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1 Division of Integrated Life Sciences, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan
2 Institute for Advanced Technology, Suntory Research Center, Osaka 618-8503, Japan
3 Division of Gene Research Center for Biological Resources and Informatics, Tokyo Institute of Technology, Yokohama 226-8501, Japan
4 School of Life Sciences and Biotechnology, Korea University, Anam-Dong, Sungbuk-ku, 136-701 Seoul, Korea
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Abstract |
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subunit are required for both maintenance and modulation of cell size in response to glucose. In the presence of glucose, mutants lacking GPR1 or GPA2 gene showed smaller cells than the wild-type strain. Physiological studies revealed that protein synthesis rate was reduced in the mutant strains indicating that reduced growth rate, while the level of mRNAs for CLN1, 2 and 3 was not affected in all strains. Gene chip analysis also revealed a down-regulation in the expression of genes related to biosynthesis of not only protein but also other cellular component in the mutant strains. We also show that GPR1 and GPA2 are required for a rapid increase in cell size in response to glucose. Wild-type cells grown in ethanol quickly increased in size by addition of glucose, while little change was observed in the mutant strains, in which glucose-dependent cell cycle arrest caused by CLN1 repression was somewhat alleviated. Our study indicates that the yeast G-protein coupled receptor system consisting of Gpr1 and Gpa2 regulates cell size by affecting both growth rate and cell division. |
Introduction |
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A number of studies suggest that the cAMP pathway regulates cell size. Mutant cells with reduced activity in the cAMP pathway were small in size. The cdc25-1 mutant strain, which has temperature-sensitive mutation in guanine nucleotide exchange factor for Ras proteins, showed a smaller critical cell size required for budding (Baroni et al. 1989). Also, the tpk1Wtpk2 tpk3 bcy1 quadruple mutant strain, which has weak but constitutive cAMP-dependent protein kinase (PKA) activity (Cameron et al. 1988), had a reduced cell volume (Tokiwa et al. 1994). In contrast, the activation of this pathway results in large cells. Constitutive activated allele of RAS2, which encodes a small GTP-binding protein known to activate adenylate cyclase in yeast S. cerevisiae, showed increased cell mass (Baroni et al. 1989). The deletion of both PDE1 and PDE2, which encode 3'-5'-cyclic nucleotide phosphodiesterases, resulted in a high concentration of cellular cAMP and increased cell volume (Mitsuzawa 1994). Moreover, exogenously added cAMP resulted in an increase in minimal budding size (Baroni et al. 1992; Tokiwa et al. 1994). Glucose has been shown to affect the cellular cAMP level. When glucose is added to cells grown in a non-fermentable carbon source, a transient rise in the intracellular cAMP level called cAMP spike is observed followed by a resetting of the basal cAMP to a higher level (Beullens et al. 1988; Russell et al. 1993; Tokiwa et al. 1994). Although it is still unclear whether the cAMP spike or increased basal cAMP level or both are critical for the cellular response to glucose, it has been thought that the cAMP pathway is activated by glucose. The activated cAMP pathway was shown to modulate cell size through the regulation of G1 cyclins. The G1-S transition in the cell cycle is largely regulated by the activity of the G1 cyclin-Cdc28 cyclin-dependent protein kinase complex, and thus an abundance of G1 cyclins is a key regulator. Among G1 cyclins, Cln1 and Cln2 appear to make a specific contribution to the initiation of the cell cycle (Hadwiger et al. 1989; Richardson et al. 1989; Wittenberg et al. 1990), while Cln3 functions for the timely expression of CLN1, CLN2 and other G1-specific genes (Tyers et al. 1993; Dirick et al. 1995; Stuart & Wittenberg 1995). The addition of glucose to cells grown in a non-fermentable carbon source represses CLN1 transcription (Tokiwa et al. 1994), and exogenously added cAMP represses both CLN1 and CLN2 expression (Baroni et al. 1994; Tokiwa et al. 1994). Thus, the glucose signal has been thought to regulate cell size mainly through the transcriptional repression of CLN1. But how cells monitor the glucose and control cell size has not yet been clarified.
Several studies revealed a novel glucose sensing machinery in S. cerevisiae consisting of Gpr1 receptor and Gpa2 G subunit. GPA2 gene was isolated on the basis of its similarity to mammalian G proteins (Nakafuku et al. 1988) and Gpr1 was identified in a two-hybrid screen via its ability to interact with Gpa2 (Yun et al. 1997; Xue et al. 1998). Also, a recent study revealed Gpa2-interacting proteins Gpb1, Gpb2, and Gpg1. Gpb1 and Gpb2 were shown as unusual proteins which contain seven kelch repeats that may mimic Gß subunit instead of seven WD-40 repeats normally found in the mammalian Gß subunits (Harashima & Heitman 2002). It has been shown that glucose signaling pathway regulates pseudohyphal development and invasive growth in S. cerevisiae. The mutant lacking either GPR1 or GPA2 gene showed defect in pseudohyphal development and invasive growth suggesting positive contribution of glucose signal on filamentation (Kubler et al. 1997; Lorenz & Heitman 1997; Lorenz et al. 2000; Tamaki et al. 2000). In contrast, Gpb1 and Gpb2 inhibit morphogenesis (Harashima & Heitman 2002). Also it was shown that homolog of Gpr1 and Gpa2 in Candida albicans were required for hyphal formation and morphogenesis (Sanchez-Martinez & Perez-Martin 2002; Miwa et al. 2004). Because these defects in morphogenesis were suppressed by exogenously added cAMP or dibutyryl-cAMP in both S. cerevisiae and C. albicans, Gpr1 and Gpa2 were shown to regulate cell differentiation by cAMP dependent mechanism.
In this study, we present evidence that glucose signaling pathway consisting of Gpr1 and Gpa2 regulates glucose-dependent cell size in S. cerevisiae.
