RESUMEN
Model organisms have emerged as suitable and reliable biological tools to study the properties of proteins whose function is altered in human disease. In the case of the PI3K and PTEN human cancer-related proteins, several vertebrate and invertebrate models, including mouse, fly, worm and amoeba, have been exploited to obtain relevant functional information that has been conserved from these organisms to humans along evolution. The yeast Saccharomyces cerevisiae is an eukaryotic unicellular organism that lacks a canonical mammalian-like PI3K/PTEN pathway and PIP3 as a physiological second messenger, PIP2 being essential for its life. The mammalian PI3K/PTEN pathway can be reconstituted in S. cerevisiae, generating growth alteration phenotypes that can be easily monitored to perform in vivo functional analysis of the molecular constituents of this pathway. Here, we review the current nonmammalian model systems to study PTEN function, summarize our knowledge of PTEN orthologs in yeast species and propose the yeast S. cerevisiae as a sensitive biological sensor of PI3K oncogenicity and PTEN tumor suppressor activity.
Asunto(s)
Genes Supresores de Tumor/fisiología , Fosfohidrolasa PTEN/fisiología , Levaduras/genética , Secuencia de Aminoácidos , Animales , Células Eucariotas/metabolismo , Células Eucariotas/fisiología , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/fisiología , Filogenia , Homología de Secuencia , Transducción de Señal/genética , Transducción de Señal/fisiología , Levaduras/fisiologíaRESUMEN
In Saccharomyces cerevisiae, entry into mitosis requires activation of the cyclin-dependent kinase Cdc28 in its cyclin B (Clb)-associated form. Clb-bound Cdc28 is susceptible to inhibitory tyrosine phosphorylation by Swe1 protein kinase. Swe1 is itself negatively regulated by Hsl1, a Nim1-related protein kinase, and by Hsl7, a presumptive protein-arginine methyltransferase. In vivo all three proteins localize to the bud neck in a septin-dependent manner, consistent with our previous proposal that formation of Hsl1-Hsl7-Swe1 complexes constitutes a checkpoint that monitors septin assembly. We show here that Hsl7 is phosphorylated by Hsl1 in immune-complex kinase assays and can physically associate in vitro with either Hsl1 or Swe1 in the absence of any other yeast proteins. With the use of both the two-hybrid method and in vitro binding assays, we found that Hsl7 contains distinct binding sites for Hsl1 and Swe1. A differential interaction trap approach was used to isolate four single-site substitution mutations in Hsl7, which cluster within a discrete region of its N-terminal domain, that are specifically defective in binding Hsl1. When expressed in hsl7Delta cells, each of these Hsl7 point mutants is unable to localize at the bud neck and cannot mediate down-regulation of Swe1, but retains other functions of Hsl7, including oligomerization and association with Swe1. GFP-fusions of these Hsl1-binding defective Hsl7 proteins localize as a bright perinuclear dot, but never localize to the bud neck; likewise, in hsl1Delta cells, a GFP-fusion to wild-type Hsl7 or native Hsl7 localizes to this dot. Cell synchronization studies showed that, normally, Hsl7 localizes to the dot, but only in cells in the G1 phase of the cell cycle. Immunofluorescence analysis and immunoelectron microscopy established that the dot corresponds to the outer plaque of the spindle pole body (SPB). These data demonstrate that association between Hsl1 and Hsl7 at the bud neck is required to alleviate Swe1-imposed G2-M delay. Hsl7 localization at the SPB during G1 may play some additional role in fine-tuning the coordination between nuclear and cortical events before mitosis.
