Glucose is necessary and sufficient for TORC1 activation
To investigate the role of TORC1 in glucose signaling, we first tested whether glucose is required for TORC1 activation. To assess the kinetics of TORC1 activation, we tagged the TORC1 substrate Sch9 with 6 copies of the hemagglutinin (HA) epitope. Phosphorylation of Sch9 can be assayed by cleaving it with NTCB (2-nitro-5-thiocyanatobenzoic acid) followed by detecting the electrophoretic mobility of the C-terminal HA-tagged Sch9 fragment by Western analysis [7]. We transferred log-phase wild type and gtr1Δ cells grown in Synthetic Complete medium containing 2% glucose (SC/D) into SC medium lacking glucose (SC-D). After 60’ following transfer, Sch9 was completely dephosphorylated in both wild type and gtr1Δ cells indicating that TORC1 activity requires the presence of glucose in the medium (Fig. 2a). We transferred the glucose-starved cells back into SC medium-containing glucose (SC/D). TORC1 was reactivated immediately upon addition of glucose in the wild type strain (Fig. 2a). However, TORC1 activation in the gtr1Δ strain was delayed by about 10 minutes in comparison to the wild type strain (Fig. 2a). These results suggest that glucose activates TORC1 via Gtr1/2-dependent and Gtr1/2-independent pathways in yeast.
We then tested whether glucose is sufficient for TORC1 activation. We subjected log-phase yeast cells to complete nutrient starvation by washing off all the nutrients and transferring them into 0.3 M sorbitol. TORC1 was inactive after 1 h of incubation in 0.3 M sorbitol (Fig. 2b). We then added either glucose (110 mM) or equimolar amounts of fructose or raffinose or glycerol to starved cells, in the presence and absence of TORC1 inhibitor rapamycin (2 μM). Addition of glucose or fructose or raffinose caused an immediate phosphorylation of Sch9 which was abolished by addition of rapamycin (2 μM) (Fig. 2b). However, addition of glycerol did not activate TORC1. Unlike glucose, both ammonium sulfate and a mixture of all amino acids failed to activate TORC1 in the assay (Fig. 2c). Our results indicate that glucose (or a rapidly fermenting carbon source like fructose/raffinose) is sufficient for TORC1 activation.
We then tested whether glucose-induced TORC1 activation requires its upstream regulator Gtr1. Addition of glucose caused phosphorylation of Sch9 immediately in wild type cells but with a 10-min delay in gtr1Δ cells (Fig. 2d). Sch9 phosphorylation was abolished by rapamycin (2 μM) in both wild type and gtr1Δ cells (Fig. 2d). Rapamycin binds to the peptidyl-prolyl cis-trans isomerase Fpr1, and Rapamycin-Fpr1 complex inhibits TORC1 by binding to Tor1/Tor2 kinase [8]. To determine whether rapamycin at 2 μM specifically inhibits TORC1, we examined the effect of rapamycin on glucose-induced Sch9 phosphorylation in wild type and gtr1Δ strains lacking FPR1 gene. As observed previously, there was a 10-min delay in the onset of Sch9 phosphorylation in the fpr1Δ gtr1Δ strain in comparison to the fpr1Δ strain. However, addition of rapamycin had no effect on Sch9 phosphorylation in both strains confirming that TORC1 is the Sch9-phosphorylating kinase. In summary, our data indicate that glucose is sufficient to activate TORC1 through Gtr1-dependent and Gtr1-independent mechanisms in yeast.
