RESUMEN
Nuclear pore complexes (NPCs) mediate communication between the nucleus and the cytoplasm, and regulate gene expression by interacting with transcription and mRNA export factors. Lysine acetyltransferases (KATs) promote transcription through acetylation of chromatin-associated proteins. We find that Esa1, the KAT subunit of the yeast NuA4 complex, also acetylates the nuclear pore basket component Nup60 to promote mRNA export. Acetylation of Nup60 recruits the mRNA export factor Sac3, the scaffolding subunit of the Transcription and Export 2 (TREX-2) complex, to the nuclear basket. The Esa1-mediated nuclear export of mRNAs in turn promotes entry into S phase, which is inhibited by the Hos3 deacetylase in G1 daughter cells to restrain their premature commitment to a new cell division cycle. This mechanism is not only limited to G1/S-expressed genes but also inhibits the expression of the nutrient-regulated GAL1 gene specifically in daughter cells. Overall, these results reveal how acetylation can contribute to the functional plasticity of NPCs in mother and daughter yeast cells. In addition, our work demonstrates dual gene expression regulation by the evolutionarily conserved NuA4 complex, at the level of transcription and at the stage of mRNA export by modifying the nucleoplasmic entrance to nuclear pores.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomycetales , Acetilación , Transporte Activo de Núcleo Celular/fisiología , Ciclo Celular , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Histona Desacetilasas/metabolismo , Poro Nuclear/genética , Poro Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismoRESUMEN
Pho85 is a multifunctional CDK that signals to the cell when environmental conditions are favorable. It has been connected to cell cycle control, mainly in Start where it promotes the G1/S transition. Here we describe that the Start repressor Whi7 is a key target of Pho85 in the regulation of cell cycle entry. The phosphorylation of Whi7 by Pho85 inhibits the repressor and explains most of the contribution of the CDK in the activation of Start. Mechanistically, Pho85 downregulates Whi7 protein levels through the control of Whi7 protein stability and WHI7 gene transcription. Whi7 phosphorylation by Pho85 also restrains the intrinsic ability of Whi7 to associate with promoters. Furthermore, although Whi5 is the main Start repressor in normal cycling cells, in the absence of Pho85, Whi7 becomes the major repressor leading to G1 arrest. Overall, our results reveal a novel mechanism by which Pho85 promotes Start through the regulation of the Whi7 repressor at multiple levels, which may confer to Whi7 a functional specialization to connect the response to adverse conditions with the cell cycle control.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomycetales , Ciclo Celular/genética , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/genética , Ciclinas/metabolismo , Regulación Fúngica de la Expresión Génica , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismoRESUMEN
The protein kinase C (PKC) family plays important regulatory roles in numerous cellular processes. Saccharomyces cerevisiae contains a single PKC, Pkc1, whereas in mammals, the PKC family comprises nine isoforms. Both Pkc1 and the novel isoform PKCδ are involved in the control of DNA integrity checkpoint activation, demonstrating that this mechanism is conserved from yeast to mammals. To explore the function of PKCδ in a non-tumor cell line, we employed CRISPR-Cas9 technology to obtain PKCδ knocked-out mouse embryonic stem cells (mESCs). This model demonstrated that the absence of PKCδ reduced the activation of the effector kinase CHK1, although it suggested that other isoform(s) might contribute to this function. Therefore, we used yeast to study the ability of each single PKC isoform to activate the DNA integrity checkpoint. Our analysis identified that PKCθ, the closest isoform to PKCδ, was also able to perform this function, although with less efficiency. Then, by generating truncated and mutant versions in key residues, we uncovered differences between the activation mechanisms of PKCδ and PKCθ and identified their essential domains. Our work strongly supports the role of PKC as a key player in the DNA integrity checkpoint pathway and highlights the advantages of combining distinct research models.
