would be of interest to the field. I have several technical and interpretation comments/concerns that should be addressed before publishing this work.Technical:1. The luciferase-based reporter is introduced into cells as a plasmid, but I do not see how maintenance of the plasmid is selected. The Methods section states that transformants were cultured in ‘normal EMM media.’ Is there an auxotrophic or antibiotic-based selection to make sure that cells have and maintain the plasmid? This is important because changes in plasmid maintenance would alter the results.2. For the luciferase-based assays, is the total luciferase signal normalized to cell number? It is important to control for changes in the growth rate of cells, so the light units should be normalized to cell number in each case.Data interpretation:1. sre1∆ cells retain some luciferase activity upon treatment with CoCl2, suggesting Sre1-independent transcription of the reporter. I do not disagree with this interpretation, but the authors should note that the response is two orders of magnitude reduced from wild type cells.2. The authors measure Sre1N-GFP levels in the nucleus, and conclude that degradation has been impacted in their mutants. However, their measured differences are also consistent with altered shuttling between the nucleus and cytoplasm. They should measure total cellular levels if they want to conclude changes in total protein levels.3. The authors could note that their results are consistent with Ssp2/Gsk3/Gsk31 acting as inhibitors of Hhp2, or alternatively acting on Sre1N activity independently of Hhp2.4. The authors’ data seem consistent with changes in Sre1N activity as well as Sre1N protein levels. They might add this possibility into the text. In particular, they have not provided data for changes in Sre1N degradation in the new kinase mutants (e.g. lines 297-299). To make this conclusion, they would have to measure degradation rates with different assays.**********6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.Reviewer #1: Yes: Shigeaki SaitohReviewer #2: No[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.8 Jan 2020January 04, 2020PLoS ONEDear Dr. Takashi Toda:Thank you for your e-mail of November 21, 2019 on our manuscript entitled “AMPKα Subunit Ssp2 and Glycogen Synthase Kinases Gsk3/Gsk31 are involved in regulation of sterol regulatory element-binding protein (SREBP) activity in fission yeast” by Hao Miao et al. (PONE-S-19-38064). We would like to thank you for your very helpful comments and suggestions, and for inviting us to revise our manuscript.In your e-mail, you’ve suggested that our revisions should address several specific points raised by two reviewers as mentioned below. Consequently, we performed a number of experiments based on the comments and were successful in obtaining new data. Thus, we address each point raised by the two reviewers.A list of the revisions which addresses reviewers' comments is as follows:Reviewer #1Comments1. In Figure 4, they measure the luciferase activity expressed form the episomally-introduced multicopy plasmid. Although the authors conclude that the reduction of the luciferase activity in the mutant cells lacking ssp2 and/or gsk3/gsk31 is caused by deficiency of Sre1 (SREBP) function, it may be possibly caused by the reduction of the plasmid copy number. Considering that the copy number of the plasmid stably maintained cells is greatly affected by various factors, the authors cannot exclude the possibility that deletion of these genes may somehow reduce the plasmid copy number as long as the episomal plasmid is used. I strongly suggest the authors to perform the experiment using cells in which the reporter construct is integrated to the chromosome. At the minimum, they should confirm that the cells used in the experiment harbor the same number of the reporter plasmid by measuring the amount of the plasmid DNA in the cells.Response:Accordingly, we constructed wild-type and Δsre1 chromosome-borne 3×SRE2::luc (R2.2) strains named CM150 and CM172, respectively (page 6, lines 127-131 / page 7, lines 150-153 / listed in table.1). The results showed that wild-type chromosome-borne 3×SRE2::luc (R2.2) cells could be activated by various concentration of CLZ, TER or CoCl2 (Fig 3A-C) in a dose-dependent manner, but in Δsre1 chromosome-borne 