Glucose-induced TORC1 activation is independent of PKA and glucose sensors Snf2 and Rgt2
As PKA is the main regulator of the transcriptional response to glucose in yeast [2], we tested whether glucose-induced TORC1 activation requires PKA activity. Catalytic subunit of PKA is encoded by three genes TPK1-3 in yeast. We constructed an analog-sensitive allele of PKA (pka-as) by deleting TPK3 and introducing gatekeeper mutations namely tpk1-M164G and tpk2-M147G [5]. 1-NM-PP1 completely inhibited the growth of the pka-as strain at 1.5 μM but did not affect the wild type strain indicating that it specifically inhibits PKA activity in the pka-as strain (Additional file 1: Fig. S1a). This was confirmed by monitoring the effect of 1-NM-PP1 on phosphorylation of PKA substrates using an anti-PKA substrate antibody. Addition of 1-NM-PP1 (Concentration range 1.5–25 μM) to pka-as but not wild type (PKA) cells inhibited PKA activity (Additional file 1: Fig. S1b). We tested whether PKA activity is required for glucose-induced TORC1 activation by adding 1-NM-PP1 to wild type and pka-as cells during complete nutrient starvation and following addition of 2% glucose. As expected, 1-NM-PP1 at 1.5 μM inhibited PKA activity in pka-as but not PKA cells (Additional file 1: Fig. S2). Rapamycin (2 μM) had no discernible effect on PKA activity (Additional file 1: Fig. S2). Glucose-induced Sch9 phosphorylation in 1-NM-PP1-treated PKA and pka-as cells were comparable but was completely inhibited by rapamycin (Fig. 2e). These results indicate that glucose-induced TORC1 activation is independent of PKA activity. We also tested whether glucose-induced TORC1 activation is dependent on glucose sensors namely Rgt2 and Snf3 that regulate the transport of glucose into yeast cells [2]. Glucose-induced TORC1 activation was comparable in wild type, rgt2Δ, snf3Δ, and rgt2Δ snf3Δ strains (Fig. 2f) precluding a role for these glucose sensors in glucose-induced TORC1 activation.
TORC1 is activated during glucose response
When glucose is added to yeast cells growing in a medium containing a non-fermentable carbon source, several genes are induced or repressed which restructure the transcriptional and metabolic state of yeast [4]. This phenomenon is referred to as the glucose response and the differentially expressed genes constitute the glucose-responsive genes. We investigated whether TORC1 activity is altered during the glucose response. We grew wild type and gtr1Δ cells to log phase in SC medium containing non-fermentable carbon sources ethanol and glycerol (SC/EG) and added glucose to a final concentration of 2% along with either DMSO or rapamycin (200 nM). Sch9 phosphorylation increased upon addition of glucose to wild type and gtr1Δ cells growing in in SC/EG medium and abolished by rapamycin treatment after 30’ (Fig. 3a). Increase in TORC1 activity upon glucose addition was delayed in gtr1Δ cells as observed previously in our glucose-induced TORC1 activity assays (Fig. 2a, d). We also performed the converse experiment in which we switched the carbon source of log-phase yeast cells from glucose to ethanol-glycerol. TORC1 activity was drastically reduced upon transfer from SC/D to SC/EG in both wild type and gtr1Δ cells (Fig. 3b).
Overlap of TORC1 targets with the glucose-responsive genes
We then explored whether there is any overlap of glucose-responsive genes with TORC1 target genes by analyzing published transcriptomic data. Based on the response to glucose and Ras activation, glucose-responsive genes (N = 3273) were classified into 8 categories I–VIII [4]. Genes belonging to categories I–IV and categories V–VII are activated and repressed by glucose, respectively [4]. Genes in class VIII (N = 804) are not regulated by glucose in wild type cells but only in pka-as cells and were therefore excluded from our analysis. We compared the remaining list of glucose-responsive genes (N = 2469) with the list of 2426 TORC1 target genes reported in a recent transcriptomic study [4, 9]. We found that 58% of glucose-responsive genes (N = 1436; upregulated = 828 and downregulated = 608) were co-regulated by TORC1 (Fig. 3c). Among the 828 genes upregulated by glucose, 807 (98%) genes were positively regulated by TORC1. Likewise, of the 608 genes negatively regulated by glucose, 578 (95%) genes were also negatively regulated by TORC1. Genes regulated by TORC1 were spread across the seven categories of glucose-responsive genes to different extents (Fig. 3d). These observations indicate that addition of glucose and TORC1 activation have similar qualitative effects on the yeast transcriptome.
