RESUMEN

The identification of an actomyosin-based contractile ring in budding yeast has recently established this organism as a general model for studying cytokinesis. Work over the past three years has provided important new insights into the conserved mechanisms underlying the assembly and regulation of the cytokinetic structures. This review covers the recent progress in studying cytokinesis in budding yeast.

Asunto(s)

Mitosis/fisiología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Huso Acromático/fisiología , Actomiosina/fisiología , Saccharomyces cerevisiae/citología

RESUMEN

A Saccharomyces cerevisiae mutant unable to grow in a cdc28-1N background was isolated and shown to be affected in the ELM1 gene. Elm1 is a protein kinase, thought to be a negative regulator of pseudo-hyphal growth. We show that Cdc11, one of the septins, is delocalised in the mutant, indicating that septin localisation is partly controlled by Elm1. Moreover, we show that cytokinesis is delayed in an elm1delta mutant. Elm1 levels peak at the end of the cell cycle and Elm1 is localised at the bud neck in a septin-dependent fashion from bud emergence until the completion of anaphase, at about the time of cell division. Genetic and biochemical evidence suggest that Elm1 and the three other septin-localised protein kinases, Hsl1, Gin4 and Kcc4, work in parallel pathways to regulate septin behaviour and cytokinesis. In addition, the elm1delta;) morphological defects can be suppressed by deletion of the SWE1 gene, but not the cytokinesis defect nor the septin mislocalisation. Our results indicate that cytokinesis in budding yeast is regulated by Elm1.

Asunto(s)

División Celular/fisiología , Proteínas del Citoesqueleto , Proteínas Quinasas/fisiología , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiología , Proteínas de Ciclo Celular/fisiología , Proteínas Fúngicas/fisiología , Mutación , Saccharomyces cerevisiae/citología

RESUMEN

We previously isolated the SKN7 gene in a screen designed to isolate new components of the G1-S cell cycle transcription machinery in budding yeast. We have now found that Skn7 associates with Mbp1, the DNA-binding component of the G1-S transcription factor DSC1/MBF. SKN7 and MBP1 show several genetic interactions. Skn7 overexpression is lethal and is suppressed by a mutation in MBP1. Similarly, high overexpression of Mbp1 is lethal and can be suppressed by skn7 mutations. SKN7 is also required for MBP1 function in a mutant compromised for G1-specific transcription. Gel-retardation assays indicate that Skn7 is not an integral part of MBF. However, a physical interaction between Skn7 and Mbp1 was detected using two-hybrid assays and GST pulldowns. Thus, Skn7 and Mbp1 seem to form a transcription factor independent of MBF. Genetic data suggest that this new transcription factor could be involved in the bud-emergence process.

Asunto(s)

Proteínas de Unión al ADN/genética , Proteínas Fúngicas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Ciclo Celular/genética , Supervivencia Celular/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Mutación , Plásmidos , Saccharomyces cerevisiae/crecimiento & desarrollo , Factores de Transcripción/metabolismo , Transformación Genética

RESUMEN

To identify potential RhoA effector proteins, we conducted a two-hybrid screen for cDNAs encoding proteins that interact with a Gal4-RhoA.V14 fusion protein. In addition to the RhoA effector ROCK-I we identified cDNAs encoding Kinectin, mDia2 (a p140 mDia-related protein), and the guanine nucleotide exchange factor, mNET1. ROCK-I, Kinectin, and mDia2 can bind the wild type forms of both RhoA and Cdc42 in a GTP-dependent manner in vitro. Comparison of the ROCK-I and Kinectin sequences revealed a short region of sequence homology that is both required for interaction in the two-hybrid assay and sufficient for weak interaction in vitro. Sequences related to the ROCK-I/Kinectin sequence homology are present in heterotrimeric G protein beta subunits and in the Saccharomyces cerevisiae Skn7 protein. We show that beta2 and Skn7 can interact with mammalian RhoA and Cdc42 and yeast Rho1, both in vivo and in vitro. Functional assays in yeast suggest that the Skn7 ROCK-I/Kinectin homology region is required for its function in vivo.