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Results |
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First, we examined the cell size distributions of either wild-type, gpr1 mutant or gpa2 mutant strain. Each strain was grown in glucose (YPD) or ethanol (YPE) medium to mid-log phase and cell size distribution was measured with a Coulter Multisizer II. The strains showed mostly the same cell size distribution when grown in YPE medium (Fig. 1B), while in the YPD medium, the size distribution of the gpr1 and gpa2 mutant cells were smaller than that of the wild-type cells (Fig. 1A).
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The regulation of cell size is chiefly a function of both growth and division, so we examined which is affected in the mutant strains. In the budding yeast, the G1-S transition is regulated by G1 cyclins. At first, we measured the message level of G1 cyclins in the wild-type and mutant strains. Total RNA was prepared from cells grown in either YPD or YPE medium to early log phase and subjected to Northern blot analysis. As described (Flick et al. 1998), the CLN1 message level was higher in YPE-grown cells than in YPD-grown cells in all strains tested (Fig. 3A,B), while no difference in the message level of CLN1, 2, and 3 was observed between the strains (Fig. 3A,B). We also measured the G1 cyclin levels. We used a triple hemagglutinin (3HA)-tagged construct to determine Cln protein by Western blot analysis. The wild-type and mutant strains carrying different 3HA-tagged proteins were grown in YPD medium to early log phase and the Cln protein levels were measured. Mostly the same protein levels of Cln1–3HA and Cln2–3HA were observed in both wild-type and mutant strains (Fig. 3C,D). However, Cln3–3HA levels were slightly higher in the mutant strains (Fig. 3C,D), suggesting a possible contribution of higher Cln3 levels to the smaller cell size in the mutant strains.
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In S. cerevisiae, when glucose is added to cells growing in a poor carbon source, the critical cell size required for cell division is quickly reset from small to large (Alberghina et al. 1998). Then, we tested using a synchronized population of G1 cells if GPR1 and GPA2 are required for a rapid response to glucose addition. Cells grown in YPE medium were harvested at early log phase and unbudded small G1 cells were fractionated by elutriation centrifuge. Harvested G1 cell cultures were divided into two portions and glucose was added to one portion at a final conc. of 2%. After two hours incubation at 30 °C, cell size was measured using Coulter Multisizer II (Fig. 5). In the wild-type strain, cell size quickly increased after glucose was added whereas it did not change in the gpr1 or gpa2 mutant strain with or without glucose. These results suggest that GPR1 and GPA2 are required for glucose-dependent rapid increase in cell size.
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Our results that GPR1 and GPA2 are required for the glucose-dependent increase in cell size led us to determine if GPR1 and GPA2 are required for glucose-dependent cell cycle arrest. Cells were grown in YPE medium to OD600 = 0.3 and G1 cells were prepared by elutriation centrifuge. The cell cultures were divided into two portions and glucose was added to one portion at a final concentration of 2% and further incubated at 30 °C with shaking. Samples were taken every 30 min and fixed in 70% ethanol at –20 °C overnight. Cells were stained with propidium iodide, then the DNA content of each cell was analyzed with a FACScan flow cytometer (Becton Dickinson). In the wild-type strain, a transient cell cycle delay was observed at 1–1.5 h after glucose addition, whereas in the gpr1 and gpa2 mutant strains, no apparent delay in cell cycle progression was observed (Fig. 6A). We also measured the budding index under a microscope (Fig. 6B). In the wild-type strain, the budding index decreased when glucose was added to the medium, while in the mutant strains, no difference was observed in the cells grown with or without glucose.
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Previous reports indicated that the activation of the cAMP pathway by glucose represses the expression of CLN1 and coregulated genes to inhibit Start (Baroni et al. 1994; Tokiwa et al. 1994). Because Gpr1 was shown to regulate cAMP levels in response to glucose, we examined the possibility that Gpr1 is involved in the glucose-dependent cell cycle regulation. Glucose was added to cells growing in raffinose medium (YPR) and transcript levels of CLN1 and CLN2 were measured. In the wild-type strain, levels of CLN1 mRNA dropped quickly in response to glucose and increased gradually as previously described (Tokiwa et al. 1994) (Fig. 7), while in the gpr1 and gpa2 mutant strains, only a slight decrease in the level of CLN1 was observed. These results suggest that the defects in glucose-dependent rapid cell size change found in the mutant strains are mainly the result of the inability of transient decrease in CLN1 transcript in response to glucose. Unlike CLN1, the levels of CLN2 mRNA were unchanged in both wild-type and the mutant strains. Also, the gene expression of SUC2, which encodes invertase also known to be repressed by glucose (Carlson & Botstein 1982), was not affected in the mutant strains.