Asunto(s)
Proteína Quinasa C , Saccharomyces cerevisiae , Animales , Ratones , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Mamíferos/metabolismo , ADN , Proteína Quinasa C-delta/genéticaRESUMEN
Start is the main decision point in the eukaryotic cell cycle at which cells commit to a new round of cell division. It involves the irreversible activation of a transcriptional programme through the inactivation of Start transcriptional repressors: the retinoblastoma family in mammals, or Whi5 and its recently identified paralogue Whi7 (also known as Srl3) in budding yeast. Here, we provide a comprehensive comparison of Whi5 and Whi7 that reveals significant qualitative differences. Indeed, the expression, subcellular localization and functionality of Whi7 and Whi5 are differentially regulated. Importantly, Whi7 shows specific properties in its association with promoters not shared by Whi5, and for the first time, we demonstrate that Whi7, and not Whi5, can be the main contributor to Start inhibition such as it occurs in the response to cell wall stress. Our results help to improve understanding of the interplay between multiple differentially regulated Start repressors in order to face specific cellular conditions.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomycetales , Ciclo Celular/genética , División Celular , Regulación Fúngica de la Expresión Génica , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismoRESUMEN
The protein kinase C (PKC) superfamily plays key regulatory roles in numerous cellular processes. Saccharomyces cerevisiae contains a single PKC, Pkc1, whose main function is cell wall integrity maintenance. In this work, we connect the Pkc1 protein to the maintenance of genome integrity in response to genotoxic stresses. Pkc1 and its kinase activity are necessary for the phosphorylation of checkpoint kinase Rad53, histone H2A and Xrs2 protein after deoxyribonucleic acid (DNA) damage, indicating that Pkc1 is required for activation of checkpoint kinases Mec1 and Tel1. Furthermore, Pkc1 electrophoretic mobility is delayed after inducing DNA damage, which reflects that Pkc1 is post-translationally modified. This modification is a phosphorylation event mediated by Tel1. The expression of different mammalian PKC isoforms at the endogenous level in yeast pkc1 mutant cells revealed that PKCδ is able to activate the DNA integrity checkpoint. Finally, downregulation of PKCδ activity in HeLa cells caused a defective activation of checkpoint kinase Chk2 when DNA damage was induced. Our results indicate that the control of the DNA integrity checkpoint by PKC is a mechanism conserved from yeast to humans.
Asunto(s)
Daño del ADN , Proteína Quinasa C/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/metabolismo , Quinasa de Punto de Control 2/metabolismo , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mutágenos/toxicidad , Mutación , Proteína Quinasa C/análisis , Proteína Quinasa C/genética , Proteína Quinasa C-delta/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/análisis , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Polar residues are present in TM (transmembrane) helices and may influence the folding or association of membrane proteins. In the present study, we use an in vivo approach to analyse the functional and structural roles for amino acids in membrane-spanning motifs using the Rot1 (reversal of Tor2 lethality 1) protein as a model. Rot1 is an essential membrane protein in Saccharomyces cerevisiae and it contains a single TM domain. An alanine insertion scanning analysis of this TM helix revealed that the integrity of the central domain is essential for protein function. We identified a critical serine residue inside the helix that plays an essential role in maintaining cell viability in S. cerevisiae. Replacement of the serine residue at position 250 with a broad variety of amino acids did not affect protein targeting and location, but completely disrupted protein function causing cell death. Interestingly, substitution of the serine residue by threonine resulted in sustained cell viability, demonstrating that the hydroxy group of the TM serine side chain plays a critical role in protein function. The results of the present study indicate that Rot1 needs the TM Ser250 to interact with other membrane components and exert its functional role, avoiding exposure of the serine hydrogen-bonding group at the lipid-exposed surface.