3×SRE2::luc (R2.2) cells, an extremely low response upon stimulation was observed (Fig 3D-F). These results are consistent with those obtained with multicopy reporter and suggested that both the episomal multicopy and the chromosome integration 3×SRE2::luc (R2.2) reporter could reflect Sre1 activity in living cells. These results are incorporated in the revised manuscript (page 12-13, lines 259-269 / Fig 3 and legend, page22, lines 449-462).2. In Figure 5, the authors claims that the reduction of GFP-Sre1N fluorescence in the nucleus in the mutant cells is caused by degradation of the GFP-Sre1N protein. However, I believe that it is equally possible that deletion of ssp2 and/or gsk3/gsk31 genes may somehow perturb translocation of the GFP-Sre1N protein from the cytoplasm to the nucleus and the protein may be dispersed throughout the cells. The authors should perform the immunoblotting experiment to show more directly that the whole amount of GFP-Sre1N is reduced in the mutants. If the accumulation of degraded product of GFP-Sre1N in the mutant could be detected in immunoblot, their conclusion would be further strengthened. If phosphorylation of Sre1N causes its band-shift, the author may be also able to see how much the Sre1N phosphorylation is affected by deletion of ssp2 and/or gsk3/gsk31 genes.Response:We thank the reviewer for giving us this very helpful comment. Accordingly, we performed the immunoblot assay to detect the total Sre1N protein levels in wild-type and mutant cells (page 10, lines 201-214). Consistent with fluorescence intensity in wild-type cells and the mutants, the whole amount of Sre1N was markedly reduced in these mutants compared with that of wild-type cells. Notably, the whole amount of Sre1N in Δssp2Δgsk3Δgsk31 triple deletion was lower than that in any of their single or double deletions assessed by immunoblot analysis (Fig 6D). These are incorporated in the revised manuscript (page 15-16, lines 313-314, 329-337 / Fig 6D and legend, page 24-25, lines 493-494, 507-514).Reviewer #2CommentsTechnical:1. The luciferase-based reporter is introduced into cells as a plasmid, but I do not see how maintenance of the plasmid is selected. The Methods section states that transformants were cultured in ‘normal EMM media.’ Is there an auxotrophic or antibiotic-based selection to make sure that cells have andmaintain the plasmid? This is important because changes in plasmid maintenance would alter the results.Response:Accordingly, there is an auxotrophic based selection for the transformants to make sure that cells have and maintain the plasmid described as follow: the multicopy 3×SRE2::luc (R2.2) reporter vector containing leucine marker can complement the S.pombe mutations leu1 (for example, wide-type cells HM123 (h- leu1-32)), then the transformants would be successfully selected in EMM media without leucine. These are incorporated in the revised manuscript (page 7-8, lines 153-156).2. For the luciferase-based assays, is the total luciferase signal normalized to cell number? It is important to control for changes in the growth rate of cells, so the light units should be normalized to cell number in each case.Response:Accordingly, for all the luciferase-based assays, we measured the cell density (OD660) of each groups at the peak time, and the luciferase light units of peak height were normalized to the corresponding cell density at the peak time. The figures and figure legends (Fig 1D-F and legend, page 21, lines 431-433 / Fig 2B and legend, page 22, lines 443-444 / Fig 3D-F and legend, page 22, lines 459-460 / Fig 4A-C and legend, page 23, lines 468-469 / Fig 5A-C and legend, page 23, lines 480-481) as well as a description of the results (page 14, lines 297-299) are modified in the revised manuscript.Data interpretation:1. sre1∆ cells retain some luciferase activity upon treatment with CoCl2, suggesting Sre1-independent transcription of the reporter. I do not disagree with this interpretation, but the authors should note that the response is two orders of magnitude reduced from wild type cells.Response:Although ∆sre1 cells transformed with 3×SRE2::luc (R2.2) multicopy plasmid reporter retain some luciferase activity upon treatment with CoCl2, it is still unclear what its physiological significance is. We further constructed wild-type and