Regulation of the G1 phase of the cell cycle by periodic stabilization and degradation of the p25rum1 CDK inhibitor
1998; Springer Nature; Volume: 17; Issue: 2 Linguagem: Inglês
10.1093/emboj/17.2.482
ISSN1460-2075
Autores Tópico(s)Cancer-related Molecular Pathways
ResumoArticle15 January 1998free access Regulation of the G1 phase of the cell cycle by periodic stabilization and degradation of the p25rum1 CDK inhibitor Javier Benito Javier Benito Instituto de Microbiología Bioquímica, Departamento de Microbiología y Genética, CSIC/Universidad de Salamanca, Edificio Departamental, Campus Miguel de Unamuno, 37007 Salamanca, Spain Search for more papers by this author Cristina Martín-Castellanos Cristina Martín-Castellanos Instituto de Microbiología Bioquímica, Departamento de Microbiología y Genética, CSIC/Universidad de Salamanca, Edificio Departamental, Campus Miguel de Unamuno, 37007 Salamanca, Spain Search for more papers by this author Sergio Moreno Corresponding Author Sergio Moreno Instituto de Microbiología Bioquímica, Departamento de Microbiología y Genética, CSIC/Universidad de Salamanca, Edificio Departamental, Campus Miguel de Unamuno, 37007 Salamanca, Spain Search for more papers by this author Javier Benito Javier Benito Instituto de Microbiología Bioquímica, Departamento de Microbiología y Genética, CSIC/Universidad de Salamanca, Edificio Departamental, Campus Miguel de Unamuno, 37007 Salamanca, Spain Search for more papers by this author Cristina Martín-Castellanos Cristina Martín-Castellanos Instituto de Microbiología Bioquímica, Departamento de Microbiología y Genética, CSIC/Universidad de Salamanca, Edificio Departamental, Campus Miguel de Unamuno, 37007 Salamanca, Spain Search for more papers by this author Sergio Moreno Corresponding Author Sergio Moreno Instituto de Microbiología Bioquímica, Departamento de Microbiología y Genética, CSIC/Universidad de Salamanca, Edificio Departamental, Campus Miguel de Unamuno, 37007 Salamanca, Spain Search for more papers by this author Author Information Javier Benito1, Cristina Martín-Castellanos1 and Sergio Moreno 1 1Instituto de Microbiología Bioquímica, Departamento de Microbiología y Genética, CSIC/Universidad de Salamanca, Edificio Departamental, Campus Miguel de Unamuno, 37007 Salamanca, Spain *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:482-497https://doi.org/10.1093/emboj/17.2.482 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info In fission yeast, the cyclin-dependent kinase (CDK) inhibitor p25rum1 is a key regulator of progression through the G1 phase of the cell cycle. We show here that p25rum1 protein levels are sharply periodic. p25rum1 begins to accumulate at anaphase, persists in G1 and is destroyed during S phase. p25rum1 is stabilized and polyubiquitinated in a mutant defective in the 26S proteasome, suggesting that its degradation normally occurs through the ubiquitin-dependent 26S proteasome pathway. Phosphorylation of p25rum1 by cdc2–cyclin complexes at residues T58 and T62 is important to target the protein for degradation. Mutation of one or both of these residues to alanine causes stabilization of p25rum1 and induces a cell cycle delay in G1 and polyploidization due to occasional re-initiation of DNA replication before mitosis. The CDK–cyclin complex cdc2–cig1, which is insensitive to p25rum1 inhibition, seems to be the main kinase that phosphorylates p25rum1. Phosphorylation of p25rum1 in S phase and G2 serves as the trigger for p25rum1 proteolysis. Thus, periodic accumulation and degradation of the CDK inhibitor p25rum1 in G1 plays a role in setting a threshold of cyclin levels important in determining the length of the pre-Start G1 phase and in ensuring the correct order of cell cycle events. Introduction Eukaryotic cells co-ordinate cell growth with cell division at a point late in G1 called Start in yeast and the restriction point in animal cells. Beyond this point, cells become committed to a new round of cell division (Hartwell et al., 1974; Pardee, 1974; Nurse, 1975). Genetic analysis in budding and fission yeast identified a single gene, CDC28/cdc2+, encoding the essential cyclin-dependent kinase (CDK) required not only for passage through Start but also to initiate mitosis (Nurse and Bisset, 