RESUMEN
Eukaryotic cells commit in G1 to a new mitotic cycle or to diverse differentiation processes. Here we show that Whi3 is a negative regulator of Cln3, a G1 cyclin that promotes transcription of many genes to trigger the G1/S transition in budding yeast. Whi3 contains an RNA-recognition motif that specifically binds the CLN3 mRNA, with no obvious effects on Cln3 levels, and localizes the CLN3 mRNA into discrete cytoplasmic foci. This is the first indication that G1 events may be regulated by locally restricting the synthesis of a cyclin. Moreover, Whi3 is also required for restraining Cln3 function in meiosis, filamentation, and mating, thus playing a key role in cell fate determination in budding yeast.
Asunto(s)
Ciclinas/química , Ciclinas/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/fisiología , Proteínas de Saccharomyces cerevisiae , Saccharomycetales/fisiología , Secuencias de Aminoácidos , Northern Blotting , Linaje de la Célula , Ciclina G , Citoplasma/metabolismo , Hibridación Fluorescente in Situ , Meiosis , Microscopía Fluorescente , Modelos Biológicos , Plásmidos/metabolismo , Unión Proteica , ARN/metabolismo , ARN Mensajero/metabolismo , Factores de TiempoRESUMEN
WHI3 is a gene affecting size control and cell cycle in the yeast Saccharomyces cerevisiae. The whi3 mutant has small cells, while extra doses of WHI3 produce large cells, and a large excess of WHI3 produces a lethal arrest in G1 phase. WHI3 seems to be a dose-dependent inhibitor of Start. Whi3 and its partially redundant homolog Whi4 have an RNA-binding domain, and mutagenesis experiments indicate that this RNA-binding domain is essential for Whi3 function. CLN3-1 whi3 cells are extremely small, nearly sterile, and largely nonresponsive to mating factor. Fertility is restored by deletion of CLN2, suggesting that whi3 cells may have abnormally high levels of CLN2 function.
Asunto(s)
Proteínas Fúngicas/genética , Genes Fúngicos/fisiología , Proteínas de Unión al ARN/genética , Saccharomyces cerevisiae/citología , Secuencia de Aminoácidos , Humanos , Datos de Secuencia Molecular , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
A novel multiple affinity purification (MAFT) or tandem affinity purification (TAP) tag has been constructed. It consists of the calmodulin binding peptide, six histidine residues, and three copies of the hemagglutinin epitope. This 'CHH' MAFT tag allows two or three consecutive purification steps, giving high purity. Active Clb2-Cdc28 kinase complex was purified from yeast cells after inserting the CHH tag into Clb2. Associated proteins were identified using mass spectrometry. These included the known associated proteins Cdc28, Sic1 and Cks1. Several other proteins were found including the 70 kDa chaperone, Ssa1.
Asunto(s)
Proteína Quinasa CDC28 de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular , Ciclina B/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Saccharomyces cerevisiae , Proteínas Adaptadoras Transductoras de Señales , Adenosina Trifosfatasas , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Secuencia de Bases , Western Blotting , Proteína Quinasa CDC28 de Saccharomyces cerevisiae/química , Proteína Quinasa CDC28 de Saccharomyces cerevisiae/genética , Proteína Quinasa CDC28 de Saccharomyces cerevisiae/aislamiento & purificación , Calmodulina/metabolismo , Proteínas de Unión a Calmodulina/genética , Proteínas de Unión a Calmodulina/metabolismo , Cromatografía de Afinidad/métodos , Ciclina B/genética , Ciclina B/aislamiento & purificación , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/aislamiento & purificación , Proteínas Fúngicas/metabolismo , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/aislamiento & purificación , Proteínas HSP70 de Choque Térmico/metabolismo , Hemaglutininas/genética , Hemaglutininas/inmunología , Histidina/genética , Histidina/metabolismo , Sustancias Macromoleculares , Espectrometría de Masas , Datos de Secuencia Molecular , Peso Molecular , Mutagénesis Insercional , Níquel/metabolismo , Pruebas de Precipitina , Unión Proteica , Proteínas Recombinantes de Fusión/aislamiento & purificación , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