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To better understand the glucose-dependent regulation of cell size, we carried out genome-wide expression analyses using DNA microarrays. The GeneChip methodology developed by Affymetrix was employed to monitor global gene expression in the wild-type, gpr1 and gpa2 strains grown in YPD medium. Approximately 7000 ORFs containing recognized and nonannotated ORFs, as suggested by SAGE analysis (Velculescu et al. 1997), were analyzed. The ORFs whose transcripts decreased or increased more than 1.5-fold (in at least three experiments out of four) in both the gpr1 and gpa2 mutant strains, compared with the wild-type strain, are listed in Table 2 in which ORFs with more than a 3-fold change in the gene expression level are underlined. There are 79 genes whose expression was reduced in both mutant strains (Table 2). The genes related to the biosynthesis of amino acids and the metabolism of either amino acid, phosphate, carbohydrate or lipid are included. Genes for protein synthesis including the transcription of rRNA, tRNA and mRNA, ribosome biogenesis, and translation were also involved. These results suggest that the reduced levels of 35S-Met and 35S-Cys incorporation into protein found in the mutant strains (Fig. 4) were caused at least in part by reduced levels of transcripts for many genes related to protein synthesis. These results are consistent with the report that the activation of the cAMP pathway induces expression of the genes encoding ribosomal protein (Klein & Struhl 1994; Neuman-Silberberg et al. 1995). There were also 100 ORFs whose expression was increased in both mutant strains. Surprisingly, 41 of these genes encode mitochondrial proteins, which are encoded by nuclear but not mitochondrial DNA (Table 2). These results are consistent with the recent finding that the activated cAMP pathway negatively regulates the gene expression of mitochondria-related proteins (Dejean et al. 2002). Our data also revealed that 10 genes related to respiration are induced in the mutant strains. It has been reported that the cAMP pathway negatively influences sporulation, carbohydrate storage, gluconeogenesis, and the glyoxylic cycle (Thevelein & de Winde 1999). Gene chip data also support these findings because five genes related to carbohydrate reserves and four genes related to gluconeogenesis are induced in the mutant strains. These results indicate that the gpr1 and gpa2 mutant strains express genes to adapt to a postdiauxic shift even in the presence of glucose, because these mutants cannot sense glucose. To confirm the results obtained by GeneChip analysis, we carried out Northern blot analysis of some of the genes listed in Table 2. Wild-type, gpr1 and gpa2 mutant strains were grown in YPD medium to early log phase and total RNA was prepared for Northern blot analysis. Both mutant strains showed reduced expression level of AQY2, CMK2, HIS4, HSP30 and PHO5 compared to the wild-type strain, while increased expression level of CLB1, GPH1, HXK1, PEX2, and QCR2 was observed in the mutant strains (Fig. 8). These results are in good agreement with our GeneChip analysis (Table 2) and support our model that Gpr1-Gpa2 signaling complex regulates many genes related to biosynthesis of proteins and other cellular components to maintain large cell size.
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Discussion |
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Cell division requires the activity of G1 cyclins complexed with the cyclin-dependent kinase and thus the amount of G1 cyclins is critical for promoting cell cycle progression (Cross 1988; Nash et al. 1988; Hadwiger et al. 1989). A previous study using synchronous cultured cells indicated that the repression of cell cycle progression by glucose was mainly the result of the transcriptional repression of CLN1 (Flick et al. 1998). Thus the repression of the CLN1 gene by glucose could be a major factor in the enlargement of cells grown in glucose. In our experiment, however, a difference in the mRNA level of G1 cyclins including CLN1, CLN2, and CLN3 was hardly observed in either the wild-type, gpr1, or gpa2 mutant strains grown in glucose (Fig. 3). Although we could not detect a difference in the G1 cyclin message levels between the wild-type and the mutants, these results do not exclude the possible involvement of Gpr1 and Gpa2 in glucose-dependent transcriptional regulation of G1 cyclins in the control of cell size, because GPR1 as well as GPA2 was shown to be required for glucose-dependent CLN1 repression when glucose was added to cells grown in YPR (Fig. 7).
It was previously reported that the levels of Cln3 protein were higher in cells growing in glucose than in cells grown in non-fermentable carbon sources (Hall et al. 1998), and it was concluded that glucose positively regulates the Cln3 protein levels. In the present study, we find that Cln3 protein level was slightly higher in the mutant strains than in the wild-type strain when grown in glucose (Fig. 3). Although it is unclear if the slightly increased Cln3 protein levels contribute to the smaller cell size found in the mutant strains, because the levels of Cln1 and Cln2 were unchanged in both wild-type and the mutant strains (Fig. 3), our results indicate that Gpr1-Gpa2 signaling pathway does not contribute to glucose-dependent increase of Cln3. This result is consistent with the recent report that the induction of CLN3 by glucose requires the transport and metabolism of glucose but does not require the nutrient signaling pathways including cAMP-pathway (Newcomb et al. 2003).
The rate of protein synthesis has also been thought to affect cell size. It was reported that cAMP activates protein synthesis by increasing the expression of the genes involved in translation in a manner dependent on the transcription factor Rap1 (Klein & Struhl 1994; Neuman-Silberberg et al. 1995). We found that protein synthesis rate in the gpr1 and gpa2 mutant strains was reduced to about 80% of that in the wild-type strain in the media containing glucose (Fig. 4), suggesting that the possible contribution of reduced activity in cAMP pathway to smaller sized cells. Also it was reported that glucose depletion rapidly inhibited protein synthesis at the translational level and this inhibition was overcome in the mutant strain with reduced PKA activity (Ashe et al. 2000). Although it is unclear whether the Gpr1-Gpa2 signaling pathway also affects this system because resistance to translational inhibition on glucose withdrawal was not observed in the gpr1 and gpa2 mutant strains (Ashe et al. 2000), it is possible that a slight decrease in translation could affect cell size in the mutant strain.
Thus, these results together with observations by others indicate that glucose signaling through Gpr1-Gpa2 might regulate the rate of both cell growth and cell division.
In this study, we also observed two different effects of glucose on cell size through Gpr1-Gpa2 signaling pathway. One is long-term effect, which was found as the maintenance of large cell size in the presence of glucose. The other is short-term effect, which was observed as a quick size adaptation of cells grown in non-fermentable carbon source to newly added glucose. In the long-term effect, we found that the CLN1 transcript level was mostly the same between wild-type and mutant strains grown in glucose (Fig. 3), but that protein synthesis rate was decreased in the mutant strains (Fig. 4). Thus, the protein synthesis rate but not CLN1 level may cause cell size difference in the long-term effect. In the short-term effect, we observed transient decrease of CLN1 transcript in the wild-type strain but not in the mutant strains when glucose was added to the cells grown in raffinose (Fig. 7), suggesting a possible contribution of CLN1 level to quick cell size change. Similar effect of glucose has been reported as the glucose-dependent cAMP spike in which transient increase of cAMP was observed when glucose was added to glucose-starved cells. Because GPR1 and GPA2 were shown to be required for glucose-dependent cAMP spike (Yun et al. 1998; Kraakman et al. 1999; Lorenz et al. 2000), we hypothesize that glucose-dependent CLN1 repression may require transient increased level of cellular cAMP. This hypothesis may be supported by the previous report that the addition of cAMP transiently represses CLN1 transcript level (Baroni et al. 1994; Tokiwa et al. 1994).