1981; Piggott et al., 1982; Reed and Wittenberg, 1990). In the budding yeast Saccharomyces cerevisiae, nine different cyclins associate with Cdc28 (Nasmyth, 1993, 1996). Six B-type cyclins (Clb1–6) are required for S phase and mitosis and three G1 cyclins (Cln1–3) are necessary for passage through Start. The three G1 cyclins have different functions. Cln3 is needed to promote the activation of Start-dependent transcription factors (SBF and MBF), whereas Cln1 and Cln2 are required to trigger Start-regulated events such as acquisition of pheromone resistance, budding and spindle pole body duplication (Dirick et al., 1995; Stuart and Wittenberg, 1995). In the fission yeast Schizosaccharomyces pombe, four cyclins, puc1, cig1, cig2 and cdc13, can form complexes with cdc2 (reviewed in Fisher and Nurse, 1995). cig1, cig2 and cdc13 are B-type cyclins that have been shown to play a role in regulating the cell cycle. cig2 regulates the G1–S transition (Obara-Ishihara and Okayama, 1994; Martín-Castellanos et al., 1996; Monderset et al., 1996), while cdc13 is the mitotic cyclin (Booher et al., 1989; Moreno et al., 1989). cig1 has been argued to make a minor contribution to the onset of S phase, because cells deleted for cig2+ and cdc13+ can still undergo S phase but a triple deletion cig1Δ cig2Δ cdc13Δ blocks the cell cycle before the initiation of DNA replication (Fisher and Nurse, 1996; Monderset et al., 1996). The role of puc1 in the fission yeast mitotic cycle has not been clearly established (Forsburg and Nurse, 1991, 1994). CDK inhibitors are negative regulators of CDK–cyclin complexes. Some CDK inhibitors, like budding yeast Far1, seem to respond to extracellular signals such as the presence of mating pheromones (Chang and Herskowitz, 1990; Peter et al., 1993; Peter and Herskowitz, 1994), but others, like budding yeast p40SIC1 (Donovan et al., 1994; Nugroho and Mendenhall, 1994; Schwob et al., 1994; Schneider et al., 1996) or fission yeast p25rum1 (Moreno and Nurse, 1994), appear to be part of the intrinsic cell cycle machinery. While Far1 inhibits Cln-type cyclins, p40SIC1 and p25rum1 are specific inhibitors of B-type cyclins. p25rum1 inhibits the cdc2–cdc13 complex, preventing the activation of this mitotic complex in G1 cells (Correa-Bordes and Nurse, 1995; Martín-Castellanos et al., 1996). p25rum1 is also an inhibitor of cig2-associated cdc2 kinase (Correa-Bordes and Nurse, 1995; Martín-Castellanos et al., 1996). It has been proposed that a transient inhibition of cdc2–cig2 complexes in G1 is important in setting the minimum cell size required to pass Start (Labib and Moreno, 1996; Martín-Castellanos et al., 1996). Thus, p25rum1 plays a central role in the regulation of the fission yeast G1 phase. It prevents the onset of mitosis in cells that have not initiated DNA replication and it determines the cell cycle timing of Start, maintaining cells in the pre-Start state until they have attained the minimal critical mass required to initiate the cell cycle. Degradation of cyclins and CDK inhibitors is important for moving from one phase of the cell cycle to the next (reviewed by Deshaies, 1995). Degradation of mitotic cyclins depends on a sequence located near the amino-terminus called the destruction box, which targets cyclins to the ubiquitin-dependent proteolytic pathway (Glotzer et al., 1991; Hershko et al., 1991). A multiprotein complex, called cyclosome or anaphase-promoting complex (APC), has been shown to contain the E3 ubiquitin–protein ligase activity that catalyses the ligation of multiple ubiquitin molecules to cyclins (Hershko et al., 1994; King et al., 1995; Sudakin et al., 1995). APC is a high molecular weight complex that contains the Cdc16, Cdc23 and Cdc27 protein members of the TPR family essential for the onset of anaphase (Irniger et al., 1995; Tugendreich et al., 1995), highly conserved among eukaryotes (Hirano et al., 1988; O′Donnell et al., 1991; Mirabito and Morris, 1993; Samejima and Yanagida, 1994; King et al., 1995; Tugendreich et al., 1995). The fission yeast cut2 protein (a protein that might be involved in holding sister chromatids together until the onset of anaphase) also contains a destruction box that is recognized by APC in mitosis and destroyed through this proteolytic