Asunto(s)
Proteínas Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiología , Alelos , Secuencia de Aminoácidos , Sitios de Unión , Ciclo Celular , Proteínas de Ciclo Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Regulación hacia Abajo , Proteínas Fúngicas/metabolismo , Genotipo , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Microscopía Inmunoelectrónica , Microtúbulos/metabolismo , Mitosis , Datos de Secuencia Molecular , Mutación , Fosforilación , Plásmidos/metabolismo , Mutación Puntual , Pruebas de Precipitina , Unión Proteica , Proteínas Serina-Treonina Quinasas , Estructura Terciaria de Proteína , Proteína-Arginina N-Metiltransferasas , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , Factores de Tiempo , Técnicas del Sistema de Dos HíbridosRESUMEN
The Saccharomyces cerevisiae CDC15, DBF2, TEM1 and CDC14 genes encode regulatory proteins that play a crucial role in the latest stages of the M phase of the cell cycle. By complementation of a S. cerevisiae cdc15-lyt1 mutant with a Candida albicans centromeric-based genomic library, we have isolated a homologue of the protein phosphatase-encoding gene CDC14. The sequence analysis of the C. albicans CDC14 gene reveals a putative open reading frame of 1626 base pairs interrupted by an intron located close to the 5' region. Analysis of C. albicans cDNA proved that the intron is processed in vivo. The CaCDC14 gene shares 49% of amino acid sequence identity with the S. cerevisiae CDC14 gene, 46% with Schizosaccharomyces pombe homologue, 35% with Caenorhabditis elegans and 37% and 38% with human CDC14A and CDC14B genes, respectively. As expected, the C. albicans CDC14 gene complemented a S. cerevisiae cdc14-1 mutant. We found that this gene was able to efficiently suppress not only a S.cerevisiae cdc15-lyt1 mutant but also a dbf2-2 mutant in a low number of copies and allowed growth, although very slightly, of a tem1 deletant. Overexpression of the human CDC14A and CDC14B genes complemented, although very poorly, S. cerevisiae cdc15-lyt1 and dbf2-2 mutants, suggesting a conserved function of these genes throughout phylogeny. The sequence of CaCDC14 was deposited in the EMBL database under Accession No. AJ243449.
Asunto(s)
Candida albicans/genética , Proteínas de Ciclo Celular/genética , Proteínas de Unión al GTP/genética , Mitosis/genética , Proteínas Quinasas/genética , Proteínas Tirosina Fosfatasas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Supresión Genética/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Dosificación de Gen , Regulación Fúngica de la Expresión Génica , Prueba de Complementación Genética , Intrones/genética , Datos de Secuencia Molecular , Proteínas Serina-Treonina Quinasas , Empalme del ARN/genética , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimología , Homología de Secuencia de AminoácidoRESUMEN
Using a hierarchical approach, 620 non-essential single-gene yeast deletants generated by EUROFAN I were systematically screened for cell-wall-related phenotypes. By analyzing for altered sensitivity to the presence of Calcofluor white or SDS in the growth medium, altered sensitivity to sonication, or abnormal morphology, 145 (23%) mutants showing at least one cell wall-related phenotype were selected. These were screened further to identify genes potentially involved in either the biosynthesis, remodeling or coupling of cell wall macromolecules or genes involved in the overall regulation of cell wall construction and to eliminate those genes with a more general, pleiotropic effect. Ninety percent of the mutants selected from the primary tests showed additional cell wall-related phenotypes. When extrapolated to the entire yeast genome, these data indicate that over 1200 genes may directly or indirectly affect cell wall formation and its regulation. Twenty-one mutants with altered levels of beta1,3-glucan synthase activity and five Calcofluor white-resistant mutants with altered levels of chitin synthase activities were found, indicating that the corresponding genes affect beta1,3-glucan or chitin synthesis. By selecting for increased levels of specific cell wall components in the growth medium, we identified 13 genes that are possibly implicated in different steps of cell wall assembly. Furthermore, 14 mutants showed a constitutive activation of the cell wall integrity pathway, suggesting that they participate in the modulation of the pathway either directly acting as signaling components or by triggering the Slt2-dependent compensatory mechanism. In conclusion, our screening approach represents a comprehensive functional analysis on a genomic scale of gene products involved in various aspects of fungal cell wall formation.
RESUMEN
The Saccharomyces cerevisiae Ygr189c, Yel040w, and Ylr213c gene products show significant homologies among themselves and with various bacterial beta-glucanases and eukaryotic endotransglycosidases. Deletion of the corresponding genes, either individually or in combination, did not produce a lethal phenotype. However, the removal of YGR189c and YEL040w, but not YLR213c, caused additive sensitivity to compounds that interfere with cell wall construction, such as Congo red and Calcofluor White, and overexpression of YEL040w led to resistance to these compounds. These genes were renamed CRH1 and CRH2, respectively, for Congo red hypersensitive. By site-directed mutagenesis we found that the putative glycosidase domain of CRH1 was critical for its function in complementing hypersensitivity to the inhibitors. The involvement of CRH1 and CRH2 in the development of cell wall architecture was clearly shown, since the alkali-soluble glucan fraction in the crh1Delta crh2Delta strain was almost twice the level in the wild-type. Interestingly, the three genes were subject to different patterns of transcriptional regulation. CRH1 and YLR213c (renamed CRR1, for CRH related) were found to be cell cycle regulated and also expressed under sporulation conditions, whereas CRH2 expression did not vary during the mitotic cycle. Crh1 and Crh2 are localized at the cell surface, particularly in chitin-rich areas. Consistent with the observed expression patterns, Crh1-green fluorescent protein was found at the incipient bud site, around the septum area in later stages of budding, and in ascospore envelopes. Crh2 was found to localize mainly at the bud neck throughout the whole budding cycle, in mating projections and zygotes, but not in ascospores. These data suggest that the members of this family of putative glycosidases might exert a common role in cell wall organization at different stages of the yeast life cycle.