The Saccharomyces cerevisiae cyclin-dependent kinase Cdc28 forms complexes with nine different cyclins to promote cell division. These nine cyclin-Cdc28 complexes have different roles, but share the same catalytic subunit; thus, it is not clear how substrate specificity is achieved. One possible mechanism is specific sub-cellular localization of specific complexes. We investigated the location of two G1 cyclins using fractionation and microscopy. In addition, we developed 'forced localization' cassettes, which direct proteins to particular locations, to test the importance of localization. Cln2 was found in both nucleus and cytoplasm. A substrate of Cln2, Sic1, was also in both compartments. Cytoplasmic Cln2 was concentrated at sites of polarized growth. Forced localization showed that some functions of Cln2 required a cytoplasmic location, while other functions required a nuclear location. In addition, one function apparently required shuttling between the two compartments. The G1 cyclin Cln3 required nuclear localization. An autonomous, nuclear localization sequence was found near the C-terminus of Cln3. Our data supports the hypothesis that Cln2 and Cln3 have distinct functions and locations, and the specificity of cyclin-dependent kinases is mediated in part by subcellular location.
Asunto(s)
Ciclinas/fisiología , Proteínas Fúngicas/fisiología , Fase G1 , Proteínas de Saccharomyces cerevisiae/fisiología , Proteína Quinasa CDC28 de Saccharomyces cerevisiae/genética , Proteína Quinasa CDC28 de Saccharomyces cerevisiae/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/fisiología , Ciclina G , Ciclinas/genética , Ciclinas/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Citoplasma/fisiología , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fase G1/genética , Mutagénesis Insercional , Señales de Localización Nuclear/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiología , Transporte de Proteínas/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
A selection of World Wide Web sites relevant to papers published in this issue of Current Opinion in Cell Biology.
RESUMEN
Microarrays have been used to characterize gene expression through the yeast cell cycle. Computational methods have been applied to the microarray data to identify coregulated clusters of genes, and motif-finding algorithms have found promoter elements characteristic of each cluster. The functional relevance of these promoter elements can be tested using chromatin immunoprecipitation, additional microarrays and other molecular techniques. The yeast forkhead proteins have been successfully identified as cell cycle transcription factors for an important cluster of genes by this and other approaches.
Asunto(s)
Ciclo Celular/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Transcripción Genética , Proteínas de Ciclo Celular/metabolismo , Ciclina B/genética , Ciclina B/metabolismo , Factores de Transcripción Forkhead , Regulación de la Expresión Génica , Familia de Multigenes/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismoRESUMEN
There are about 800 genes in Saccharomyces cerevisiae whose transcription is cell-cycle regulated. Some of these form clusters of co-regulated genes. The 'CLB2' cluster contains 33 genes whose transcription peaks early in mitosis, including CLB1, CLB2, SWI5, ACE2, CDC5, CDC20 and other genes important for mitosis. Here we find that the genes in this cluster lose their cell cycle regulation in a mutant that lacks two forkhead transcription factors, Fkh1 and Fkh2. Fkh2 protein is associated with the promoters of CLB2, SWI5 and other genes of the cluster. These results indicate that Fkh proteins are transcription factors for the CLB2 cluster. The fkh1 fkh2 mutant also displays aberrant regulation of the 'SIC1' cluster, whose member genes are expressed in the M-G1 interval and are involved in mitotic exit. This aberrant regulation may be due to aberrant expression of the transcription factors Swi5 and Ace2, which are members of the CLB2 cluster and controllers of the SIC1 cluster. Thus, a cascade of transcription factors operates late in the cell cycle. Finally, the fkh1 fkh2 mutant displays a constitutive pseudohyphal morphology, indicating that Fkh1 and Fkh2 may help control the switch to this mode of growth.