Δsre1 chromosome-borne 3×SRE2::luc (R2.2) strains, and found that CoCl2-induced increase in SRE2 reporter activity was almost abolished in Δsre1 cells (Fig 3). Therefore, we removed the related description of Sre1-independent activation as well as partially enlarged part in Fig 1 and Fig 2. According to the reviewer’s comment, we noted that, when sre1+ was knocked out, Sre1 activity stimulated with CoCl2 decreased to less than 1% compared to that of wild-type cells. These are included in the revised manuscript (page 12, line 248, 254-256 / Fig 1D-F / Fig 2B and legend, page 21, line 437, 440-441).2. The authors measure Sre1N-GFP levels in the nucleus, and conclude that degradation has been impacted in their mutants. However, their measured differences are also consistent with altered shuttling between the nucleus and cytoplasm. They should measure total cellular levels if they want to conclude changes in total protein levels.Response:Accordingly, immunoblot analysis was performed to detect the total Sre1N protein levels in wild-type cells and the mutants (page 10, lines 201-214). Consistent with their decreased fluorescence intensity of GFP-Sre1N in the nucleus, the total cellular protein levels of GST-Sre1N was also markedly reduced in the mutants. These results were included in the revised manuscript (page 15-16, lines 313-314, 329-337 / Fig 6D and legend, page 24-25, lines 493-494, 507-514).3. The authors could note that their results are consistent with Ssp2/Gsk3/Gsk31 acting as inhibitors of Hhp2, or alternatively acting on Sre1N activity independently of Hhp2.Response:We thank this reviewer for giving us these helpful comments. Accordingly, we added some comments on these issues. These are incorporated in the revised manuscript (page 18, lines 380-382).4. The authors’ data seem consistent with changes in Sre1N activity as well as Sre1N protein levels. They might add this possibility into the text. In particular, they have not provided data for changes in Sre1N degradation in the new kinase mutants (e.g. lines 297-299). To make this conclusion, they would have to measure degradation rates with different assays.Response:Accordingly, we added the possibility that our data are consistent with changes in Sre1N activity in the text (page15, line 328-329). To test whether the changes in Sre1N activity were consistent with Sre1N protein levels in the kinase mutants, we performed the immunoblot analysis to detect the total protein levels of Sre1N in wild-type cells and the deletion mutants. The results showed that the whole amount of GST-Sre1N was markedly reduced in the mutants except that in Δgsk3 or Δgsk31 cells. Notably, similar to the changes in Sre1N activity detected by luciferase reporter assay, the whole amount of Sre1N in Δssp2Δgsk3Δgsk31 triple deletion was lower than that in any of their single or double deletions. These results suggested that the changes in Sre1N activity were consistent with Sre1N protein levels. These are incorporated in the revised manuscript (page17-18, lines 374-380)We hope that these revisions would satisfy your requirements. Thank you very much for your time and consideration.Sincerely,Yue Fang, M.D, Ph.D.Department of Microbial and Biochemical Pharmacy,School of Pharmacy, China Medical University,No.77 Puhe Road, Shenyang North New Area, Shenyang 110112, ChinaTEL: +86-18900910820, FAX: +86-24-31939448Email: yfang@cmu.edu.cnSubmitted filename: response to reviewers file.docClick here for additional data file.22 Jan 2020PONE-D-19-30640R1AMPKα Subunit Ssp2 and Glycogen Synthase Kinases Gsk3/Gsk31 are involved in regulation of sterol regulatory element-binding protein (SREBP) activity in fission yeastPLOS ONEDear Dr Fang,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.==============================ACADEMIC EDITOR:Dr Fang,Thank you for submitting the revised manuscript, which was now evaluated by the two original referees.Both referees acknowledged the revision; however, they raised the same point, that is the interpretation of the immunoblotting data shown in Figure 6D. They pointed out the possibility that Ssp2 and Gsk31/32 may regulate GFP-Sre1N in a distinct manner; Ssp2 mainly regulates its translocation (nuclear import/retention), while Gsk31/32 regulate overall protein levels. Having seen the data myself, I agree with these referees’ point. Therefore, I strongly encourage you rephrasing/adding some discussion which incorporates this interesting point.I am looking forward to receiving your new revised manuscript.