TORC1 is required for establishment and maintenance of TGC gene expression
We investigated whether TORC1 is required for the transcriptional response to glucose.
We chose 7 TORC1 target genes GFD2 (I), DHR2 (II), CIT1(V), CRC1(V), UGA1(VI), RME1(VI), and GPG1(VII) spread across the top 5 expression categories as representative of “TORC1 and Glucose Co-regulated (TGC)” genes for analysis. The choice of these 7 genes was also informed by transcriptome analysis of TORC1 targets during spore germination (see below). We tested whether TORC1 activity is necessary for glucose-induced changes in expression of the seven TGC genes. For comparison, we tested the role of PKA activity in the transcriptional response to glucose using the pka-as strain. We added glucose (to a final concentration of 2%) to log-phase wild type (PKA) and pka-as cells growing in SC/EG (SC medium containing 2% ethanol and 2% glycerol), in the presence of either DMSO or rapamycin (200 nM) or 1-NM-PP1 (1.5 μM). Expression of all the 7 TGC genes was significantly altered after 30 min following addition of glucose (Fig. 4 and Additional file 1: Fig. S3). As expected, GFD2 and DHR2 genes (belonging to clusters I and II respectively) were upregulated in the presence of glucose (Fig. 4, Additional file 1: Fig. S3 and Additional file 2: Table S1) in both PKA and pka-as cells. Genes GPG1, UGA1, RME1, CIT1, and CRC1 (belonging to clusters V–VII) were downregulated in the presence of glucose (Fig. 4, Additional file 1: Fig. S3 and Additional file 2: Table S1). Glucose-induced changes in the TGC genes were inhibited by 1-NM-PP1 in pka-as cells but not in wild type cells (Fig. 4, Additional file 1: Fig. S3, and Additional file 2: Table S1). There was a non-specific effect of 1-NM-PP1 on expression of DHR2 and GFD2 (Fig. 4 and Additional file 1: Fig. S3). However, 1-NM-PP1 still affected DHR2 and GFD2 expression much more strongly in pka-as cells compared to wild type cells (Fig. 4 and Additional file 1: Fig. S3). These results confirm PKA’s role in the transcriptional response to glucose. Importantly, glucose-induced changes in expression of the 7 TGC genes were also inhibited by addition of rapamycin (Fig. 4, Additional file 1: Fig. S3 and Additional file 2: Table S1). Timing of change in transcript levels in the presence of rapamycin (30’) agrees with timing of TORC1 inhibition observed previously (Fig. 3a). These results indicate that TORC1 and PKA co-regulate the transcriptional response to glucose in yeast.
We then tested whether maintenance of TGC gene expression in glucose-containing medium is dependent on TORC1 activity. We treated log-phase yeast cells growing in YPD medium (with 2 % glucose) with either DMSO or rapamycin (200 nM) and analyzed the expression of the 7 TGC genes by real-time qRT-PCR. Addition of rapamycin affected the expression of all the 7 genes with the transcript levels shifting towards the corresponding expression level in SC/EG medium (Additional file 3: Fig. S4). These results indicate that TORC1 activity is also required for maintaining the expression status of TGC genes during growth in glucose-containing medium (Additional file 3: Fig. S4).