Asunto(s)
Genes Fúngicos , Genes cdc , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Sitios de Unión , Ciclina B/genética , ADN de Hongos/metabolismo , Factores de Transcripción Forkhead , Regulación Fúngica de la Expresión Génica , Familia de Multigenes , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Pruebas de Precipitina , Regiones Promotoras Genéticas , Unión Proteica , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
Many transcription factors, particularly those involved in the control of cell growth, are unstable proteins destroyed by ubiquitin-mediated proteolysis. In a previous study of sequences targeting the transcription factor Myc for destruction, we observed that the region in Myc signaling ubiquitin-mediated proteolysis overlaps closely with the region in Myc that activates transcription. Here, we present evidence that the overlap of these two activities is not unique to Myc, but reflects a more general phenomenon. We show that a similar overlap of activation domains and destruction elements occurs in other unstable transcription factors and report a close correlation between the ability of an acidic activation domain to activate transcription and to signal proteolysis. We also show that destruction elements from yeast cyclins, when tethered to a DNA-binding domain, activate transcription. The intimate overlap of activation domains and destruction elements reveals an unexpected convergence of two very different processes and suggests that transcription factors may be destroyed because of their ability to activate transcription.
Asunto(s)
Glicoproteínas de Membrana , Chaperonas Moleculares , Proteínas de Saccharomyces cerevisiae , Transducción de Señal , Activación Transcripcional/genética , Ubiquitinas/metabolismo , Ciclinas/metabolismo , Proteínas Fúngicas/metabolismo , Células HeLa , Humanos , Hidrólisis , Saccharomyces cerevisiae/genética , Transactivadores/metabolismoRESUMEN
Est1 is a component of yeast telomerase, and est1 mutants have senescence and telomere loss phenotypes. The exact function of Est1 is not known, and it is not homologous to components of other telomerases. We previously showed that Est1 protein coimmunoprecipitates with Tlc1 (the telomerase RNA) as well as with telomerase activity. Est1 has homology to Ebs1, an uncharacterized yeast open reading frame product, including homology to a putative RNA recognition motif (RRM) of Ebs1. Deletion of EBS1 results in short telomeres. We created point mutations in a putative RRM of Est1. One mutant was unable to complement either the senescence or the telomere loss phenotype of est1 mutants. Furthermore, the mutant protein no longer coprecipitated with the Tlc1 telomerase RNA. Mutants defective in the binding of Tlc1 RNA were nevertheless capable of binding single-stranded TG-rich DNA. Our data suggest that an important role of Est1 in the telomerase complex is to bind to the Tlc1 telomerase RNA via an RRM. Since Est1 can also bind telomeric DNA, Est1 may tether telomerase to the telomere.
Asunto(s)
Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Telomerasa/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas Fúngicas/genética , Datos de Secuencia Molecular , Mutación , ARN/genética , ARN/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Telomerasa/genéticaAsunto(s)
Genética Microbiana/educación , Sistemas de Computación , Educación Continua , Diseño de Equipo , Genes Fúngicos , Genética Microbiana/instrumentación , Análisis de Secuencia por Matrices de Oligonucleótidos , Robótica/educación , Robótica/instrumentación , Saccharomyces cerevisiae/genéticaRESUMEN
The transcription complexes SBF and MBF mediate the G(1)-S transition in the cell cycle of Saccharomyces cerevisiae. In late G(1), SBF and MBF induce a burst of transcription in a number of genes, including G(1)- and S-phase cyclins. Activation of SBF and MBF depends on the G(1) cyclin Cln3 and a largely uncharacterized protein called Bck2. We show here that the induction of SBF/MBF target genes by Bck2 depends partly, but not wholly, on SBF and MBF. Unlike Cln3, Bck2 is capable of inducing its transcriptional targets in the absence of functional Cdc28. Our results revealed promoter-specific mechanisms of regulation by Cln3, Bck2, SBF, and MBF. We isolated high-copy suppressors of the cln3 bck2 growth defect; all of these had the ability to increase CLN2 expression. One of these suppressors was the negative regulator of meiosis RME1. Rme1 induces CLN2, and we show that it has a haploid-specific role in regulating cell size and pheromone sensitivity. Genetic analysis of the cln3 bck2 defect showed that CLN1, CLN2, and other SBF/MBF target genes have an essential role in addition to the degradation of Sic1.