The GeneChip analysis confirmed that the Gpr1-Gpa2 signaling pathway positively regulates protein synthesis at the transcriptional level (Table 2). Also, many other genes were shown to be regulated by this pathway. The expression of AQY2, which encodes aquaporin permeable to water, was reduced more than 3-fold in the mutant strains. There are two homologous genes, AQY1 and AQY2, found in S. cerevisiae. Although both Aqy1 and Aqy2 are nonfunctional in the laboratory strain S288c and W303-1A, this result is in agreement with the report that the expression level of the AQY2 gene was reduced in the tpk2 mutant strain (Robertson et al. 2000).
The GeneChip experiment also revealed that the Gpr1-Gpa2 signaling pathway negatively regulates gene expression involved in the postdiauxic shift. Many genes for mitochondrial biogenesis, respiration, carbohydrate storage, and gluconeogenesis were up-regulated in the mutant strains, which agrees with the reports that the cAMP pathway negatively regulates these genes (Thevelein & de Winde 1999; Dejean et al. 2002).
The experimental data obtained in this study indicates that the Gpr1-Gpa2 signaling pathway regulates the glucose-dependent change in cell size. However, this is not the only factor responsible for the large cell size on glucose, because even in the gpr1 and gpa2 mutant strains, cells grown with glucose were larger than those with ethanol (Fig. 1). These results suggest that the cell size modulation by nutrient is regulated by several pathways in a redundant manner. Glucose is the preferred carbon source for producing energy, and its incorporation is strictly regulated by the gene expression of many hexose transporters. The incorporation of glucose into cells instantly represses the expression of genes related to the metabolism of other carbon sources and so the glucose is metabolized preferentially, which is known as glucose repression (Gancedo 1998; Carlson 1999). Recent global profiling of the yeast transcriptome confirmed that another glucose sensing pathway consisting of Snf3 and Rgt2 glucose sensors mainly regulates gene expression of HXTs encode glucose transporters (Kaniak et al. 2004). The fact that glucose is the most preferred carbon source, which activates many metabolic pathways in yeast, supports the idea that the activated metabolic pathways also regulate cell size. This idea is consistent with our findings that the rate of protein synthesis was higher in cells grown in glucose than those in ethanol in both wild-type and mutant strains (Fig. 4).
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Experimental procedures |
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The basic culture medium used for S. cerevisiae was YPD medium containing 1% Bacto-yeast extract, 2% peptone and 2% glucose (Sherman 1991). Other carbon sources were also used. YPE medium contains 2% ethanol and YPR medium contains 2% raffinose instead of glucose. The synthetic medium contains 0.67% yeast nitrogen base without amino acids (Difco) and either 2% glucose (SD) or ethanol (SE) and amino acids as required (Sherman 1991). Synthetic complete medium, SCD or SCE, contains SD or SE, respectively, supplemented with adenine, uracil and amino acids. The media were solidified with 2% Bacto-agar for plates.
Plasmid construction
An integration vector pRS406 was cleaved with EcoRI and HindIII to remove the EcoRV site in the multicloning site, then it was blunted and self ligated to produce pRS406-1. A 3.8kb PstI fragment containing the GPR1 gene from GPR1/pUC119 (Yun et al. 1998) was blunted and ligated into EcoRI-HindIII site (blunted) of pRS406 to produce GPR1/pRS406. A GPA2 genomic clone (GPA2/pUC19) was isolated from the SmaI-digested genomic library constructed in pUC19, which contained the 2kb SmaI fragment. The SmaI fragment containing GPA2 was also ligated into PvuII site of pRS406 and SmaI site of pRS413 to produce GPA2/pRS406 and GPA2/pRS413, respectively. To construct GPA2 disruption vector (gpa2::HIS3/pUC19), a 1.3 kb KpnI-SphI fragment of GPA2/pUC19 was replaced by HIS3 cassette.
The dominant active mutation in GPA2 was made by changing arginine-273 to alanine using a Quick Change Site-Directed Mutagenesis Kit (Stratagene). Site-directed mutagenesis was performed using GPA2/pRS413 as a template with the following primers in which the altered nucleotides are underlined: 5'-AGATCGGCACAGATGACGTCAG-3' and 5'-CTGACGTCATCTGTGCCGATCT-3'. The GPA2R273A open reading frame (ORF) was amplified with the primers (5'-AAGAATTCCATGGGTCTCTGCGCATCTTCAG-3' and 5'-TTGCGGCCGCGCATTCATTGTAACACTCCAGAGTC-3') to introduce EcoRI and NotI sites then ligated into the SmaI site of pUC19 to produce GPA2R273A ORF/pUC19. The integrative over-expression vector was constructed as follows. A 2.2kb HindIII fragment from pYE2211 (Ashikari et al. 1989) containing TDH3 encoding glyceraldehyde 3-phosphate dehydrogenase, in which an EcoRI site was introduced just before the start codon, was cloned into the HindIII site of pUC19 in which SacI and EcoRI sites were removed. The plasmid was subjected to digestion with SacI followed by blunting and an EcoRI-NotI adaptor was ligated to both ends. Then the plasmid was further digested with EcoRI to remove the TDH3 ORF and self-ligated to produce GAp/pUC19. A 1.2kb HindIII fragment containing the TDH3 promoter and terminator from GAp/pUC19 was blunted and ligated to the PvuII site of pRS406 to produce pRS406-GAp. A 1.4kb EcoRI-NotI fragment from GPA2R273A ORF/pUC19 was ligated to pRS406-GAp to produce GPA2R273A/pRS406-GAp. The SalI-EcoRV fragment containing loxP-kanMX-loxP cassette of pUG6 (Guldener et al. 1996) was replaced by LEU2 to form pUGL, which was used to remove HA-tag from CLN3.