TORC1 regulates the expression of glucose-responsive genes independently of Bcy1 phosphorylation
TORC1 could regulate the expression of glucose-responsive genes indirectly by activating PKA. Indeed, TORC1 has been shown to promote PKA activity by inhibiting phosphorylation of PKA inhibitor Bcy1 at T129 [10]. Phospho-mimetic mutation of T129 (bcy1-T129D) was shown to have an inhibitory effect on PKA activity [10]. If TORC1’s effect on PKA activity via regulating T129 phosphorylation is important for the transcriptional response to glucose, then bcy1-T129D should block TORC1’s role in the glucose response. We added glucose (final concentration = 2%) to BCY1 and bcy1-T129D cultures growing in SC/EG medium and monitored the expression of three TGC genes DHR2, CIT1, and RME1 by real-time qRT-PCR. Rapamycin treatment affected the expression of 3 TGC genes to comparable extents in both wild type and bcy1-T129D strains (Additional file 3: Fig. S5) indicating that TORC1 regulates the glucose-responsive genes independently of Bcy1 phosphorylation at T129.
TORC1 inhibition does not affect PKA activity
TORC1 and PKA have been shown to exert mutually antagonistic effects on their activities [11]. Our previous data indicated TORC1 inhibition had no effect on PKA activity in yeast cells treated with 2% glucose following complete starvation (Fig. 2e and Additional file 1: Fig. S2). To check if this result also extends to actively growing cells, we grew wild type and pka-as cells to logarithmic phase and treated them with either DMSO or rapamycin or 1-NM-PP1. We assayed PKA activity by Western blotting of whole cell extracts using a phospho-specific antibody directed against phosphorylated PKA substrates [10]. As expected, 1-NM-PP1 inhibited phosphorylation of PKA substrates in pka-as cells but not in wild type cells (Additional file 3: Fig. S6a). In contrast, rapamycin treatment did not affect phosphorylation of PKA substrates (Additional file 3: Fig. S6a). However, rapamycin treatment activated expression of TORC1-repressed genes DIP5 and GAP1 confirming inhibition of TORC1 (Additional file 3: Fig. S6b). Our data suggest that TORC1 inhibition does not affect PKA activity.
TORC1 regulates expression of glucose-responsive genes independently of Sch9
As TORC1 appears to regulate the transcriptional response to glucose independently of PKA, we focused our attention on its two downstream effectors Sch9 and Tap42. As PKA and TORC1 co-regulate the glucose response, inactivating the TORC1 effector involved in glucose response is not expected to have a major effect on glucose-induced gene expression. However, rapamycin treatment will have a reduced effect on expression of glucose-responsive genes in the effector mutant strain in comparison to the wild type strain. To assess the role of Sch9 in TORC1-mediated regulation of the TGC genes, we added glucose (2% final) to wild type and sch9Δ cultures growing in SC/EG medium. We assayed the expression of the 7 TGC genes in the presence and absence of rapamycin by real-time qRT-PCR. The 7 TGC genes were either upregulated (GFD2 and DHR2) or downregulated (CIT1, CRC1, UGA1, RME1, and GPG1) in both wild type and sch9Δ strains following the addition of glucose to the medium (Fig. 5, Additional file 2: Table S1 and Additional file 3: Fig. S7). Importantly, addition of rapamycin reversed the glucose-induced changes in TGC gene expression albeit to different extents in wild type and sch9Δ strains. Doubling time of sch9Δ cells is twice that of wild type cells (Additional file 3: Fig. S8). Quantitative differences in the effect of rapamycin on TGC gene expression could be caused by pleiotropic growth defects of sch9Δ. Taken together, our data indicate that TORC1 regulates the glucose-responsive genes independently of Sch9. However, we cannot exclude the possibility that Sch9 facilitates the regulation of glucose-responsive genes either via TORC1 or independently of TORC1.