Asunto(s)
Pared Celular/fisiología , Regulación Fúngica de la Expresión Génica , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/fisiología , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Alelos , Secuencia de Aminoácidos , Bencenosulfonatos/farmacología , Pared Celular/metabolismo , Rojo Congo/farmacología , Glucanos/metabolismo , Glicósido Hidrolasas/metabolismo , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/metabolismo , Microscopía Confocal , Datos de Secuencia Molecular , Familia de Multigenes , Mutagénesis Sitio-Dirigida , Sistemas de Lectura Abierta , Fenotipo , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Factores de Tiempo , Transcripción GenéticaRESUMEN
The complete yeast genome contains a large number of genes of unknown biological function. Simple, rapid and reliable specific screens are valuable tools in exploring gene function via systematic phenotypic analysis of large mutant collections. This report provides a new approach for monitoring changes in cell wall strength, based on the deleterious effects caused by ultrasound on the yeast cell surface. Sonication can thus be used for the screening of mutants affected in the architecture or stability of the cell wall, since such mutants are expected to have an altered sensitivity to this treatment compared to that of a wild-type. The experimental procedure, consisting in the quantification of damaged cells after a mild sonication treatment, by means of flow cytometry, can be applied on a large scale. The usefulness of the sonication assay as a primary screen for cell wall-related mutants is evaluated on the collection of calcofluor white-hypersensitive and -resistant mutants obtained by Lussier et al. (1997). A further phenotypic characterization of the sonication-hypersensitive mutants within the calcofluor white collection is also presented.
Asunto(s)
Pared Celular/fisiología , Genes Fúngicos , Mutación , Levaduras/genética , Bencenosulfonatos/farmacología , Cafeína/farmacología , Calcio/farmacología , Pared Celular/química , Citometría de Flujo , Fenotipo , Sonicación , Levaduras/efectos de los fármacos , Levaduras/ultraestructuraRESUMEN
The Candida albicans XOG1 gene, previously shown to be a good reporter gene in Saccharomyces cerevisiae and C. albicans, was tested in Schizosaccharomyces pombe. Unlike the budding yeast, S. pombe does not produce exoglucanase activity and hence this system would be applicable to any given strain of this organism. The XOG1 gene was located under the control of the nmt1 promoter and its functionality could be demonstrated even at high temperatures (37 degrees C). The exoglucanase activity can be measured both in vivo and in vitro by either a simple biochemical reaction (on cells or media) or by flow cytometry, because the cells remain viable after the assay.
Asunto(s)
Candida albicans/genética , Genes Reporteros/genética , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética , Transformación Genética , beta-Glucosidasa/genética , Candida albicans/enzimología , Medios de Cultivo , Citometría de Flujo , Genes Fúngicos , Glucano 1,3-beta-Glucosidasa , Regiones Promotoras Genéticas , Schizosaccharomyces/crecimiento & desarrollo , Temperatura , Tiamina/metabolismo , beta-Glucosidasa/metabolismoRESUMEN
Exit from mitosis in the budding yeast Saccharomyces cerevisiae cell cycle is regulated by a regulatory network that involves, among other proteins, the small GTPase Tem1, the protein phosphatase Cdc14, and the protein kinases Dbf2 and Cdc15. Using a fusion to jellyfish green fluorescent protein (GFP), here we report that Cdc15 costains with the microtubular-organizing apparatus and that this localization is precluded in a mutant lacking the outer plaque of the spindle pole body (SPB). The appearance of Cdc15 in the SPB is asymmetric and cell-cycle-regulated, preferentially marking the daughter cell SPB at anaphase and eventually disappearing at cytokinesis. Overproduction of GFP-tagged Cdc15 led to an accumulation of the fusion protein in both mother and daughter cells SPBs and, transiently, in small budded cells and shmoos. The Cdc15 localization pattern was maintained in dbf2, cdc14 and anaphase-promoting complex (cdc16) mutants, suggesting that the function of these proteins is not related to the localization of Cdc15 to the SPB but rather, at least in the case of Cdc14, to its timely removal from this structure. Tem1-depleted cells kept alive by Cdc15-GFP overexpression still display a proper localization of Cdc15. The results presented here suggest that the transient cell-cycle-dependent localization of Cdc15 to the SPB plays a role in the regulation of the latest stages of the cell cycle.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas Tirosina Fosfatasas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Anafase , Secuencia de Bases , Ciclo Celular , Proteínas de Ciclo Celular/genética , Cartilla de ADN/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Unión al GTP/genética , Eliminación de Gen , Genes Fúngicos , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestructura , Huso Acromático/metabolismoRESUMEN