pathway (Funabiki et al., 1996). Degradation of the mitotic cyclins occurs in an interval from anaphase until passage through Start or the restriction point in late G1 (Amon et al., 1994; Brandeis and Hunt, 1996). This prevents the accumulation of mitotic cyclins before the formation of the G1 complexes, and this period of low CDK activity is thought to be important for proper assembly of pre-initiation DNA replication complexes (Adachi and Laemmli, 1994; Dahmann et al., 1995; Nasmyth, 1996; Stern and Nurse, 1996; Wuarin and Nurse, 1996). During the G1 phase, the right balance of cyclins and CDK inhibitors is necessary to produce a co-ordinate Start to the cell cycle. In budding yeast, G1 cyclins have very short half-lives and are destroyed as cells enter S phase. Degradation of the CDK inhibitor p40SIC1 is required for cells to initiate S phase (Schwob et al., 1994; Schneider et al., 1996). Proteolysis of G1 cyclins and of p40SIC1 also occurs through the ubiquitin-dependent proteolytic pathway and requires the participation of a ubiquitin–protein ligase complex, different from APC, formed by a multiprotein complex containing the product of the CDC34, CDC4, SKP1 and CDC53 genes (Schwob et al., 1994; Deshaies, 1995; Bai et al., 1996; Connelly and Hieter, 1996; Schneider et al., 1996; Willems et al., 1996; Verma et al., 1997). CDC34 encodes an E2 ubiquitin-conjugating enzyme (Goebl et al., 1988), CDC4 a protein with WD40 repeats (Yochem and Byers, 1987), SKP1 a protein that interacts with Cdc4 (Bai et al., 1996; Connelly and Hieter, 1996) and CDC53 a member of the cullin protein family conserved in yeast, Caenorhabditis elegans, Drosophila and humans (Kipreos et al., 1996; Mathias et al., 1996). Mutations in any of these genes lead to stabilization of p40SIC1 and G1 cyclins. G1 cyclins are unstable because they contain PEST sequences (Rogers et al., 1986). CDK phosphorylation of residues located near the PEST sequence seems to be the signal that targets Cln2 and Cln3 to degradation (Yaglom et al., 1995; Lanker et al., 1996). CDK–cyclin complexes not only play positive roles in the cell cycle, determining when cells initiate S phase or mitosis, but are also required to inhibit the initiation of a unscheduled cell cycle event. In fission yeast, inactivation of the mitotic cdc2–cdc13 complex causes cells to undergo repeated rounds of DNA replication without intervening mitoses (Broek et al., 1991; Hayles et al., 1994; Fisher and Nurse, 1996). Budding yeast mutants with low levels of Cdc28–Clb5 kinase re-replicate (Dahmann et al., 1996), Drosophila mutants in cyclin A also undergo re-replication (Sauer et al., 1995) and, in starfish oocytes, cyclin B suppresses DNA replication between meiosis I and meiosis II (Adachi and Laemmli, 1994; Picard et al., 1996). These results suggest that CDK–cyclin complexes play a dual role in the cell cycle. They are required to promote the initiation of S phase and mitosis and, in addition, in G2 they prevent extra rounds of DNA replication within the same cell cycle (Nurse, 1994; Nasmyth, 1996; Stern and Nurse, 1996). In fission yeast, high level expression of the rum1+ gene blocks mitosis but allows repeated rounds of DNA replication (Moreno and Nurse, 1994). This result is explained because p25rum1 strongly inhibits the cdc2–cdc13 complex leading to a phenotype which is similar to the cdc13+ deletion (Hayles et al., 1994). Thus, transit from G2 to G1 in fission yeast occurs by inactivation of the cdc2–cdc13 kinase complex, either by destruction of the mitotic cdc13 cyclin in mitosis, or by production of high levels of the p25rum1 CDK inhibitor. According to this idea, the presence of cdc2–cdc13 activity defines a cell in G2, and destruction of this complex in mitosis resets the cell to G1, allowing a new S phase (Nurse, 1994; Stern and Nurse, 1996). Therefore, G1 is defined as a period of low CDK–cyclin activity prior to the initiation of a new cell cycle. Here we have studied the regulation of the p25rum1 protein. We have found that p25rum1 levels sharply oscillate during the fission yeast cell cycle. This oscillation is due mainly to changes in protein stability. p25rum1 is stabilized in G1, a period were there is low CDK–cyclin activity, and is destroyed as cells enter