==============================We would appreciate receiving your revised manuscript in 30 days. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocolsPlease include the following items when submitting your revised manuscript:A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.We look forward to receiving your revised manuscript.Kind regards,Takashi Toda PhDAcademic EditorPLOS ONE[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: (No Response)Reviewer #2: (No Response)**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.Reviewer #1: YesReviewer #2: Partly**********3. Has the statistical analysis been performed appropriately and rigorously?Reviewer #1: YesReviewer #2: Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.Reviewer #1: YesReviewer #2: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)Reviewer #1: The criticisms raised against the original version are properly responded, and the manuscript is satisfactorily revised. I have one minor comment regarding the interpretation of the new result of immunoblotting in Figure 6.Minor point:While nuclear accumulation of GFP-Sre1N appears to be diminished to a similar extent in the ssp2 mutant and the gsk3 gsk31 double mutant, the total amount of GST-Sre1N was markedly different in these two mutants (Figure 6B and D). Thus, it seems possible that Ssp2 regulates not only the degradation of Sre1N but also its translocation to the nucleus, whereas Gsks regulate mainly its degradation. It may be better to discuss this possibility.Reviewer #2: The authors have done a nice job revising the manuscript and adding new data to address the reviewer comments. I have one lingering concern related to the new immnunoblot experiment in Figure 6D. This new experiment was added in response to both reviewers, who noted that the authors should test total cellular levels of GST-Sre1N. The authors conclude that levels are decreased in a similar pattern to the nuclear levels that were determined by microscopy (e.g. page 16, lines 335-337; and stated again in the Discussion). I disagree and suggest changing the text to better reflect the actual results. The immunoblot shows a dramatic decrease in Sre1N levels in the gsk3 gsk31 double mutant cells. Deletion of ssp2 has a minor (if any) effect on these levels, and the authors would need to quantify a better exposure to make this conclusion. This is different from the nuclear localization, where ssp2 mutant had a major effect that was similar to the gsk3 gsk31 double mutant. In comparing the gsk3 gsk31 double mutant versus the ssp2 gsk3 gsk31 triple mutant, there is no additive defect in Sre1N levels and the two strains look identical. These results suggest that Gsk3 and Gsk31 play the primary role in regulating Sre1N protein levels, whereas Ssp2 might regulate nuclear localization of Sre1N. The authors can address this comment by editing the text without additional experiments. The fact that this immunoblot does not perfectly repeat the microscopy experiment is actually quite interesting and reveals the potential for some specific roles for Ssp2 versus Gsk3/31 in regulating Sre1N. I encourage the authors to revise their interpretation to better reflect this nice result.**********7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.Reviewer #1: Yes: Shigeaki SaitohReviewer #2: No[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.23 Jan 2020January 24, 2020PLoS ONEDear Dr. Takashi Toda:Thank you for your e-mail of January 23, 2020 on our manuscript entitled “AMPKα Subunit Ssp2 and Glycogen Synthase Kinases Gsk3/Gsk31 are involved in regulation of sterol regulatory element-binding protein (SREBP) activity in fission yeast” by Hao Miao et al. (PONE-D-19-30640R1). We would like to thank you for your very helpful comments and suggestions, and for inviting us to revise our manuscript. Both reviewers raised the same point that is the interpretation of the immunoblotting data shown in Figure 6D. Accordingly, we revised our manuscript.The revision which addresses reviewer's comment is as follows:Reviewer #1CommentThe criticisms raised against the original version are properly responded, and