Asunto(s)
Membrana Celular/genética , Supervivencia Celular/fisiología , Proteínas de la Membrana/genética , Chaperonas Moleculares/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Serina/genética , Secuencia de Aminoácidos , Membrana Celular/fisiología , Proteínas de la Membrana/fisiología , Chaperonas Moleculares/fisiología , Datos de Secuencia Molecular , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Serina/fisiologíaRESUMEN
The yeast ß-karyopherin Msn5 controls the SBF cell-cycle transcription factor, responsible for the periodic expression of CLN2 cyclin gene at G1/S, and the nuclear export of Cln2 protein. Here we show that Msn5 regulates Cln2 by an additional mechanism. Inactivation of Msn5 causes a severe reduction in the cellular content of Cln2. This occurs by a post-transcriptional mechanism, since CLN2 mRNA level is not importantly affected in asynchronous cultures. Cln2 stability is not significantly altered in msn5 cells and inactivation of Msn5 causes a reduction in protein level even when Cln2 is stabilized. Therefore, the reduced amount of Cln2 in msn5 cells is mainly due not to a higher rate of protein degradation but to a defect in Cln2 synthesis. In fact, analysis of polysome profiles indicated that Msn5 inactivation causes a shift of CLN2 and SWI5 mRNAs from heavy-polysomal to light-polysomal and non-polysomal fractions, supporting a defect in Cln2 and Swi5 protein synthesis in the msn5 mutant. The analysis of truncated versions of Cln2 and of chimeric cyclins combining distinct domains from Cln2 and the related Cln1 cyclin identified an internal region in Cln2 from 181 to 225 residues that when fused to GFP is able to confer Msn5-dependent regulation of protein cellular content. Finally, we showed that a high level of Cln2 is toxic in the absence of Msn5. In summary, we described that Msn5 is required for the proper protein synthesis of specific proteins, introducing a new level of control of cell cycle regulators.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ciclinas/metabolismo , Carioferinas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Actinas/genética , Actinas/metabolismo , Proteínas de Ciclo Celular/genética , Ciclinas/genética , Regulación Fúngica de la Expresión Génica , Carioferinas/genética , Mutagénesis , Polirribosomas/metabolismo , Biosíntesis de Proteínas , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genéticaRESUMEN
Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer. Although most cSCCs have good prognosis, a subgroup of high-risk cSCC has a higher frequency of recurrence and mortality. Therefore, the identification of molecular risk factors associated with this aggressive subtype is of major interest. In this work we carried out a global-scale approach to investigate the DNA-methylation profile in patients at different stages, from premalignant actinic keratosis to low-risk invasive and high-risk non-metastatic and metastatic cSCC. The results showed massive non-sequential changes in DNA-methylome and identified a minimal methylation signature that discriminates between stages. Importantly, a direct comparison of low-risk and high-risk stages revealed epigenetic traits characteristic of high-risk tumours. Finally, a prognostic prediction model in cSCC patients identified a methylation signature able to predict the overall survival of patients. Thus, the analysis of DNA-methylation in cSCC revealed changes during the evolution of the disease through the different stages that can be of great value not only in the diagnosis but also in the prognosis of the disease.
Asunto(s)
Carcinoma de Células Escamosas/genética , Epigénesis Genética/genética , Neoplasias Cutáneas/genética , Anciano , Anciano de 80 o más Años , Carcinoma de Células Escamosas/patología , Dermatoglifia del ADN/métodos , Metilación de ADN/genética , Progresión de la Enfermedad , Epigenómica/métodos , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Estudio de Asociación del Genoma Completo/métodos , Humanos , Queratosis Actínica/patología , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/patología , Estadificación de Neoplasias/métodos , Pronóstico , Factores de Riesgo , Neoplasias Cutáneas/patologíaRESUMEN
This article describes a laboratory exercise designed for undergraduate students in the subject of "Regulation of cell proliferation" which allows the students to carry out a research experiment in an important field such as cell cycle control, and to be introduced to a widely used technique in molecular biology laboratories such as the western blot. The cell cycle is regulated by the succession of cyclin-CDK kinase activities. Activation and inactivation of different cyclin-CDK complexes depend on the control of their positive and negative regulators, cyclins and CDK inhibitors (CKIs), respectively. In this experiment, fluctuations in the level of mitotic cyclin Clb2 and CDK inhibitor Sic1 throughout the cell cycle of Saccharomyces cerevisiae are analyzed, particularly in the context of the control of mitotic exit and Start, two of the most important cell cycle transitions. In order to do this, a cdc15 mutant strain is used to block cells in telophase and, upon release from this blocking, the variation in the levels of Clb2 and Sic1 proteins are analyzed by western blot. Progress along the cell cycle is also evaluated by microscopic analysis of cell morphology and nuclear staining. This practical illustrates the experimental basis of theoretical concepts worked in the classroom and it is a good framework for an in-depth discussion of these concepts based on experimental data analysis. © 2018 International Union of Biochemistry and Molecular Biology, 46(5):527-535, 2018.