Strain construction
Yeast strains used in this study are listed in Table 1. The deletion of GPR1 allele in strain W303–1 A was made by transformation of cells with the 3.9 kb PstI fragment from gpr1::LEU2/pUC119 as described before (HTY5) (Yun et al. 1997). Also GPR1 gene disruption was performed to produce strain HTY189 using PCR-derived loxP-kanMX-loxP cassette containing the kanamycin resistance gene (Guldener et al. 1996) with the following primers: 5'-ATCCGAAGTGTGACGAATAAAGCAAACTCTCCAACTCAAAATGATACAGCTGAAGCTTCGTACGC-3' and 5'-CCTTACTTTCCATTTTCAAACATCGCGATACAAAAACTTTATAATGGGCATAGGCCACTAGTGGATCTG-3'. The deletion of GPA2 allele in strain W303–1 A was made by transformation of cells with the 1.9 kb SmaI fragment from gpa2::HIS3/pUC119 to construct strain HTY26. Also the PCR-derived loxP-kanMX-loxP cassette was used to create strain HTY190 with the following primers: 5'-TGTTACAGCACAAATCACGCGTATTTTCAAGCAAATATCATGGGTCAGCTGAAGCTTCGTACGC-3' and 5'-GCATGCAGTTTTGTCTCTGTTTTAGCTGTGCATTCATTGTAACACGCATAGGCCACTAGTGGATCTG-3'. The GPR1 or GPA2 null allele in strain CML128 derivatives was made by PCR-mediated gene disruption using Candida glabrata TRP1 gene as a selectable marker (Kitada et al. 1995; Sakumoto et al. 1999). The gene disruption cassettes for GPR1 and GPA2 were PCR-amplified using pCgTRP1 as a template with the following primer pairs: 5'-ATCCGAAGTGTGACGAATAAAGCAAACTCTCCAACTCAAAATGATACGCCAGGGTTTTCCCAGTCACGAC-3' and 5'-CCTTACTTTCCATTTTCAAACATCGCGATACAAAAACTTTATAATGGAGCGGATAACAATTTCACACAGGAAAC-3'. (GPR1); 5'-TGTTACAGCACAAATCACGCGTATTTTCAAGCAAATATCATGGGTCGCCAGGGTTTTCCCAGTCACGAC-3' and 5'-GCATGCAGTTTTGTCTCTGTTTTAGCTGTGCATTCATTGTAACACAGCGGATAACAATTTCACACAGGAAAC-3' (GPA2). The strains CML326, CML204, and CML203 carry the 3HA-tagged CLN1, CLN2, and CLN3 genes, respectively. Strains CML326, CML204, and CML203 were transformed with the gene disruption cassette for GPR1 to obtain strains HTY215, HTY216, and HTY217, respectively. The same strains were also transformed with the gene disruption cassette for GPA2 to create strains HTY218, HTY219, and HTY220, respectively. Strains W303–1A, HTY5, and HTY26 were transformed with EcoRV-digested pRS406-1, which integrates the URA3 gene into ura3-1 locus, to obtain strains HTY207, HTY204, and HTY206, respectively.
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Cell size measurement
Cells were grown to early log phase and small unbudded cells were fractionated by centrifugal elutriation and subjected to incubation at 30 °C with shaking. Cells were collected at appropriate time points and photographed under a Zeiss Axioskop 2 microscope with a x 40 objective, equipped with a Fujix Digital camera system HC-2500. The data were recorded as TIFF files. Micrographs were measured using Image-Pro Plus (Media Cybernetics) to determine cell volume just before bud emergence with the formula: volume = (
/6)ab2, where a is the length of the major axis, and b is the length of the minor axis (Lord & Wheals 1981). For both determinations, samples with 70–200 cells were scored. The distribution of cell size was measured with a Coulter Multisizer II (Beckman Coulter) using a 50-µm aperture calibrated with 5-µm latex beads.
Flow cytometry and the budding index
Yeast cells were grown in YPE medium to early log phase and small G1 cells were isolated by centrifugal elutriation. Glucose was added to the G1 cells of a final conc. of 2% and incubation was conducted at 30 °C with reciprocal shaking. After a period of time, aliquots were taken and washed with ice-cold water then fixed in ice-cold 95% ethanol. The distribution of DNA content was obtained by propidium iodide staining as described (Nash et al. 1988) with a FACScan flow cytometer (Becton Dickinson).
A budding index was also obtained by inspecting a minimum of 500 fixed cells under the microscope.