S phase, when CDK–cyclin activity begins to rise. We have also found that phosphorylation of p25rum1 by cdc2–cyclin complexes at the end of G1 is important for targeting p25rum1 for degradation. Results p25rum1 level oscillates through the cell cycle In order to study the regulation of the rum1+ gene through the cell cycle, we determined the levels of p25rum1 and of rum1+ mRNA in synchronous cultures. Rapidly growing wild-type fission yeast cells have a very short G1 and lack the pre-Start G1 interval. Since rum1+ function is required in G1, we used the temperature-sensitive mutant wee1-50 where the pre-Start G1 interval is extended when incubated at the restrictive temperature of 36°C (Nurse, 1975) (Figure 1A). Cells of the wee1-50 strain were grown at 25°C, and a synchronous culture was made using an elutriator rotor. Small cells in early G2 were selected and incubated at 36°C. These cells proceeded synchronously into mitosis. Cell cycle position and the degree of synchrony were monitored by determining the mitotic index and the percentage of cells in G1 after flow cytometry analysis, as a function of time. Protein extracts were prepared every 20 min for two cell cycles, and p25rum1 levels were measured by Western blotting using an anti-p25rum1 affinity-purified polyclonal antibody. p25rum1 levels were sharply periodic, rising to a peak 40 min after the shift as cells were undergoing anaphase and decreasing at 100 min when the cells where exiting G1 (Figure 1B). In the same experiment, p56cdc13 levels were exactly the opposite to those of p25rum1. p56cdc13 levels dropped at the onset of anaphase and started to accumulate during the next S phase. This experiment shows that p25rum1 protein levels oscillate though the cell cycle and that the levels are maximal in G1, consistent with previous observations where p25rum1 was shown to accumulate in cells arrested in G1 but not in S phase or G2 (Correa-Bordes and Nurse, 1995). rum1+ mRNA levels were also determined in a similar experiment and found to oscillate through the cell cycle with a peak of expression at the end of the G2 phase, 40–60 min earlier than the protein (Figure 1C). Levels of rum1+ mRNA only changed 2- to 3-fold (when normalized to the ura4+ mRNA) compared with a 10-fold oscillation in the protein levels, suggesting that additional post-transcriptional mechanisms are involved in regulating p25rum1 levels. Figure 1.Periodicity of p25rum1 and rum1+ mRNA levels during the cell cycle. p25rum1 protein and rum1+ mRNA levels were measured in a synchronous culture of the temperature-sensitive wee1-50 strain. (A) Wild-type or wee1-50 cells at 25°C have a very short G1. wee1-50 cells at 36°C enter mitosis with a reduced cell size and, as a consequence, they have an extended G1 to meet the minimal cell size requirement to pass Start. (B) A homogeneous population of cells in early G2 was selected by elutriation of the wee1-50 strain at 25°C. This culture was incubated for 20 min at 25°C and then shifted up to 36°C. Samples were taken every 20 min to determine cdc13 and rum1 protein levels, the percentage of G1 cells and the mitotic index. rum1 and cdc13 protein levels were measured by Western blot using affinity-purified R3 anti-rum1 polyclonal antibody and affinity-purified SP4 anti-cdc13 polyclonal antibody, respectively. The percentage of G1 cells was measured by flow cytometry and the mitotic index by counting the number of cells in anaphase after DAPI staining (see Materials and methods). p25rum1 levels increase as cells undergo anaphase and decrease as cells enter S-phase. In contrast, levels of cdc13 decrease in anaphase and increase at the end of G1. We used a rum1Δ (Δ) and purified p25rum1 (rum1) as negative and positive controls. (C) rum1+ mRNA levels determined in a similar experiment also oscillate during the cell cycle. The time of the peak of transcription was 40 min earlier than the time of the peak of the protein. We observed a 10-fold oscillation in the levels of protein compared with a 2- to 3-fold in the level of the transcript. The blot was probed with ura4+ as a loading control and with cdc18+ as a gene known to be transcribed in G1/S (Kelly et al., 1993). rum1+ mRNA levels were normalized using the ura4+ gene. Download figure