the manuscript is satisfactorily revised. I have one minor comment regarding the interpretation of the new result of immunoblotting in Figure 6.Minor point:While nuclear accumulation of GFP-Sre1N appears to be diminished to a similar extent in the ssp2 mutant and the gsk3 gsk31 double mutant, the total amount of GST-Sre1N was markedly different in these two mutants (Figure 6B and D). Thus, it seems possible that Ssp2 regulates not only the degradation of Sre1N but also its translocation to the nucleus, whereas Gsks regulate mainly its degradation. It may be better to discuss this possibility.Response:Accordingly, we added some comments on this issue in the revised manuscript (pages 3, lines 46-50 / page 5, lines 99-101 / page 15, line 310 / page 16, lines 334-344 / page 17, lines 353-354 / page 18, lines 379-384, 395 / page 25, lines 512-515).Reviewer #2CommentThe authors have done a nice job revising the manuscript and adding new data to address the reviewer comments. I have one lingering concern related to the new immnunoblot experiment in Figure 6D. This new experiment was added in response to both reviewers, who noted that the authors should test total cellular levels of GST-Sre1N. The authors conclude that levels are decreased in a similar pattern to the nuclear levels that were determined by microscopy (e.g. page 16, lines 335-337; and stated again in the Discussion). I disagree and suggest changing the text to better reflect the actual results. The immunoblot shows a dramatic decrease in Sre1N levels in the gsk3 gsk31 double mutant cells. Deletion of ssp2 has a minor (if any) effect on these levels, andthe authors would need to quantify a better exposure to make this conclusion. This is different from the nuclear localization, where ssp2 mutant had a major effect that was similar to the gsk3 gsk31 double mutant. In comparing the gsk3 gsk31 double mutant versus the ssp2 gsk3 gsk31 triple mutant, there is no additive defect in Sre1N levels and the two strains look identical. These results suggest that Gsk3 and Gsk31 play the primary role in regulating Sre1N protein levels, whereas Ssp2 might regulate nuclear localization of Sre1N. The authors can address this comment by editing the text without additional experiments. The fact that this immunoblot does not perfectly repeat the microscopy experiment is actually quite interesting and reveals the potential for some specific roles for Ssp2 versus Gsk3/31 in regulating Sre1N. I encourage the authors to revise their interpretation to better reflect this nice result.Response:Accordingly, we added these points in the revised manuscript (pages 3, lines 46-50 / page 5, lines 99-101 / page 15, line 310 / page 16, lines 334-344 / page 17, lines 353-354 / page 18, lines 379-384, 395 / page 25, lines 512-515).We hope that these revisions would satisfy your requirements. Thank you very much for your time and consideration.Sincerely,Yue Fang, M.D, Ph.D.Department of Microbial and Biochemical Pharmacy,School of Pharmacy, China Medical University,No.77 Puhe Road, Shenyang North New Area, Shenyang 110112, ChinaTEL: +86-18900910820, FAX: +86-24-31939448Email: yfang@cmu.edu.cnSubmitted filename: Response to Reviewers.docClick here for additional data file.27 Jan 2020AMPKα Subunit Ssp2 and Glycogen Synthase Kinases Gsk3/Gsk31 are involved in regulation of sterol regulatory element-binding protein (SREBP) activity in fission yeastPONE-D-19-30640R2Dear Dr. Fang,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Congratulations.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. 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Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.With kind regards,Takashi Toda PhDAcademic EditorPLOS ONEAdditional Editor Comments (optional):Reviewers' comments:30 Jan 2020PONE-D-19-30640R2AMPKα Subunit Ssp2 and Glycogen Synthase Kinases Gsk3/Gsk31 are involved in regulation of sterol regulatory element-binding protein (SREBP) activity in fission yeastDear Dr. Fang:I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.For any other questions or concerns, please email plosone@plos.org.Thank you for submitting your work to PLOS ONE.With kind regards,PLOS ONE Editorial Office Staffon behalf ofProf. Takashi Toda PhDAcademic EditorPLOS ONE
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