TORC1 regulates expression of glucose-responsive genes via Tap42
To test the role of Tap42 in the expression of glucose-responsive genes, we used tap42-11 a temperature-sensitive allele of TAP42 [12]. We grew wild type and tap42-11 cells to mid-log phase at 25 °C (permissive temperature) in SC/EG medium and transferred them to 37 °C (non-permissive temperature) for 30’ to inactivate Tap42. We then added 2% glucose to the cultures along with either rapamycin or DMSO and assayed the expression of the 7 TGC genes by real-time qRT-PCR. Induction of GFD2 and DHR2 expression after 30’ following addition of glucose was severely inhibited in tap42-11 cells (Fig. 6, Additional file 3: Fig. S9 and Additional file 2: Table S1). Furthermore, addition of rapamycin had a reduced effect on GFD2 and DHR2 expression in tap42-11 cells in comparison to wild type cells suggesting that TORC1 regulates GFD2 and DHR2 expression mainly via Tap42. For the remaining 5 TGC genes, addition of glucose affected their expression to comparable extents in both wild type and tap42-ts strains (Fig. 6, Additional file 2: Table S1 and Additional file 3: Fig. S9). Interestingly, addition of rapamycin had little or no effect on expression of GPG1, UGA1, RME1, and CIT1 genes in tap42-ts cells (Fig. 6, Additional file 2: Table S1 and Additional file 3: Fig. S9). In addition, the effect of rapamycin on CRC1 expression in tap42-11 cells was reduced by 3-10-fold at 120’ in comparison to wild type cells (Additional file 2: Table S1). These results indicate that TORC1 regulates glucose-driven changes in the TGC genes through Tap42.
Rrd1/Rrd2 and Sit4 proteins are required for TORC1’s role in the transcriptional response to glucose
Tap42 interacts with the catalytic subunit of PP2A phosphatases (PP2Ac) and PP2A-like phosphatase (Sit4) in log-phase cells but not stationary phase cells and keeps them inactive and localized to the vacuole [13]. Upon starvation (or rapamycin treatment), the phosphatases dissociate from Tap42 and dephosphorylate their target proteins in collaboration with two phosphotyrosyl phosphatase activator (PTPA) proteins Rrd1 and Rrd2 [14]. We tested whether the TORC1-mediated regulation of TGC genes is dependent on PP2A-like phosphatase Sit4, Rrd1, and Rrd2. As sit4Δ and rrd1Δ rrd2Δ strains cannot grow in the presence of glycerol as a carbon source, we tested the roles of Sit4 and Rrd1/2 in the maintenance of TGC gene expression. We treated wild type, sit4Δ, rrd1Δ, rrd2Δ, and rrd1Δ rrd2Δ cells growing in YPD (with 2% glucose) medium with either DMSO or rapamycin and assessed the expression of TGC genes by real-time qRT-PCR. As expected, rapamycin treatment decreased the expression of GFD2 and DHR2 genes and increased the expression of CIT1, CRC1, UGA1, RME1, and GPG1 genes in wild type cells. However, rapamycin-induced changes in expression of TGC genes were reduced considerably in rrd1Δ rrd2Δ and sit4Δ cells (Fig. 7, Additional file 2: Table S1 and Additional file 3: Fig. S10 ). Rapamycin-induced changes in TGC gene expression were slightly reduced in the rrd1Δ strain but unaffected in the rrd2Δ strain. However, the rapamycin-induced changes in the rrd1Δ rrd2Δ strain were further reduced compared to the rrd1Δ strain (Fig. 7, Additional file 2: Table S1 and Additional file 3: Fig. S10) suggesting that Rrd1 and Rrd2 proteins play overlapping roles in TORC1-mediated regulation of TGC genes. Taken together, our results are consistent with the hypothesis that TORC1 regulates the glucose-responsive genes by inhibiting the activities of PP2A/Sit4/Rrd1/Rrd2 phosphatases via Tap42.