Asunto(s)
Ciclo Celular/genética , Ciclinas/metabolismo , Proteínas Fúngicas/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Transcripción Genética , Ciclinas/genética , Replicación del ADN , Proteínas de Unión al ADN , Proteínas Fúngicas/genética , Fase G1 , Regulación Fúngica de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular , Factor de Apareamiento , Péptidos/fisiología , Fosfoproteínas/genética , Fase S , Saccharomyces cerevisiae/crecimiento & desarrollo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
In this study, we examined yeast proteins by two-dimensional (2D) gel electrophoresis and gathered quantitative information from about 1,400 spots. We found that there is an enormous range of protein abundance and, for identified spots, a good correlation between protein abundance, mRNA abundance, and codon bias. For each molecule of well-translated mRNA, there were about 4,000 molecules of protein. The relative abundance of proteins was measured in glucose and ethanol media. Protein turnover was examined and found to be insignificant for abundant proteins. Some phosphoproteins were identified. The behavior of proteins in differential centrifugation experiments was examined. Such experiments with 2D gels can give a global view of the yeast proteome.
Asunto(s)
Proteínas Fúngicas/aislamiento & purificación , Perfilación de la Expresión Génica , ARN de Hongos/aislamiento & purificación , ARN Mensajero/aislamiento & purificación , Saccharomyces cerevisiae/genética , Codón , Electroforesis en Gel Bidimensional , Genes Fúngicos , Código Genético , Procesamiento de Imagen Asistido por Computador , Biosíntesis de Proteínas , Saccharomyces cerevisiae/químicaRESUMEN
The yeast Saccharomyces cerevisiae has been an excellent model system for cell cycle studies. Many such studies require cells synchronized in some particular portion of the cell cycle. Here, methods are described for obtaining and examining synchronized cells as they pass through one or more rounds of the cell cycle. The methods are of two types. First, block-and-release methods, where cells are initially synchronized by blocking them at some particular cell cycle stage, then releasing them from the block under conditions suitable for growth, and taking samples at different times after the release, thereby obtaining samples representing different cell cycle stages. The second type of method is elutriation. Centrifugal elutriation can be used to obtain samples of uniformly sized cells, and because cell size is correlated with cell cycle stage, these cells are synchronized with respect to their position in the cycle. Because elutriation is a very different method from block-and-release, it is ideal as a second method of synchronization to ensure that results achieved by block-and-release are not artefactual. Here, block-and-release experiments with the mating pheromone alpha factor, and with the cdc15-2 mutation, are described in detail, as are some elutriation methods.
Asunto(s)
Genes cdc/fisiología , Fenómenos Fisiológicos de las Plantas , Saccharomyces cerevisiae/fisiología , Factores de TiempoRESUMEN
We sought to create a comprehensive catalog of yeast genes whose transcript levels vary periodically within the cell cycle. To this end, we used DNA microarrays and samples from yeast cultures synchronized by three independent methods: alpha factor arrest, elutriation, and arrest of a cdc15 temperature-sensitive mutant. Using periodicity and correlation algorithms, we identified 800 genes that meet an objective minimum criterion for cell cycle regulation. In separate experiments, designed to examine the effects of inducing either the G1 cyclin Cln3p or the B-type cyclin Clb2p, we found that the mRNA levels of more than half of these 800 genes respond to one or both of these cyclins. Furthermore, we analyzed our set of cell cycle-regulated genes for known and new promoter elements and show that several known elements (or variations thereof) contain information predictive of cell cycle regulation. A full description and complete data sets are available at http://cellcycle-www.stanford.edu
Asunto(s)