Download PowerPoint p25rum1 is polyubiquitinated and degraded by the proteasome pathway Protein degradation plays a crucial role in the regulation of the cell cycle. Destruction of mitotic cyclins controls exit from mitosis into G1 (Murray and Kirschner, 1989; Murray et al., 1989). In the budding yeast and animal cells, G1 cyclins and the CDK inhibitors p40SIC1 and p27KIP1 are destroyed at the end of G1 by the ubiquitin-dependent proteasome pathway (Wittenberg et al., 1990; Tyers et al., 1992; Donovan et al., 1994; Schwob et al., 1994; Deshaies et al., 1995; Pagano et al., 1995; Bai et al., 1996; Clurman et al., 1996; Willems et al., 1996; Won and Reed, 1996; Diehl et al., 1997). To establish whether p25rum1 is a substrate of the proteasome degradation pathway, we determined p25rum1 levels in the fission yeast temperature-sensitive mutant mts3-1, defective in subunit 14 of the 26S proteasome (Gordon et al., 1996). p25rum1 levels were measured in this mutant at 25°C and 2, 4 and 6 h after the shift to 36°C. p25rum1 protein was more abundant in the mts3-1 mutant at the restrictive temperature than in the wild-type strain (Figure 2, lanes 1–6), suggesting that the proteasome degradation pathway is involved in regulating p25rum1 levels. In this experiment, we used affinity-purified R4 rum1 antibody that reacts with a single band in wild-type cells. This antibody recognized several high molecular weight bands in the mts3-1 mutant at 36°C that could be p25rum1 ubiquitin conjugates (Figure 2, lanes 4, 5 and 6). To investigate this possibility, we expressed in fission yeast a His6-tagged version of ubiquitin. Extracts from the rum1 deletion, the wild-type and the mts3-1 mutant expressing His6-ubiquitin were purified using Ni2+-NTA resin (Treier et al., 1994), separated on a polyacrylamide gel and then Western blotted with anti-rum1 R4 antibody. Reactive high molecular weight bands were detected in the mts3-1 mutant expressing His6-ubiquitin at the restrictive temperature (Figure 2, lane 9). These bands were absent both in wild-type and rum1+-deleted cells expressing His6-ubiquitin grown under identical conditions (Figure 2, lanes 7 and 8). This result clearly shows that p25rum1 is polyubiquitinated and degraded through the ubiquitin-dependent 26S proteasome pathway. Figure 2.p25rum1 is stabilized in the mts3-1 mutant defective in subunit 14 of the 26S proteasome. Cells of the rum1+ deletion (Δ, lane 1) wild-type (wt, lane 2) and the temperature-sensitive mts3-1 mutant (mts3-1, lanes 3–6) were grown at 25°C and shifted to 36°C for 6 h. Samples were taken at 0, 2, 4 and 6 h after the shift, and levels of p25rum1 and p34cdc2, as a loading control, were determined by Western blot using affinity-purified R4 anti-rum1 and PN24 anti-cdc2 antibodies. His6-ubiquitin was expressed from the nmt1 promoter for 22 h at 25°C and then shifted to 36°C for a further 4 h in the rum1+ deletion (Δ, lane 7), wild-type (wt, lane 8) and in the mts3-1 mutant cells (mts3-1, lane 9). The His6-ubiquitin conjugates were purified on Ni2+-NTA columns and analysed by Western blot using affinity-purified R4 anti-rum1 antibody (1:50). Download figure Download PowerPoint Phosphorylation of p25rum1 is important for its stability In budding yeast, phosphorylation of several Cdc28-specific sites in the carboxy-terminus of the G1 cyclins Cln2 and Cln3 is important in promoting their degradation (Yaglom et al., 1995; Lanker et al., 1996). We have found in p25rum1 eight putative CDK phosphorylation sites containing the minimal consensus sequence of S/T.P (Figure 3A). Three of these sites, at positions 58, 62 and 212, correspond exactly to the full consensus cdc2 phosphorylation site of S/T.;P.;X.;K/R (where X is any amino acid) (Moreno and Nurse, 1990). We have mutated all eight serines and threonines to alanine and expressed each mutant using the nmt1 promoter in S.pombe. Mutants rum1-A58 and rum1-A62 showed a phenotype consistent with hyperactivation or stabilization of p25rum1. In both cases, very few transformants were obtained even when we used the weakest modified nmt1 promoter (pREP81X) to transform yeast cells in the presence of thiamine, compared with a control pREP3X plasmid with the rum1+ gene (data not shown, see below). The transformants