The budding yeast lyt1 mutation causes cell lysis. We report here that lyt1 is an allele of cdc15, a gene which encodes a protein kinase that functions late in the cell cycle. Neither cdc15-1 nor cdc15-lyt1 strains are able to septate at 37 degreesC, even though they may manage to rebud. Cells lyse after a shmoo-like projection appears at the distal pole of the daughter cell. Actin polarizes towards the distal pole but the septins remain at the mother-daughter neck. This morphogenetic response reflects entry into a new round of the cell cycle: the preference for polarization from the distal pole was lost in bud1 cdc15 double mutants; double cdc15-lyt1 cdc28-4 mutants, defective for START, did not develop apical projections and apical polarization was accompanied by DNA replication. The same phenomena were caused by mutations in the genes CDC14, DBF2, and TEM1, which are functionally related to CDC15. Apical polarization was delayed in cdc15 mutants as compared with budding in control cells and this delay was abolished in a septin mutant. Our results suggest that the delayed M/G1 transition in cdc15 mutants is due to a septin-dependent checkpoint that couples initiation of the cell cycle to the completion of cytokinesis.
Asunto(s)
Ciclo Celular/fisiología , Proteínas Fúngicas/genética , Genes Fúngicos , Proteínas de Unión al GTP Monoméricas , Proteínas Tirosina Fosfatasas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/genética , División Celular , Mapeo Cromosómico , Clonación Molecular , Replicación del ADN , Proteínas de Unión al GTP/genética , Genotipo , Cinética , Mitosis , Modelos Biológicos , Morfogénesis , Mutagénesis , Sistemas de Lectura Abierta , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas , Transducción de SeñalRESUMEN
We have deleted six different ORFs of unknown function located on the right arm of Saccharomyces cerevisiae chromosome VII; namely, YGR187c/HGH1, YGR189c, YGR194c, YGR195w, YGR196c and YGR198w. No basic phenotypes could be attributed to the strains deleted in any of genes YGR187c/HGH1, YGR189c, YGR194c and YGR196c. These deletants did not show mating, sporulation or growth defects under any of the conditions tested. However, spores bearing deletions in either the YGR195w or YGR198w genes were unable to develop into macroscopical colonies. The YGR195w gene product shows significant homology with bacterial ribonuclease PH, an enzyme hitherto undescribed in yeasts, and its deletion causes a loss of viability after one to three rounds of cell division. Overexpression of this gene, using a tetracycline-regulatable promoter system, did not cause any effect on the cells. Contrary to what has been reported for prokaryotic homologs, this enzyme could play an essential role in yeast cell biology. The product encoded by the other essential ORF, YGR198w, shows no significant homology with any protein of known function in the databases. Spores bearing the deletion usually germinate and give rise to microcolonies of 50-100 non-viable cells.
Asunto(s)
Genes Fúngicos/genética , Sistemas de Lectura Abierta/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Cromosomas Fúngicos/genética , Exorribonucleasas/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma , Eliminación de Gen , Genes Esenciales , Fenotipo , Saccharomyces cerevisiae/fisiología , Esporas Fúngicas/fisiologíaRESUMEN
We report the finding of a Saccharomyces cerevisiae gene necessary for growth in culture media with D-xylulose as the sole carbon source. This gene corresponds to the YGR194c open reading frame that we have previously described, and it is renamed now XKS1. Data bank comparisons of the protein encoded by the XKS1 gene showed significant homology with different xylulokinases, indicating a possible role in xylulose phosphorylation. The wild-type gene in a centromeric plasmid complemented defective growth of xks1 S. cerevisiae mutant strains in xylulose. By contrast, overexpression negatively influenced cell growth in this carbon source.