Immunoprecipitation and Western blot analysis
Yeast cells grown to OD600 = 0.5 were pelleted, washed with ice-cold water and resuspended in 1–2 vol. of buffer (50 mM Tris-HCl pH 7.5, 0.25 M NaCl, 0.1% NP-40) containing Complete protease inhibitor (Roche). Cells were lysed by using FastPrep FP120 (Bio 101) in the presence of glass beads for 30 s and cell-free extracts were obtained by centrifugation at 3000 x g for 5 min. For Western blot, cell-free extracts (75 µg of total protein) were electrophoresed on a 10% SDS-PAGE and then electro blotted onto polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were blocked with TBS-T (Tris-buffered saline, pH 7.6, 0.1% Tween 20) containing 5% skim milk for 1 h at room temperature then incubated with a 0.1 µg/mL of 12CA5 monoclonal antibody for 1 h at room temperature. After washing with TBS-T, the membranes were incubated with a 1: 5000 dilution of anti-mouse IgG peroxidase-linked whole antibody (from sheep) (Amersham Biosciences) for 1 h at room temperature. After washing with TBS-T, HA-tagged protein was detected by Super Signal West Pico detection reagent (Pierce). Quantification of each protein was done with lumino-image analyser LAS1000 (Fuji Film). For immunoprecipitation, 1 µL of 12CA5 monoclonal antibody (1 µg/µL) was added to the cell free extract (10 mg of total protein) and stood on ice for 1 h and then rocked in the presence of protein A sepharose beads at 4 °C for 1 h. Protein sepharose beads were collected by centrifugation (3000 x g) for 5 s and washed four times with the buffer, and immunoprecipitated samples were subjected to Western blot analysis as described above.
RNA preparation and Northern blot analysis
Yeast total RNA was extracted as described (Cross & Tinkelenberg 1991) with a slight modification (Tamaki et al. 2000). Cells were grown in YPR liquid medium to OD600 = 1, then glucose was added to a final conc. of 2% and incubation was conducted at 30 °C with reciprocal shaking. Cells were collected at each time point before and after the addition of glucose, washed with ice-cold water and immediately subjected to RNA extraction.
Ten micrograms of total RNA was subjected to formaldehyde-agarose gel electrophoresis followed by a capillary transfer to a Hybond-N+ membrane (Amersham Biosciences). Hybridization with the 32P-labeled probe was done at 42 °C for 16 h. A PCR product corresponding to 69–1634 bp of the CLN1 ORF, 104–1491 bp of the CLN2 ORF, 202–1367 bp of the CLN3 ORF, 8–1982 bp of the SUC2 ORF, 30–791 bp of the AQY2 ORF, 2–745 bp of the CMK2 ORF, 296–2090 bp of the HIS4 ORF, 42–935 bp of the HSP30 ORF, 181–1023 bp of the PHO5 ORF, 233–1244 bp of the CLB1 ORF, 1,236–2240 bp of the GPH1 ORF, 106–1140 bp of the HXK1 ORF, 105–780 bp of the PEX2 ORF, and 59–1072 bp of the QCR2 ORF was used to probe for each message, and an 1055 bp PCR product internal to 3' exon of ACT1 was used to probe for the ACT1 message as a loading control. The quantitation of mRNA was performed using a bio-imaging analyser BAS2000 (Fuji Film). The amount of each message in each strain was standardized to the amount of ACT1 message from the same strain and then normalized to the amount of each message in the wild-type strain.
Incorporation of 35S-Met and 35S-Cys
The rate of 35S-Met and 35S-Cys incorporation into protein was determined as described (Hall et al. 1998). Cells were grown in SCD or SCE medium lacking Met and Cys to OD600 = 1 and 250 µL aliquots of cell culture, which contain equal numbers of cells, were prepared. Cells were labeled in microfuge tubes at 30 °C with 5 µCi of Trans 35S-Label (ICN) for 2 min and the labeling was stopped with 250 µL of ice-cold 10% TCA. After incubation for more than 1 h, precipitates were recovered on GF/C glass filters (Whatman) and washed 2 times with 5% ice-cold TCA and 2 times with ice-cold ethanol then air dried. The incorporated 35S-Met and 35S-Cys were measured with a liquid scintillation counter. The incorporation was determined to be linear for 2 min in the reaction.
GeneChip expression analysis
Cells were grown to early log phase in YPD medium at 30 °C. Total RNA was extracted as described above and poly(A)+ RNA was purified from total RNA using the Oligotex-dt30 mRNA purification kit (Takara). Poly(A)+RNA was amplified, biotin-labeled and hybridized to oligonucleotide arrays (GeneChip S98 arrays, Affymetrix); and hybridization intensities were analyzed and normalized as described by Jelinsky and Samson (1999). Genes whose expression increased or decreased were listed, mostly based on the following criteria: a change of more than 1.5-fold or 2-fold if the change in values was above the background level in both comparisons. The reliability of the binding ratio was also judged based on the change in P-value (increase; P < 0.0025, decrease; P > 0.9975). The raw data can be obtained from GEO (http://www.ncbi.nlm.nih.gov/geo) with accession number GSE970 [NCBI GEO] .
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Acknowledgements |
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* Correspondence: E-mail: noritama{at}kais.kyoto-u.ac.jp
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
Ashe, M.P., De Long, S.K. & Sachs, A.B. (2000) Glucose depletion rapidly inhibits translation initiation in yeast. Mol. Biol. Cell
11, 833–848.
Ashikari, T., Kiuchi-Goto, N., Tanaka, Y., Shibano, Y., Amachi, T. & Yoshizumi, H. (1989) High expression and efficient secretion of Rhizopus oryzae glucoamylase in the yeast Saccaromyces cerevisiae. Appl. Microbiol. Biotechnol. 30, 515–520.
Baroni, M.D., Martegani, E., Monti, P. & Alberghina, L. (1989) Cell size modulation by CDC25 and RAS2 genes in Saccharomyces cerevisiae. Mol. Cell. Biol.
9, 2715–2723.