Ciclo Celular/genética , Ciclina B , Genes Fúngicos , Hibridación de Ácido Nucleico/métodos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Secuencia de Bases , Sitios de Unión/genética , Ciclinas/genética , Cartilla de ADN/genética , Reparación del ADN/genética , Replicación del ADN/genética , ADN de Hongos/genética , ADN de Hongos/metabolismo , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Familia de Multigenes , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/metabolismo , Transcripción GenéticaRESUMEN
In most eukaryotes, commitment to cell division occurs in late G1 phase at an event called Start in the yeast Saccharomyces cerevisiae, and called the restriction point in mammalian cells. Start is triggered by the cyclin-dependent kinase Cdc28 and three rate-limiting activators, the G1 cyclins Cln1, Cln2 and Cln3. Cyclin accumulation in G1 is driven in part by the cell-cycle-regulated transcription of CLN1 and CLN2, which peaks at Start. CLN transcription is modulated by physiological signals that regulate G1 progression, but it is unclear whether Cln protein stability is cell-cycle-regulated. It has been suggested that once cells pass Start, Cln proteolysis is triggered by the mitotic cyclins Clb1, 2, 3 and 4. But here we show that G1 cyclins are unstable in G1 phase, and that Clb-Cdc28 activity is not needed fgr G1 cyclin turnover. Cln instability thus provides a means to couple Cln-Cdc28 activity to transcriptional regulation and protein synthetic rate in pre-Start G1 cells.
Asunto(s)
Ciclinas/metabolismo , Fase G1 , Saccharomyces cerevisiae/metabolismo , Complejos de Ubiquitina-Proteína Ligasa , Ciclosoma-Complejo Promotor de la Anafase , Ligasas/metabolismo , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae , Ubiquitina-Proteína LigasasRESUMEN
The ubiquitin conjugating (ubc) E2 enzyme ubc-9 conjugates the ubiquitin-like peptide sentrin/SUMO-1/PIC1 to target proteins which include the Fas antigen. We show that the mouse genome contains four copies of the ubc-9 gene. These include a structural ubc-9 gene consisting of seven exons which encode a protein identical to human ubc-9, and three intronless processed pseudogenes. The open reading frames (ORF) of two of the pseudogenes, ubc9-psi1 and ubc9-psi2, correspond to the cDNA of ubc-9 and encode for proteins which differ from ubc9 by three and one amino acid substitutions respectively. The third pseudogene, ubc9-psi3, contains many mutations and stop codons. ubc9-psi1 and ubc9-psi2 are flanked by 5'- and 3'-untranslated (UT) regions homologous to those of the structural ubc-9 gene. Both genes contain a polyA tail and direct repeats at both ends suggesting that they arose by mRNA retroposition. Both ubc9-psi1 and ubc9-psi2 are transcribed into mRNA in murine cells. In contrast to ubc9, the protein products of ubc9-psil and ubc9-psi2 fail to bind Fas and to complement an yeast conditional ubc9 mutant. These results suggest that ubc9-psi1 and ubc9-psi2 encode for proteins that may interact with targets that differ from those recognized by ubc-9.
Asunto(s)
Ligasas/genética , Seudogenes , Enzimas Ubiquitina-Conjugadoras , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cartilla de ADN/genética , Exones , Expresión Génica , Genes , Prueba de Complementación Genética , Genoma , Humanos , Intrones , Ligasas/metabolismo , Ratones , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Mapeo Restrictivo , Retroelementos , Proteína SUMO-1 , Saccharomyces cerevisiae/genética , Ubiquitinas/metabolismo , Receptor fas/metabolismoRESUMEN
Cell cycle progression is mediated by waves of specific cyclin dependent kinases (CDKs) in all eukaryotes. Cyclins are degraded by the ubiquitin pathway of proteolysis. The recent identification of several components of the cyclin proteolysis machinery has highlighted both the importance of proteolysis at multiple transition points in the cell cycle and the involvement of other substrates degraded by the same machinery. In this study, we have investigated the effects of DNA damage on the cyclin proteolytic machinery in Saccharomyces cerevisiae. We find that the half-life of the B-type cyclin Clb5 is markedly increased following DNA damage while that of G1 cyclins is not. This effect is independent of cell cycle phase. Clb5 turnover requires p34CDC28 activity. Stabilisation of Clb5 correlates with an increase in tyrosine phosphorylation of p34CDC28, but stabilisation does not require this tyrosine phosphorylation. The stabilisation is independent of the checkpoint genes Mec1 and Rad53. These observations establish a new link between the regulation of proteolysis and DNA damage.