grew very slowly into small colonies containing many elongated cells with a phenotype similar to the phenotype of cells overexpressing the wild-type rum1+ gene (Moreno and Nurse, 1994). A double mutant rum1-A58A62 showed a more severe phenotype than each of the single mutants. Cells transformed with the rest of the alanine mutants (A5, A13A16A19, A110 and A212) showed a phenotype identical to cells expressing wild-type rum1+ plasmid control (data not shown). Figure 3.rum1+ mutants in two putative CDK phosphorylation sites, T58 and T62, show a phenotype consistent with a hyperactivation or stabilization of p25rum1. (A) Schematic representation of p25rum1 showing eight serine or threonine residues that occur in S/T.;P sequences. T58, T62 and S212 are contained in the sequence S/T.;P.;X.;K/R, a full CDK phosphorylation consensus site. Each of these residues was mutated to alanine. Single mutants in residues T58 and T62 showed a gain-of-function phenotype consistent with the mutant protein being either a better inhibitor or more stable than the wild-type protein. NLS, nuclear localization sequence. (B) Transformation of pIRT2-rum1+ (top panel) and pIRT2-rum1-A58A62 (bottom panel) into a rum1Δ leu1-32 strain. (C) Flow cytometric analysis of wild-type and rum1-A58A62 integrant strains growing exponentially in minimal media. Download figure Download PowerPoint Expression of the rum1+ gene driven by its own promoter using the multicopy plasmid pIRT2 gives a fully wild-type phenotype, with no signs of cell elongation or diploidization. When we expressed the rum1-A58A62 mutant allele driven by the rum1+ promoter using the pIRT2 plasmid, we obtained many small microcolonies and a few normal size colonies, all of which were integrants (Figure 3B). Flow cytometry analysis of these integrants showed a high frequency of diploids (Figure 3C). The phenotype of the cells expressing the rum1-A58A62 mutant could be explained either because the mutant protein is more active as a CDK inhibitor or because it is more stable, or both. To distinguish between these possibilities, we expressed wild-type rum1+ and mutant rum1-A58A62 in Escherichia coli and purified both proteins to homogeneity as judged by Coomassie blue staining (Figure 4A). Different amounts of purified p25rum1 and p25rum1-A58A62 were added to cdc13, cig2 and cig1 immunoprecipitates, and protein kinase activity was assayed using histone H1 as a substrate (Figure 4B). Both p25rum1 and p25rum1-A58A62 were able to inhibit the cdc13- and the cig2-associated H1 kinase activity to the same extent, indicating that at least in vitro there is no difference in the activity of these two proteins. In contrast, cdc2–cig1 kinase activity was not significantly inhibited by either p25rum1 or by p25rum1-A58A62 (Figure 4B), suggesting that this complex is insensitive to p25rum1 inhibition. Figure 4.p25rum1-A58A62 has the same activity as a CDK inhibitor but is more stable than p25rum1. (A) Coomassie blue-stained SDS–PAGE of purified p25rum1 and p25rum1-A58A62. Both proteins migrated with an Mr of 34 kDa. (B) Wild-type fission yeast extracts were immunoprecipitated with anti-cdc13, anti-cig2 and anti-cig1 antibodies. The immunoprecipitates were pre-incubated with different concentrations of either p25rum1 or p25rum1-A58A62 and then assayed for histone H1 kinase activity. As negative controls (Δ), extracts of cig1Δ and cig2Δ were immunoprecipitated with anti-cig1 and anti-cig2 antibodies and assayed for H1 kinase activity. (C) Western blot of exponentially growing rum1Δ, cdc10–129 rum1Δ int::rum1+, cdc10-129 rum1Δ int::rum1-A58A62, rum1Δ int::rum1+ and rum1Δ int::rum1-A58A62. Levels of p25rum1 and p34cdc2, as a loading control, were determined by Western blot of total extracts (100 μg) using affinity-purified R4 anti-rum1 and PN24 anti-cdc2 antibodies. Download figure Download PowerPoint As p25rum1 and p25rum1-A58A62 behave similarly as CDK inhibitors, a possible explanation for the phenotype of the rum1-A58A62 integrants is that the two proteins have different stabilities in vivo. We have compared p25rum1 levels in single copy integrants of rum1+ and rum1-A58A62 obtained in a rum1Δ background strain. Steady-state levels of p25rum1-A58A62 were at least 4-fold higher than wild-type