Asunto(s)

Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas de la Membrana , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , Secuencia Conservada , ADN Complementario , Proteínas de Unión al ADN/biosíntesis , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas de Unión al GTP/biosíntesis , Proteínas de Unión al GTP/química , Péptidos y Proteínas de Señalización Intracelular , Sustancias Macromoleculares , Ratones , Datos de Secuencia Molecular , Proteínas Oncogénicas/química , Proteínas Serina-Treonina Quinasas/química , Receptores de Superficie Celular/química , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Factores de Transcripción/biosíntesis , Quinasas Asociadas a rho , Proteína de Unión al GTP rhoA

RESUMEN

We previously described a mutation feeB1 conferring a temperature-sensitive filamentation phenotype and resistance to the calmodulin inhibitor 48/80 in Escherichia coli, which constitutes a single base change in the acceptor stem of the rare tRNA3Leu recognizing CUA codons. We now describe a second mutant, feeA1, unlinked to feeB, but displaying a similar phenotype, 48/80 resistance and a reduced growth rate at the permissive temperature, 30 degrees C, and temperature-sensitive, forming short filaments at 42 degrees C. In the feeA mutant, tRNA3Leu expression (but not that of tRNA1Leu) was reduced approximately fivefold relative to the wild type. We previously showed that the synthesis of beta-galactosidase, which unusually requires the translation of 6-CUA codons, was substantially reduced, particularly at 42 degrees C, in feeB mutants. The feeA mutant also shows drastically reduced synthesis of beta-galactosidase at the non-permissive temperature and reduced levels even at the permissive temperature. We also show that increased copy numbers of the abundant tRNA1Leu, which can also read CUA codons at low efficiency, suppressed the effects of feeA1 under some conditions, providing further evidence that the mutant was deficient in CUA translation. This, and the previous study, demonstrates that mutations which either reduce the activity of tRNA3Leu or the cellular amount of tRNA3Leu confer resistance to the drug 48/80, with concomitant inhibition of cell division at 42 degrees C.

Asunto(s)

Escherichia coli/genética , ARN de Transferencia de Leucina/biosíntesis , p-Metoxi-N-metilfenetilamina/farmacología , Codón , Farmacorresistencia Microbiana , Mapeo Restrictivo , Temperatura

Asunto(s)

Fenómenos Fisiológicos Bacterianos , Ciclinas/biosíntesis , Proteínas de Unión al ADN/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiología , Transducción de Señal , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/metabolismo , Fase G1 , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Factores de Transcripción/genética

RESUMEN

Until recently, two-component signal-transduction pathways were thought to be exclusively found in bacteria. Some eukaryotic examples have now been characterized but, at least in the budding yeast Saccharomyces cerevisiae, it appears that this type of signal-transduction pathway is not utilized as extensively as in bacteria. Further, the few eukaryotic examples described suggest that two-component signal-transduction pathways might not be freestanding, as in prokaryotes, but might effect gene expression by regulating eukaryotic mitogen-activated protein (MAP) kinase pathways.

RESUMEN

The transcription factors SBF and DSC1/MBF bind SCB and MCB promoter elements, respectively, and are essential for the cell cycle progression of Saccharomyces cerevisiae through the control of G1 cyclin gene expression. We isolated a gene (BRY1; Bacterial Response regulator in Yeast) able to activate either MCB or SCB promoter elements on a reporter plasmid which, when overexpressed, can bypass the normally essential requirement for SBF and DSC1/MBF by the stimulation of CLN1 and CLN2 expression. In the case of CLN2 at least, this expression depends upon the MCB and SCB promoter elements. In wild-type yeast, the disruption of BRY1 has no apparent phenotype, but under conditions where the activities of SBF and DSC1/MBF are reduced, BRY1 becomes essential. Our data imply the existence of a third pathway affecting cyclin expression. BRY1 is the same gene as SKN7 which has significant sequence homology to the receiver domains found in response regulator proteins from the bacterial two-component signal transduction pathways. SKN7 is thought to affect cell wall structure, and when highly overexpressed we find that BRY1/SKN7 is lethal perhaps because of perturbations in cell wall biosynthesis. The lethality is partially rescued by genes from the protein kinase C pathway, but genetic data imply that BRY1/SKN7 and protein kinase C are not in the same pathway. Our results suggest that Bry1/Skn7 can influence the expression of MCB- and SCB-driven gene expression in budding yeast, perhaps including genes involved in cell wall metabolism, via a two-component signal transduction pathway which activates Bry1/Skn7 in response to an unidentified signal.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Factores de Transcripción/metabolismo , Ciclinas/genética , Ciclinas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/genética , Fase G1 , Genes Fúngicos , Factores de Transcripción del Choque Térmico , Regiones Promotoras Genéticas , Proteína Quinasa C/metabolismo , Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Factores de Transcripción/genética

RESUMEN

A mutant, tfpA1, resistant to the calmodulin inhibitor trifluoroperazine (TFP) at 30 degrees C, was isolated in Escherichia coli. The mutant showed a reduced growth rate at 30 degrees C and was temperature sensitive (ts) at 42 degrees C for growth, forming short filaments. The mutation was mapped to the 24 min region of the chromosome and the gene was cloned by complementation of the ts defect. Subsequent subcloning, complementation analysis, marker rescue mapping and sequencing, identified tfpA as fabD, encoding the 35 kDa, malonyl-coenzyme A transacylase (MCT) enzyme, required for the initial step in the elongation cycle for fatty acid biosynthesis. Resistance to TFP may result from altered permeability of the cell envelope, although the mutant remained sensitive to other calmodulin inhibitors and to other antibacterial agents. Alternatively, resistance may be more indirect, resulting from alterations in intracellular Ca++ levels which affect the activity of the TFP target in some way.