To understand how Tap42/PP2A/Sit4 module regulates the transcriptional response to glucose, we evaluated the roles of RTG1, NNK1, GAT1, and GLN3 that are targets of the Tap42/PP2A pathway in yeast [15]. RTG1 is a member of the basic helix-loop-helix–leucine zipper (bHLH/Zip) family of transcription factors that is required for activation of the retrograde response pathway [16]. Rtg1 localizes to the nucleus in the presence of a poor nitrogen source [16]. Activation of RTG1 target genes requires Tap42 and Sit4 function [17]. Gln3 and Gat1 belong to GATA family of zinc-finger transcriptional activators that localize to the cytoplasm under nitrogen-rich conditions. TORC1 favors the cytoplasmic retention of Gln3 and Gat1 by promoting their phosphorylation and interaction with a cytosolic protein called Ure2. Under nitrogen-limiting conditions, TORC1 activity is reduced leading to activation of Sit4. Gln3 and Gat1 are dephosphorylated by Sit4 resulting in dissociation from Ure2 and translocation to the nucleus and expression of nitrogen catabolite repression (NCR) genes [18]. Nnk1 is a protein kinase that physically interacts with TORC1 and Ure2 and overexpression of Nnk1 results in constitutive targeting of Gln3 to the nucleus [19].
We treated wild type, rtg1Δ, nnk1Δ, gat1Δ, gln3Δ, and rrd1Δ rrd2Δ cells growing in YPD (with 2% glucose) with either DMSO or rapamycin and assessed the expression of TGC genes by real-time qRT-PCR. As expected, rapamycin-induced changes in TGC gene expression were severely reduced in rrd1Δ rrd2Δ cells in comparison to wild type cells (Additional file 3: Fig. S11). TORC1-regulated expression of UGA1 was inhibited in gat1Δ and gln3Δ cells (Additional file 3: Fig. S11). TORC1-regulated expression of CRC1 was reduced in rtg1Δ cells. nnk1Δ had no effect on TORC1-regulated expression of TGC genes. These results suggested that Gat1, Gln3, and Rtg1 could regulate a subset of glucose-responsive genes.
To test if Gln3, Gat1, and Rtg1 are directly involved in the glucose response, we examined their localization in SC-EG medium and upon addition of glucose (2%) to SC-EG medium. GFP-tagged Gln3, Gat1, and Rtg1 cells were grown to logarithmic phase in SC-URA/EG medium and then glucose (2% final concentration) was added to the cultures in the presence of either rapamycin (200 nM) or DMSO. Nuclear and cytoplasmic distribution of GFP-tagged Gln3, Gat1, and Rtg1 was determined by fluorescence microscopy. A small proportion of cells (~ 10%) in SC-URA/EG medium contained nuclear/nucleocytoplasmic staining of Rtg1. However, Gln3, Gat1, and Rtg1 were cytoplasmic in the majority of cells growing in SC-URA/EG medium and remained cytoplasmic upon addition of the glucose to the cultures (Additional file 3: Fig. S12). In contrast, Gln3, Gat1, and Rtg1 localized to the nucleus upon rapamycin treatment (Additional file 3: Fig. S12). Growth of yeast cells in the absence of glucose and with ammonia as the nitrogen source has been shown to trigger weak nitrogen stress [20] which could account for a few cells with nuclear Rtg1 staining in SC-URA/EG grown cultures (Additional file 3: Fig. S12). Taken together, these results indicate that Gln3, Gat1, and Rtg1 do not directly regulate the transcriptional response to glucose. The observed effect of deleting the 3 transcription factor genes on expression of a subset of glucose-responsive genes (Additional file 3: Fig. S11) is indirect.
TORC1 is activated during spore germination
To test the physiological importance of TORC1’s role in glucose signaling, we investigated the function of TORC1 in spore germination. Return of spores into vegetative growth cycle upon their transfer to favorable nutrient medium is referred to as spore germination. Glucose (or a fermentable carbon source) is essential for efficient spore germination [21]. Glucose alone is sufficient to trigger spore germination [21]. Moreover, spores are only responsive to glucose during the early stages of germination. Consistent with this, transcriptional changes induced upon transfer of spores into rich medium and glucose are strikingly similar during the initial stages of spore germination [1, 22].