Asunto(s)
Biología Celular/educación , Quinasas Ciclina-Dependientes/genética , Regulación Enzimológica de la Expresión Génica/genética , Laboratorios , Biología Molecular/educación , Estudiantes , Universidades , Ciclo Celular , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/química , Ciclinas/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/enzimologíaRESUMEN
The control of the subcellular localization of cell cycle regulators has emerged as a crucial mechanism in the regulation of cell division. In the present work, we have characterized the function of the karyopherin Msn5p in the control of the cell cycle of Saccharomyces cerevisiae. Phenotypic analysis of the msn5 mutant revealed an increase in cell size and a functional interaction between Msn5p and the cell cycle transcription factor SBF (composed of the Swi4p and Swi6p proteins), indicating that Msn5p is involved in Start control. In fact, we have shown that the level of Cln2p protein is drastically reduced in an msn5 mutant. The effect on CLN2 expression is mediated at a transcriptional level, Msn5p being necessary for proper SBF-dependent transcription. On the contrary, loss of MSN5 has no effect on the closely related transcription factor MBF (composed of the Mbp1p and Swi6p proteins). Regulation of SBF by Msn5p is exerted by control of the localization of the regulatory subunit Swi6p. Swi6p shuttles between the nucleus and the cytoplasm during the cell cycle, and we have found that Msn5p is required for Swi6p export from the nucleus during the G(2)-M phase. What is more important, we have demonstrated that export of Swi6p to the cytoplasm is required for SBF activity, providing evidence for a functional switch of Swi6p linked to its nucleocytoplasmic shuttling during the cell cycle.
Asunto(s)
Proteínas Portadoras/metabolismo , Ciclo Celular/fisiología , Núcleo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Carioferinas , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Transporte Activo de Núcleo Celular , Proteínas Portadoras/genética , Proteínas Cromosómicas no Histona/genética , Ciclinas/genética , Citoplasma/metabolismo , Proteínas de Unión al ADN , Regulación Fúngica de la Expresión Génica , Mutación , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
The yeast cyclins Cln1 and Cln2 are very similar in both sequence and function, but some differences in their functionality and localization have been recently described. The control of Cln1 and Cln2 cellular levels is crucial for proper cell cycle initiation. In this work, we analyzed the degradation patterns of Cln1 and Cln2 in order to further investigate the possible differences between them. Both cyclins show the same half-life but, while Cln2 degradation depends on ubiquitin ligases SCFGrr1 and SCFCdc4, Cln1 is affected only by SCFGrr1. Degradation analysis of chimeric cyclins, constructed by combining fragments from Cln1 and Cln2, identifies the N-terminal sequence of the proteins as responsible of the cyclin degradation pattern. In particular, the N-terminal region of Cln2 is required to mediate degradation by SCFCdc4. This region is involved in nuclear import of Cln1 and Cln2, which suggests that differences in degradation may be due to differences in localization. Moreover, a comparison of the cyclins that differ only in the presence of the Cln2 nuclear export signal indicates a greater instability of exported cyclins, thus reinforcing the idea that cyclin stability is influenced by their localization.