Asunto(s)

Aciltransferasas/genética , Farmacorresistencia Microbiana/genética , Escherichia coli/genética , Genes Bacterianos/genética , Trifluoperazina/farmacología , S-Maloniltransferasa de la Proteína Transportadora de Grupos Acilo , Proteínas Bacterianas/análisis , Proteínas Bacterianas/química , Secuencia de Bases , Calmodulina/antagonistas & inhibidores , Membrana Celular/metabolismo , Mapeo Cromosómico , Clonación Molecular , Escherichia coli/efectos de los fármacos , Proteínas de Escherichia coli , Acido Graso Sintasa Tipo II , Ácidos Grasos/biosíntesis , Prueba de Complementación Genética , Datos de Secuencia Molecular , Peso Molecular , Mutación/genética , Análisis de Secuencia de ADN , Temperatura

RESUMEN

We have isolated several classes of spontaneous mutants resistant to the calmodulin inhibitor 48/80 which inhibits cell division in Escherichia coli K12. Several mutants were also temperature sensitive for growth and this property was exploited to clone a DNA fragment from an E. coli gene library restoring growth at 42 degrees C and drug sensitivity at 30 degrees C in one such mutant. Physical and genetic mapping confirmed that both the mutation and the cloned DNA were located at 15.5 min on the E. coli chromosome at a locus designated feeB. By subcloning, complementation analysis and sequencing, the feeB locus was identified as identical to the tRNA(CUALEU) gene. When the mutant locus was isolated and sequenced, the mutation was confirmed as a single base change, C to A, at position 77 in the acceptor stem of this rare Leu tRNA. In other studies we obtained evidence that this mutant tRNA, recognizing the rare Leu codon, CUA, was defective in translation at both permissive and non-permissive temperatures. The feeB1 mutant is defective in division and shows a reduced growth rate at non-permissive temperature. We discuss the possibility that the mutant tRNA(3Leu) is limiting for the synthesis of a polypeptide(s), requiring several CUA codons for translation which in turn regulates in some way the level or activity of the drug target, a putative cell cycle protein.

Asunto(s)

Calmodulina/antagonistas & inhibidores , Escherichia coli/genética , ARN de Transferencia de Leucina/genética , p-Metoxi-N-metilfenetilamina/farmacología , Secuencia de Bases , Southern Blotting , Mapeo Cromosómico , Cromosomas Bacterianos , Clonación Molecular , Codón , Sondas de ADN , ADN Bacteriano/biosíntesis , Farmacorresistencia Microbiana/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Genes Bacterianos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , Biosíntesis de Proteínas , Mapeo Restrictivo , Temperatura , beta-Galactosidasa/metabolismo

RESUMEN

For a number of years now, we have argued that current models for the control of initiation of DNA synthesis, chromosomal partitioning and septum formation in Escherichia coli are unsatisfactory. Indeed, we could argue that despite considerable efforts, with the possible exception of dnaA and ftsZ, no genes specifically implicated in these control processes have been identified. In the cases of DnaA and FtsZ, no evidence has appeared to indicate how such molecules might be regulated to act once per cycle. In 1988, we formulated a specific proposal that the timing of cell cycle events in E. coli might be determined by a Ca++ flux, mediated by calcium-binding proteins and protein kinases and culminating, in the case of chromosome segregation and division, in the action of force-generating proteins such as myosin (Norris et al., 1988). In formulating this proposal, we took the view that the fundamental elements of cell cycle regulation are likely to be highly conserved across all species including prokaryotes. In this presentation, we shall describe the approaches we have been taking in order to test this hypothesis and to summarize the data obtained, in particular in relation to new genes identified which may play a role in the E. coli cell cycle. We shall also briefly indicate recent data from other laboratories consistent with our general hypothesis.

Asunto(s)

Proteínas Bacterianas/metabolismo , Calcio/metabolismo , Escherichia coli/metabolismo , Ciclo Celular/genética , Ciclo Celular/fisiología , División Celular/genética , División Celular/fisiología , Farmacorresistencia Microbiana/genética , Escherichia coli/citología , Escherichia coli/genética , Genes Bacterianos , Mutación , Miosinas/metabolismo