We first tested whether TORC1 is activated upon transfer of spores into nutrient medium. We transferred stationary phase wild type and gtr1Δ diploid cells expressing HA-tagged Sch9 pre-grown in nutrient medium (YPD) grown for 16–20 h into sporulation medium. After 84 h of incubation at 30 °C in sporulation medium, more than 90% of diploid cells had sporulated. We purified wild type and gtr1Δ spores and transferred them into nutrient medium (YPD) in the presence of rapamycin (2 μM) or DMSO. Sch9 was completely dephosphorylated in spores indicating that TORC1 is inactive in the spore (Fig. 8a). Sch9 was phosphorylated immediately after transfer of wild type spores into nutrient medium but with a delay in gtr1Δ spores (Fig. 8a). Addition of rapamycin to the nutrient medium inhibited Sch9 phosphorylation in both wild type and gtr1Δ cells (Fig. 8a) indicating that TORC1 is activated following transfer of spores into nutrient medium.
TORC1 regulates the glucose-responsive genes during spore germination
To test whether TORC1 regulates the glucose-responsive genes during spore germination, we performed transcriptomic analysis of germinating spores transferred to nutrient medium in the presence and absence of rapamycin. In addition to purified spores, we collected germinating yeast spores at different time points (0’, 10’, 30’, 60’, 120’, 240’ and 360’) following their transfer into nutrient medium (in the presence and absence of rapamycin) and analyzed their transcriptomes by RNA-Seq (Fig. 8b). Our RNA-Seq results are largely consistent with previous transcriptomic analyses of spore germination [1, 22]. Ten classes of genes namely protein synthesis, rRNA processing, gluconeogenesis, TCA sub-cycle, stress, oxidative phosphorylation, proteasome subunits, mating, and cell cycle G1 and cell cycle G2/M were reported to be differentially expressed during spore germination [1] (Additional file 4: Table S2). We found that the aforementioned 10 classes of genes were also differentially expressed following the onset of germination in our experiment (Additional file 5: Fig. S13, Additional file 6: Table S3 and Additional file 7: Table S4).
To determine whether TORC1 regulates the transcriptome during spore germination, we compared the transcriptomes of DMSO- and rapamycin-treated spore germination cultures (Additional file 8: Table S5). There was no significant difference between the transcriptomes at t = 30’ between the two cultures (Fig. 8c and Additional file 8: Table S5). However, a significant difference in gene expression between the two cultures was observed after 1 h following transfer of spores into rich medium (Fig. 8c). Around 1102 genes (503 upregulated and 599 downregulated) were found to be differentially expressed (by at least 2-fold) between the two cultures (Fig. 8c and Additional file 8: Table S5). This timing is consistent with our observation that TORC1 activity as measured by Sch9 phosphorylation is inhibited by rapamycin after 1 h following transfer of spores into nutrient medium (Fig. 8c).
We compared our list of 1102 TORC1 targets with the list of TORC1 targets obtained from a microarray analysis-based study [23] and a RNA-Seq study [9] both performed with vegetative cells. Only 90 (18%) of the TORC1 targets identified in the microarray-based study [23] were in our set (Additional file 9: Fig. S14). However, about 935 (84.8%) of the TORC1 targets identified by RNA-Seq [9] were in our target list (Additional file 9: Fig. S14) indicating that the targets of TORC1 are largely similar during spore germination and vegetative growth. We do not know the reason for differences in the TORC1 targets predicted by microarray and RNA-Seq methods but the differences in sensitivities of the two transcriptomic approaches [24] could be a contributing factor.
Expression of the 7 TGC genes during spore germination was affected by rapamycin treatment (Additional file 8: Table S5). To confirm the RNA-seq data, we compared the expression of 7 TGC genes in DMSO- and rapamycin-treated germinating spore cultures after 0’, 30’, 60’, and 120’ following transfer to nutrient medium by real-time qRT-PCR. DHR2 and GFD2 genes were upregulated following spore germination, and the genes CIT1, GPG1, RME1, CRC1, and UGA1 were downregulated following transfer of spores into nutrient medium (Additional file 9: Fig. S15). These changes in gene expression mirrored the transcriptional changes that occurred when glucose was added to yeast cells growing in medium containing ethanol and glycerol. Importantly, changes in expression of 7 TGC genes were inhibited by addition of rapamycin (Additional file 9: Fig. S15). These results indicate that TORC1 regulates the glucose-responsive genes during spore germination.