Baroni, M.D., Monti, P., Marconi, G. & Alberghina, L. (1992) cAMP-mediated increase in the critical cell size required for the G1 to S transition in Saccharomyces cerevisiae. Exp. Cell Res. 201, 299–306.[CrossRef][Medline]
Baroni, M.D., Monti, P. & Alberghina, L. (1994) Repression of growth-regulated G1 cyclin expression by cyclic AMP in budding yeast. Nature 371, 339–342.[CrossRef][Medline]
Beullens, M., Mbonyi, K., Geerts, L., et al. (1988) Studies on the mechanism of the glucose-induced cAMP signal in glycolysis and glucose repression mutants of the yeast Saccharomyces cerevisiae. Eur. J. Biochem. 172, 227–231.[Medline]
Cameron, S., Levin, L., Zoller, M. & Wigler, M. (1988) cAMP-independent control of sporulation, glycogen metabolism, and heat shock resistance in S. cerevisiae. Cell 53, 555–566.[CrossRef][Medline]
Carlson, M. (1999) Glucose repression in yeast. Curr. Opin. Microbiol. 2, 202–207.[CrossRef][Medline]
Carlson, M. & Botstein, D. (1982) Two differentially regulated mRNAs with different 5' ends encode secreted with intracellular forms of yeast invertase. Cell 28, 145–154.[CrossRef][Medline]
Cross, F.R. (1988) DAF1, a mutant gene affecting size control, pheromone arrest, and cell cycle kinetics of Saccharomyces cerevisiae. Mol. Cell. Biol.
8, 4675–4684.
Cross, F.R. & Tinkelenberg, A.H. (1991) A potential positive feedback loop controlling CLN1 and CLN2 gene expression at the start of the yeast cell cycle. Cell 65, 875–883.[CrossRef][Medline]
Dejean, L., Beauvoit, B., Bunoust, O., Guerin, B. & Rigoulet, M. (2002) Activation of Ras cascade increases the mitochondrial enzyme content of respiratory competent yeast. Biochem. Biophys. Res. Commun. 293, 1383–1388.[CrossRef][Medline]
Dirick, L., Bohm, T. & Nasmyth, K. (1995) Roles and regulation of Cln-Cdc28 kinases at the start of the cell cycle of Saccharomyces cerevisiae. EMBO J. 14, 4803–4813.[Medline]
Flick, K., Chapman-Shimshoni, D., Stuart, D., Guaderrama, M. & Wittenberg, C. (1998) Regulation of cell size by glucose is exerted via repression of the CLN1 promoter. Mol. Cell. Biol.
18, 2492–2501.
Gallego, C., Gari, E., Colomina, N., Herrero, E. & Aldea, M. (1997) The Cln3 cyclin is down-regulated by translational repression and degradation during the G1 arrest caused by nitrogen deprivation in budding yeast. EMBO J. 16, 7196–7206.[CrossRef][Medline]
Gancedo, J.M. (1998) Yeast carbon catabolite repression. Microbiol. Mol. Biol. Rev.
62, 334–361.
Guldener, U., Heck, S., Fielder, T., Beinhauer, J. & Hegemann, J.H. (1996) A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res.
24, 2519–2524.
Hadwiger, J.A., Wittenberg, C., Richardson, H.E., de Barros Lopes, M. & Reed, S.I. (1989) A family of cyclin homologs that control the G1 phase in yeast. Proc. Natl. Acad. Sci. USA
86, 6255–6259.
Hall, D.D., Markwardt, D.D., Parviz, F. & Heideman, W. (1998) Regulation of the Cln3-Cdc28 kinase by cAMP in Saccharomyces cerevisiae. EMBO J. 17, 4370–4378.[CrossRef][Medline]
Harashima, T. & Heitman, J. (2002) The Galpha protein Gpa2 controls yeast differentiation by interacting with kelch repeat proteins that mimic Gbeta subunits. Mol. Cell 10, 163–173.[CrossRef][Medline]
Jelinsky, S.A. & Samson, L.D. (1999) Global response of Saccharomyces cerevisiae to an alkylating agent. Proc. Natl. Acad. Sci. USA
96, 1486–1491.
Johnston, G.C., Ehrhardt, C.W., Lorincz, A. & Carter, B.L. (1979) Regulation of cell size in the yeast Saccharomyces cerevisiae. J. Bacteriol.
137, 1–5.
Kaniak, A., Xue, Z., Macool, D., Kim, J.H. & Johnston, M. (2004) Regulatory network connecting two glucose signal transduction pathways in Saccharomyces cerevisiae. Eukaryot. Cell
3, 221–231.
Kitada, K., Yamaguchi, E. & Arisawa, M. (1995) Cloning of the Candida glabrata TRP1 and HIS3 genes, and construction of their disruptant strains by sequential integrative transformation. Gene 165, 203–206.[CrossRef][Medline]
Klein, C. & Struhl, K. (1994) Protein kinase A mediates growth-regulated expression of yeast ribosomal protein genes by modulating RAP1 transcriptional activity. Mol. Cell. Biol.
14, 1920–1928.
Kraakman, L., Lemaire, K., Ma, P., et al. (1999) A Saccharomyces cerevisiae G-protein coupled receptor, Gpr1, is specifically required for glucose activation of the cAMP pathway during the transition to growth on glucose. Mol. Microbiol. 32, 1002–1012.[CrossRef][Medline]
Kubler, E., Mosch, H.U., Rupp, S. & Lisanti, M.P. (1997) Gpa2p, a G-protein alpha-subunit, regulates growth and pseudohyphal development in Saccharomyces cerevisiae via a cAMP-dependent mechanism. J. Biol. Chem.
272, 20321–20323.
Lord, P.G. & Wheals, A.E. (1981) Variability in individual cell cycles of Saccharomyces cerevisiae. J. Cell Sci. 50, 361–376.[Abstract]
Lorenz, M.C. & Heitman, J. (1997) Yeast pseudohyphal growth is regulated by GPA2, a G protein alpha homolog. EMBO J. 16, 7008–7018.[CrossRef][Medline]
Lorenz, M.C., Pan, X., Harashima, T., et al. (2000) The G protein-coupled receptor gpr1 is a nutrient sensor that regulates pseudohyphal differentiation in Saccharomyces cerevisiae. Genetics
154, 609–622.