Asunto(s)
Proteínas de Ciclo Celular , Ciclina B , Ciclinas/metabolismo , Daño del ADN , Proteínas Fúngicas/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Proteína Quinasa CDC28 de Saccharomyces cerevisiae/metabolismo , Ciclo Celular , Quinasa de Punto de Control 2 , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina , Ciclinas/genética , ADN de Hongos/efectos de los fármacos , ADN de Hongos/efectos de la radiación , Proteínas Fúngicas/fisiología , Semivida , Péptidos y Proteínas de Señalización Intracelular , Mutación , Fosforilación , Fosfotirosina/metabolismo , Proteínas Quinasas/metabolismo , Fase S , Saccharomyces cerevisiae/genéticaRESUMEN
Two-dimensional (2-D) gel electrophoresis can now be coupled with protein identification techniques and genome sequence information for direct detection, identification, and characterization of large numbers of proteins from microbial organisms. 2-D electrophoresis, and new protein identification techniques such as amino acid composition, are proteome research techniques in that they allow direct characterization of many proteins at the same time. Another new tool important for yeast proteome research is the Yeast Protein Database (YPD), which provides the sequence-derived protein properties needed for spot identification and tabulations of the currently known properties of the yeast proteins. Studies presented here extend the yeast 2-D protein map to 169 identified spots based upon the recent completion of the yeast genome sequence, and they show that methods of spot identification based on predicted isoelectric point, predicted molecular mass, and determination of partial amino acid composition from radiolabeled gels are powerful enough for the identification of at least 80% of the spots representing abundant proteins. Comparison of proteins predicted by YPD to be detectable on 2-D gels based on calculated molecular mass, isoelectric point and codon bias (a predictor of abundance) with proteins identified in this study suggests that many glycoproteins and integral membrane proteins are missing from the 2-D gel patterns. Using the 2-D gel map and the information available in YDP, 2-D gel experiments were analyzed to characterize the yeast proteins associated with: (i) an environmental change (heat shock), (ii) a temperature-sensitive mutation (the prp2 mRNA splicing mutant), (iii) a mutation affecting post-translational modification (N-terminal acetylation), and (iv) a purified subcellular fraction (the ribosomal proteins). The methods used here should allow future extension of these studies to many more proteins of the yeast proteome.
Asunto(s)
Electroforesis en Gel Bidimensional/métodos , Proteínas Fúngicas/genética , Proteínas Fúngicas/aislamiento & purificación , Genoma Fúngico , Mapeo Peptídico/métodos , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Bases de Datos Factuales , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/aislamiento & purificación , Punto Isoeléctrico , Peso Molecular , Mutación , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/aislamiento & purificación , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
A novel human ubiquitin conjugating enzyme (UBC) was found to associate with Fas (CD95). The mRNA for this UBC Fas-associated protein (FAP) was widely expressed in human tissues, and the protein was identified in several mammalian cell lines. UBC-FAP shows strong homology to two recently identified UBCs, Hus5 and Ubc9, which control yeast cell cycle progression. UBC-FAP, but not an active site mutant, complemented ubc9-1(ts) mutants. This suggests that UBC-FAP is a human homologue of Ubc9, possesses ubiquitin conjugating activity, and may play an important role in mammalian cell cycle regulation. A single amino acid substitution in the death domain of Fas that abolishes Fas-mediated apoptosis also abolished Fas association with UBC-FAP, suggesting that UBC-FAP may play a role in Fas signal transduction. The sequence of UBC-FAP is identical to that of HsUbc9, a UBC recently shown to interact with Rad51.