TORC1 is essential for spore germination
Glucose is essential for efficient germination of yeast spores [21]. As TORC1 regulates the glucose-responsive genes during spore germination, we tested whether TORC1 is required for spore germination. We transferred wild type spores into nutrient medium in the presence of either rapamycin (2 μM) or DMSO (mock-treated) and followed the kinetics of spore germination by bright-field microscopy (Fig. 9a, b). After 2–3 h following transfer into nutrient medium, spores increased in volume and adopted a pear-shaped appearance (Fig. 9a, b). The spores elongated further and displayed a distinct constriction after 4 h. The bud emerged from the mother cell subsequently and detached from the mother cell resulting in the first mitotic division after about 7–8 h following transfer to nutrient medium (Fig. 9a, b). However, in the presence of rapamycin, spore germination was severely inhibited. After 6 to 8 h following rapamycin treatment, 50% of spores had become enlarged but they did not form a constriction seen in untreated cultures and failed to undergo the first mitotic division (Fig. 9a, b). To test whether the effect of rapamycin on spore germination is due to inhibition of the TORC1 complex, we introduced the TOR1-1 mutation that confers resistance to rapamycin [8]. Rapamycin had no effect on germination of TOR1-1 spores indicating that the effect of rapamycin on spore germination was due to specific inhibition of TORC1 (Fig. 9b). In contrast to wild type spores, both fpr1Δ and TOR1-1 spores germinated efficiently in the presence of rapamycin (Additional file 9: Fig. S16).
We tested whether TORC1 is required for DNA replication, an event that occurs during final stages of spore germination [1]. We transferred purified wild type spores into nutrient medium in presence and absence of rapamycin and monitored DNA replication by flow cytometry. The intensity of propidium iodide (PI) staining of spores was lower than that of mitotically growing haploid cells presumably because of reduced permeability to PI caused by the presence of spore wall. However, the wild type spores replicated their DNA between 6 and 8 h as indicated by the appearance of cells with increased PI signals. In contrast, the PI signals of rapamycin-treated spores remained unchanged during the course of the experiment indicating that they did not undergo DNA replication (Fig. 9c). We also confirmed that the expression of G1 cell cycle genes (CLN1, CLN2, CLB6, EGT2, PCL1, PCL9, CHS2, and CTS1) expressed after 4 h of germination was inhibited by rapamycin (Additional file 9: Fig. S17). Taken together, our data indicate that TORC1 is required for spore germination in budding yeast.
Effect of TORC1 inhibition on spore germination is dependent on Rrd1
If TORC1’s role in regulating the glucose-responsive genes is essential for spore germination, then inactivating the Tap42/Sit4/Rrd1/Rrd2 branch should allow spores germinate even in the presence of rapamycin. As tap42-11, rrd1Δ rrd2Δ, and sit4Δ mutants failed to sporulate, we tested the effect of rapamycin treatment on germination of rrd1Δ spores. We took wild type and rrd1Δ spores and transferred them to nutrient medium in the presence and absence of rapamycin. Interestingly, rrd1Δ spores germinated earlier in comparison to wild type spores as indicated by the formation of budded cells (Additional file 9: Fig. S18). While the germination of wild type spores was inhibited by rapamycin treatment, rrd1Δ mutant spores were able to form budded cells in the presence of rapamycin (Additional file 9: Fig. S18). Our results demonstrate that TORC1’s role in the regulation of glucose-responsive genes via the Tap42/ Sit4/Rrd1/2 pathway is essential for spore germination.