RESUMEN
Start is the main decision point in eukaryotic cell cycle in which cells commit to a new round of cell division. It involves the irreversible activation of a transcriptional program by G1 CDK-cyclin complexes through the inactivation of Start transcriptional repressors, Whi5 in yeast or Rb in mammals. Here we provide novel keys of how Whi7, a protein related at sequence level to Whi5, represses Start. Whi7 is an unstable protein, degraded by the SCFGrr1 ubiquitin-ligase, whose stability is cell cycle regulated by CDK1 phosphorylation. Importantly, Whi7 associates to G1/S gene promoters in late G1 acting as a repressor of SBF-dependent transcription. Our results demonstrate that Whi7 is a genuine paralog of Whi5. In fact, both proteins collaborate in Start repression bringing to light that yeast cells, as occurs in mammalian cells, rely on the combined action of multiple transcriptional repressors to block Start transition.The commitment of cells to a new cycle of division involves inactivation of the Start transcriptional repressor Whi5. Here the authors show that the sequence related protein Whi7 associates to G1/S gene promoters in late G1 and collaborates with Whi5 in Start repression.
Asunto(s)
Proteínas Represoras/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/genética , Puntos de Control del Ciclo Celular , Ciclinas/análisis , Ciclinas/genética , Ciclinas/metabolismo , Regulación Fúngica de la Expresión Génica , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/análisis , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcripción Genética/fisiologíaRESUMEN
Epitope-tagging of proteins has become a widespread technique for the analysis of protein function, protein interactions and protein localization among others. Tagging of genes by chromosomal integration of PCR amplified cassettes is a widely used and fast method to label proteins in vivo. Different systems have been developed during years in the yeast Saccharomyces cerevisiae. In the present study, we analysed systematically a set of yeast proteins that were fused to different tags. Analysis of the tagged proteins revealed an unexpected general effect on protein level when some specific tagging module was used. This was due in all cases to a destabilization of the proteins and caused a reduced protein activity in the cell that was only apparent in particular conditions. Therefore, an extremely cautious approach is required when using this strategy.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Inestabilidad Genómica , Proteínas Recombinantes de Fusión/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Artefactos , Epítopos/genética , Epítopos/metabolismo , Marcación de Gen , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hemaglutininas Virales/genética , Hemaglutininas Virales/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Coloración y Etiquetado/métodosRESUMEN
The rsf12 mutation was isolated in a synthetic lethal screen for genes functionally interacting with Swi4. RSF12 is CLB5. The clb5 swi4 mutant cells arrest at G(2)/M due to the activation of the DNA-damage checkpoint. Defects in DNA integrity was confirmed by the increased rates of chromosome loss and mitotic recombination. Other results suggest the presence of additional defects related to morphogenesis. Interestingly, genes of the PKC pathway rescue the growth defect of clb5 swi4, and pkc1 and slt2 mutations are synthetic lethal with clb5, pointing to a connection between Clb5, the PKC pathway, and Swi4. Different observations suggest that like Clb5, the PKC pathway and Swi4 are involved in the control of DNA integrity: there is a synthetic interaction between pkc1 and slt2 with rad9; the pkc1, slt2, and swi4 mutants are hypersensitive to hydroxyurea; and the Slt2 kinase is activated by hydroxyurea. Reciprocally, we found that clb5 mutant is hypersensitive to SDS, CFW, latrunculin B, or zymolyase, which suggests that, like the PKC pathway and Swi4, Clb5 is related to cell integrity. In summary, we report numerous genetic interactions and phenotypic descriptions supporting a close functional relationship between the Clb5 cyclin, the PKC pathway, and the Swi4 transcription factor.