Asunto(s)
Genes Fúngicos/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Clonación Molecular , Expresión Génica , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiología , Saccharomyces cerevisiae/crecimiento & desarrollo , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Xilulosa/metabolismoRESUMEN
In fungi and many other organisms, a thick outer cell wall is responsible for determining the shape of the cell and for maintaining its integrity. The budding yeast Saccharomyces cerevisiae has been a useful model organism for the study of cell wall synthesis, and over the past few decades, many aspects of the composition, structure, and enzymology of the cell wall have been elucidated. The cell wall of budding yeasts is a complex and dynamic structure; its arrangement alters as the cell grows, and its composition changes in response to different environmental conditions and at different times during the yeast life cycle. In the past few years, we have witnessed a profilic genetic and molecular characterization of some key aspects of cell wall polymer synthesis and hydrolysis in the budding yeast. Furthermore, this organism has been the target of numerous recent studies on the topic of morphogenesis, which have had an enormous impact on our understanding of the intracellular events that participate in directed cell wall synthesis. A number of components that direct polarized secretion, including those involved in assembly and organization of the actin cytoskeleton, secretory pathways, and a series of novel signal transduction systems and regulatory components have been identified. Analysis of these different components has suggested pathways by which polarized secretion is directed and controlled. Our aim is to offer an overall view of the current understanding of cell wall dynamics and of the complex network that controls polarized growth at particular stages of the budding yeast cell cycle and life cycle.
Asunto(s)
Pared Celular/metabolismo , Proteínas Fúngicas/biosíntesis , Proteínas de la Membrana/biosíntesis , Saccharomyces cerevisiae/crecimiento & desarrollo , Pared Celular/enzimología , Pared Celular/ultraestructura , Quitina/biosíntesis , Proteínas Fúngicas/fisiología , Glucanos/biosíntesis , Mananos/biosíntesis , Proteínas de la Membrana/fisiología , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestructuraRESUMEN
Yeast exo-1,3-beta-glucanases are secretable proteins whose function is basically trophic and may also be involved in cell wall glucan hydrolytic processes. Since fluorescein di(beta-D-glucopyranoside) is a fluorogenic substrate detectable and quantifiable by flow cytometry, it was used for testing the ability of the EXG1 gene product of Saccharomyces cerevisiae and its homologous gene in Candida albicans to function as reporter genes. These open reading frames were coupled to different promoters in multicopy plasmids, and exoglucanase activity quantified at flow cytometry. Exoglucanases were found to be useful tools for the study of promoter regions in S. cerevisiae. This technique has the advantage over other reporter gene systems--such as beta-galactosidase fusions--that it does not require permeabilization of yeast cells and therefore it allows the recovery of viable cells--by sorting--after flow cytometry analysis.
Asunto(s)
Genes Fúngicos , Genes Reporteros , Saccharomyces cerevisiae/genética , beta-Glucosidasa/genética , Citometría de Flujo , Glucano 1,3-beta-Glucosidasa , Saccharomyces cerevisiae/enzimología , beta-Glucosidasa/análisisRESUMEN
In order to carry out a systematic search for mutants affected in cell integrity, the diploid strain Saccharomyces cerevisiae D1 was subjected to mutagenesis with ethyl methane sulphonate (EMS), and mutant clones were screened for thermosensitive autolytic phenotypes. The screening was based on examination of cell populations, from individual mutant clones, stained with propidium iodide to establish the proportion of cells lysing under non-permissive conditions by means of flow cytometry. Osmotic remediation of the autolytic phenotype in the presence of 1 M sorbitol was also checked. Out of 13,300 clones surviving mutagenesis, 34 were confirmed to be thermosensitive autolytic and 7 of them showed some osmotic complementation with regard to growth and cell lysis. The osmotic remediation in the other strains was negligible or affected only one of the two parameters. The expression of the mutant phenotype in the strains isolated led to a sporulation defect (40% of the strains) and significant alterations in morphology, such as cells in chains (35%), altered buds (25%) that eventually might elongate, round unbudded and highly vacuolated cells (12%) and large-sized cells (12%). These observations show that alterations in functions related to cell integrity can be correlated with an altered morphology. Genetic analysis of the mutant strains that could sporulate showed that in many instances the mutant phenotype was the result of more than one mutation, the mutations being individually recessive. However, at least one mutant strain, 933, carried a single mendelian mutation that was dominant in the diploid but haploid segregants were non-viable. Dominance of this mutation was also confirmed in tetraploids obtained by means of protoplast fusion.