Lorincz, A.T. & Carter, B.L.A. (1979) Control of cell size at bud initiation in Saccharomyces cerevisiae. J. General Microbiol. 113, 287–295.
Mitsuzawa, H. (1994) Increases in cell size at START caused by hyperactivation of the cAMP pathway in Saccharomyces cerevisiae. Mol. Gen. Genet. 243, 158–165.[Medline]
Miwa, T., Takagi, Y., Shinozaki, M., et al. (2004) Gpr1, a putative G-protein-coupled receptor, regulates morphogenesis and hypha formation in the pathogenic fungus Candida albicans. Eukaryot. Cell
3, 919–931.
Nakafuku, M., Obara, T., Kaibuchi, K., et al. (1988) Isolation of a second yeast Saccharomyces cerevisiae gene (GPA2) coding for guanine nucleotide-binding regulatory protein: Studies on its structure and possible functions. Proc. Natl. Acad. Sci. USA
85, 1374–1378.
Nash, R., Tokiwa, G., Anand, S., Erickson, K. & Futcher, A.B. (1988) The WHI1+ gene of Saccharomyces cerevisiae tethers cell division to cell size and is a cyclin homolog. EMBO J. 7, 4335–4346.[Medline]
Neuman-Silberberg, F.S., Bhattacharya, S. & Broach, J.R. (1995) Nutrient availability and the RAS/cyclic AMP pathway both induce expression of ribosomal protein genes in Saccharomyces cerevisiae but by different mechanisms. Mol. Cell. Biol.
15, 3187–3196.
Newcomb, L.L., Diderich, J.A., Slattery, M.G. & Heideman, W. (2003) Glucose regulation of Saccharomyces cerevisiae cell cycle genes. Eukaryot. Cell
2, 143–149.
Richardson, H.E., Wittenberg, C., Cross, F. & Reed, S.I. (1989) An essential G1 function for cyclin-like proteins in yeast. Cell 59, 1127–1133.[CrossRef][Medline]
Robertson, L.S., Causton, H.C., Young, R.A. & Fink, G.R. (2000) The yeast A kinases differentially regulate iron uptake and respiratory function. Proc. Natl. Acad. Sci. USA
97, 5984–5988.
Russell, M., Bradshaw-Rouse, J., Markwardt, D. & Heideman, W. (1993) Changes in gene expression in the Ras/adenylate cyclase system of Saccharomyces cerevisiae: Correlation with cAMP levels and growth arrest. Mol. Biol. Cell 4, 757–765.[Abstract]
Sakumoto, N., Mukai, Y., Uchida, K., et al. (1999) A series of protein phosphatase gene disruptants in Saccharomyces cerevisiae. Yeast 15, 1669–1679.[CrossRef][Medline]
Sanchez-Martinez, C. & Perez-Martin, J. (2002) Gpa2, a G-protein alpha subunit required for hyphal development in Candida albicans. Eukaryot. Cell
1, 865–874.
Sherman, F. (1991) Getting Started with Yeast. San Diego, CA: Academic Press.
Stuart, D. & Wittenberg, C. (1995) CLN3, not positive feedback, determines the timing of CLN2 transcription in cycling cells. Genes Dev.
9, 2780–2794.
Tamaki, H., Miwa, T., Shinozaki, M., et al. (2000) GPR1 regulates filamentous growth through FLO11 in yeast Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 267, 164–168.[CrossRef][Medline]
Thevelein, J.M. & de Winde, J.H. (1999) Novel sensing mechanisms and targets for the cAMP-protein kinase A pathway in the yeast Saccharomyces cerevisiae. Mol. Microbiol. 33, 904–918.[CrossRef][Medline]
Tokiwa, G., Tyers, M., Volpe, T. & Futcher, B. (1994) Inhibition of G1 cyclin activity by the Ras/cAMP pathway in yeast. Nature 371, 342–345.[CrossRef][Medline]
Tyers, M., Tokiwa, G. & Futcher, B. (1993) Comparison of the Saccharomyces cerevisiae G1 cyclins: Cln3 may be an upstream activator of Cln1, Cln2 and other cyclins. EMBO J. 12, 1955–1968.[Medline]
Velculescu, V.E., Zhang, L., Zhou, W., et al. (1997) Characterization of the yeast transcriptome. Cell 88, 243–251.[CrossRef][Medline]
Wallis, J.W., Chrebet, G., Brodsky, G., Rolfe, M. & Rothstein, R. (1989) A hyper-recombination mutation in S. cerevisiae identifies a novel eukaryotic topoisomerase. Cell 58, 409–419.[CrossRef][Medline]
Wittenberg, C., Sugimoto, K. & Reed, S.I. (1990) G1-specific cyclins of S. cerevisiae: Cell cycle periodicity, regulation by mating pheromone, and association with the p34CDC28 protein kinase. Cell 62, 225–237.[CrossRef][Medline]
Xue, Y., Batlle, M. & Hirsch, J.P. (1998) GPR1 encodes a putative G protein-coupled receptor that associates with the Gpa2p Galpha subunit and functions in a Ras-independent pathway. EMBO J. 17, 1996–2007.[CrossRef][Medline]
Yun, C.W., Tamaki, H., Nakayama, R., Yamamoto, K. & Kumagai, H. (1997) G-protein coupled receptor from yeast Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 240, 287–292.[CrossRef][Medline]
Yun, C.W., Tamaki, H., Nakayama, R., Yamamoto, K. & Kumagai, H. (1998) Gpr1p, a putative G-protein coupled receptor, regulates glucose-dependent cellular cAMP level in yeast Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 252, 29–33.[CrossRef][Medline]
Received: 15 November 2004
Accepted: 28 November 2004
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gpr1 CLN1-3HA), HTY218 (