Asunto(s)
Ciclo Celular/genética , Cromosomas Fúngicos/genética , Ciclina B/metabolismo , ADN de Hongos/química , Proteína Quinasa C/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Western Blotting , Ciclo Celular/fisiología , Ciclina B/genética , Proteínas de Unión al ADN , Citometría de Flujo , Técnica del Anticuerpo Fluorescente Indirecta , Hidroxiurea , Inmunoprecipitación , Mutación/genética , Proteína Quinasa C/genética , Recombinación Genética/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genéticaRESUMEN
Cln1p and Cln2p are considered as equivalent cyclins on the basis of sequence homology, regulation, and functional studies. Here we describe a functional distinction between the Cln1p and Cln2p cyclins in the control of the G1/S transition. Inactivation of CLN2, but not of CLN1, leads to a larger-than-normal cell size, whereas overexpression of CLN2, but not of CLN1, results in smaller-than-normal cells. Furthermore, mild ectopic expression of CLN2, but not of CLN1, suppresses the lethality of swi4swi6 and cdc28 mutant strains. In the absence of Cln1p, the kinetics of budding, initiation of DNA replication, and activation of the Start-transcription program are not affected; by contrast, loss of Cln2p causes a delay in bud emergence. A primary role for Cln2p but not for Cln1p in budding is reinforced by the observation that only the cln2 mutation is synthetic lethal with a cdc42 mutation, and only the cln2 mutant strain is hypersensitive to latrunculin B. In addition, we found that Cln1p showed a more prominent nuclear staining than Cln2p. Finally, chimeric proteins composed of Cln1p and Cln2p revealed that Cln2p integrity is required for its functional specificity.
Asunto(s)
Ciclinas/fisiología , Mitosis/fisiología , Fase S/fisiología , Saccharomyces cerevisiae/fisiología , Northern Blotting , Western Blotting , Compuestos Bicíclicos Heterocíclicos con Puentes , Tamaño de la Célula , Ciclinas/genética , Citometría de Flujo , Técnica del Anticuerpo Fluorescente Indirecta , Inmunoprecipitación , Mitosis/genética , Mutación/genética , Fase S/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae , Tiazoles , TiazolidinasRESUMEN
BACKGROUND: The maintenance of genomic integrity is essential for cell viability. Complex signalling pathways (DNA integrity checkpoints) mediate the response to genotoxic stresses. Identifying new functions involved in the cellular response to DNA-damage is crucial. The Saccharomyces cerevisiae SLT2 gene encodes a member of the mitogen-activated protein kinase (MAPK) cascade whose main function is the maintenance of the cell wall integrity. However, different observations suggest that SLT2 may also have a role related to DNA metabolism. RESULTS: This work consisted in a comprehensive study to connect the Slt2 protein to genome integrity maintenance in response to genotoxic stresses. The slt2 mutant strain was hypersensitive to a variety of genotoxic treatments, including incubation with hydroxyurea (HU), methylmetanosulfonate (MMS), phleomycin or UV irradiation. Furthermore, Slt2 was activated by all these treatments, which suggests that Slt2 plays a central role in the cellular response to genotoxic stresses. Activation of Slt2 was not dependent on the DNA integrity checkpoint. For MMS and UV, Slt2 activation required progression through the cell cycle. In contrast, HU also activated Slt2 in nocodazol-arrested cells, which suggests that Slt2 may respond to dNTP pools alterations. However, neither the protein level of the distinct ribonucleotide reductase subunits nor the dNTP pools were affected in a slt2 mutant strain. An analysis of the checkpoint function revealed that Slt2 was not required for either cell cycle arrest or the activation of the Rad53 checkpoint kinase in response to DNA damage. However, slt2 mutant cells showed an elongated bud and partially impaired Swe1 degradation after replicative stress, indicating that Slt2 could contribute, in parallel with Rad53, to bud morphogenesis control after genotoxic stresses. CONCLUSIONS: Slt2 is activated by several genotoxic treatments and is required to properly cope with DNA damage. Slt2 function is important for bud morphogenesis and optimal Swe1 degradation under replicative stress. The MAPK Slt2 appears as a new player in the cellular response to genotoxic stresses.