p25rum1 levels (Figure 4C), indicating that the more likely explanation for the phenotypes associated with the rum1-A58A62 mutant allele is that the mutant protein is more stable in vivo than the wild-type protein. Stabilization of p25rum1 causes a cell cycle delay in G1 To confirm this hypothesis, we studied the stability of p25rum1 and p25rum1-A58A62 proteins through the cell cycle using the cdc10-129 mutant to synchronize cells in G1. This was investigated by integrating the wild-type rum1+ gene and the rum1-A58A62 mutant allele in a cdc10-129 rum1Δ strain. Exponentially growing cultures of cdc10-129 rum1Δ int-rum1+ and cdc10-129 rum1Δ int-rum1-A58A62 were incubated for 4 h at 36°C and then released to 25°C. Wild-type p25rum1 disappeared by 90 min after the release as cells were entering S phase (Figure 5A and C). In contrast, the p25rum1-A58A62 levels were high and stable during the course of the experiment (Figure 5B). These levels of p25rum1-A58A62 were reduced compared with the levels required to block mitosis in cells overproducing rum1+ from the nmt1 promoter (Figure 5B, ovp). Interestingly, the cdc10-129 rum1Δ strain with the integrated rum1-A58A62 allele underwent S phase and mitosis even in the presence of considerable amounts of p25rum1-A58A62 mutant protein (Figure 5B and C). Flow cytometry analysis showed that both strains underwent S phase after the release, though with a delay of ∼15 min in the strain containing the rum1-A58A62 allele (Figure 5C). We also observed a delay of ∼30 min in the accumulation of cdc13 cyclin in cells expressing rum1-A58A62 compared with the control, indicating that once cells initiate DNA replication the cdc13 protein levels increase independently of the presence or absence of p25rum1 protein. Figure 5.p25rum1-A58A62 is stable throughout the cell cycle. Cultures of cdc10-129 rum1Δ int::rum1+ (A) and cdc10-129 rum1Δ int::rum1-A58A62 (B) were grown at 25°C to mid-exponential phase in minimal medium, shifted to 36°C for 4 h and then released at 25°C. Samples for Western blot and flow cytometry were taken before the shift to 36°C (ASN), 4 h after the shift to 36°C (t = 0) and every 30 min after the release at 25°C. Levels of p25rum1 and p56cdc13 were determined in both strains. (A) p25rum1 levels were high at the block point (t = 0) and disappeared just before the onset of S phase (t = 60). (B) p25rum1-A58A62 levels were high and constant throughout the experiment. p56cdc13 levels were undetectable at the cdc10–129 arrest point and then increased in both strains as cells underwent S phase. A delay of ∼30 min in the accumulation of p56cdc13 and in the onset of S phase was observed in the strain expressing rum1-A58A62 compared with the control strain. rum1Δ (Δ) and rum1+ overexpressor (OVP) strains were used as negative and positive controls. (C) The percentage of cells in G1 after the release to 25°C. Asn, asynchronous cells grown at 25°C. White bars, cdc10-129 rum1Δ int::rum1+ cells. Black bars, cdc10-129 rum1Δ int::rum1-A58A62 cells. Download figure Download PowerPoint If p25rum1 plays a role in determining the length of G1, stabilization of this protein should delay the onset of S phase. Flow cytometry analysis of haploid integrants of the rum1-A58A62 allele in the rum1 deletion (rum1Δ) and in the cdc10-129 rum1Δ strains grown at 25°C clearly revealed a G1 population (Figure 6A). To quantify this delay, we selected the smallest cells in the cdc10-129 rum1Δ int-rum1-A58A62 culture, that were in G1, S and early G2 phases, by elutriation at 25°C. Half of these cells were incubated at 25°C and samples for flow cytometry analysis were taken every 20 min for 6 h (Figure 6B, left). All the G1 cells underwent S phase by 40 min. In the next cell cycle, these cells remained in G1 for ∼90 min after cytokinesis. Considering that the cell cycle length in this experiment was 260 min, then the onset of S phase was delayed by as much as 0.3 of a cell cycle. In a synchronous culture of the cdc10-129 control strain at 25°C, we did not observe any delay in G1 (data not shown, see Moreno and Nurse 1994 Figure 4D). The other half of the cells were shifted at time 0 after elutriation to 36°C. Ninety percent of these cells u
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