RESUMEN
The Saccharomyces cerevisiae Start repressor Whi5, the functional analogue of mammalian pRB, shuttles between the nucleus and the cytoplasm throughout the cell cycle: enters into the nucleus at the end of mitosis and remains nuclear until Start. We studied the mechanisms involved in this spatial regulation. The nuclear import depends on the beta-karyopherins of the classical import pathway Kap95 and Cse1. Whi5 contains a monopartite and a bipartite classical NLS localized in its N-terminal region which are functionally redundant. A fragment of Whi5 containing these NLSs is able to constitutively accumulate a GFP(4) protein inside the nucleus throughout the cell cycle, which suggests that the Whi5 nuclear import is not cell cycle-regulated. The nuclear export of Whi5 is assisted by beta-karyopherin Msn5. A two-hybrid assay indicates a physical interaction between Whi5 and Msn5. We identified a fragment of Whi5 with export activity from amino acids 51 to 167. Interestingly, this region drives the export of a chimeric nuclear protein in a cell cycle-regulated pattern similarly to that observed for Whi5. Moreover, the nuclear export driven by Whi5(51-167) depends on the phosphorylation of specific Ser residues. Finally, we identified Cdc14 as the phosphatase required for the nuclear accumulation of Whi5.
Asunto(s)
Transporte Activo de Núcleo Celular , Ciclo Celular , Carioferinas/metabolismo , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular , Fosforilación , Unión Proteica , Proteínas Tirosina Fosfatasas , Saccharomyces cerevisiae , beta Carioferinas/metabolismoRESUMEN
ROT1 is an essential gene whose inactivation causes defects in cell cycle progression and morphogenesis in budding yeast. Rot1 affects the actin cytoskeleton during the cell cycle at two levels. First, it is required for the maintenance of apical growth during bud growth. Second, Rot1 is necessary to polarize actin cytoskeleton to the neck region at the end of mitosis; because of this defect, rot1 cells do not properly form a septum to complete cell division. The inability to polarize the actin cytoskeleton at the end of mitosis is not due to a defect in the recruitment of the polarisome scaffold protein Spa2 or the actin cytoskeleton regulators Cdc42 and Cdc24 in the neck region. Previous results indicate a connection between Rot1 and the cyclin Clb2. In fact, overexpression of CLB2 is toxic when ROT1 is partially inactivated, and reciprocally, deletion of CLB2 suppresses the lethality of the rot1 mutant, which indicates a functional antagonism between Clb2 and Rot1. Several genetic interactions suggest a link between Rot1 and the ubiquitin-proteasome system and we show that the Clb2 cyclin is not properly degraded in rot1 cells.
Asunto(s)
Actinas/metabolismo , Ciclina B/metabolismo , Citoesqueleto/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Actinas/aislamiento & purificación , Quinasas CDC2-CDC28/genética , Quinasas CDC2-CDC28/aislamiento & purificación , Quinasas CDC2-CDC28/metabolismo , Ciclo Celular , Polaridad Celular , Ciclina B/aislamiento & purificación , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Proteínas de la Membrana/genética , Chaperonas Moleculares , Mutación , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/aislamiento & purificaciónRESUMEN
Analysis of a three-member gene family in the yeast Saccharomyces cerevisiae has allowed the discovery of a new gene that comprises two contiguous open reading frames previously annotated as YML034w and YML033w. The gene contains a small intron with two alternative 5' splicing sites. It is specifically transcribed during G(2)/M in the cell cycle and after several hours of meiosis induction. Splicing of the mRNA is partially dependent on NAM8 but does not vary during meiosis or the cell cycle. Deletion of the gene induces a shortening of the anaphase and aggravates the phenotype of scc1 and esp1 conditional mutants, which suggests a direct role of the protein in sister chromatid separation.