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1.
PLoS One ; 6(10): e25644, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22022426

RESUMO

Gene transcription is constrained by the nucleosomal nature of chromosomal DNA. This nucleosomal barrier is modulated by FACT, a conserved histone-binding heterodimer. FACT mediates transcription-linked nucleosome disassembly and also nucleosome reassembly in the wake of the RNA polymerase II transcription complex, and in this way maintains the repression of 'cryptic' promoters found within some genes. Here we focus on a novel mutant version of the yeast FACT subunit Spt16 that supplies essential Spt16 activities but impairs transcription-linked nucleosome reassembly in dominant fashion. This Spt16 mutant protein also has genetic effects that are recessive, which we used to show that certain Spt16 activities collaborate with histone acetylation and the activities of a Bur-kinase/Spt4-Spt5/Paf1C pathway that facilitate transcription elongation. These collaborating activities were opposed by the actions of Rpd3S, a histone deacetylase that restores a repressive chromatin environment in a transcription-linked manner. Spt16 activity paralleling that of HirC, a co-repressor of histone gene expression, was also found to be opposed by Rpd3S. Our findings suggest that Spt16, the Bur/Spt4-Spt5/Paf1C pathway, and normal histone abundance and/or stoichiometry, in mutually cooperative fashion, facilitate nucleosome disassembly during transcription elongation. The recessive nature of these effects of the mutant Spt16 protein on transcription-linked nucleosome disassembly, contrasted to its dominant negative effect on transcription-linked nucleosome reassembly, indicate that mutant FACT harbouring the mutant Spt16 protein competes poorly with normal FACT at the stage of transcription-linked nucleosome disassembly, but effectively with normal FACT for transcription-linked nucleosome reassembly. This functional difference is consistent with the idea that FACT association with the transcription elongation complex depends on nucleosome disassembly, and that the same FACT molecule that associates with an elongation complex through nucleosome disassembly is retained for reassembly of the same nucleosome.


Assuntos
Histonas/metabolismo , Nucleossomos/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Transcrição Gênica , Alelos , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Testes Genéticos , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas Mutantes/metabolismo , Mutação , Regiões Promotoras Genéticas/genética , Subunidades Proteicas/metabolismo , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Transdução de Sinais , Fatores de Elongação da Transcrição/metabolismo
2.
Mol Biol Cell ; 22(13): 2337-47, 2011 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-21562219

RESUMO

Small monomeric G proteins regulated in part by GTPase-activating proteins (GAPs) are molecular switches for several aspects of vesicular transport. The yeast Gcs1 protein is a dual-specificity GAP for ADP-ribosylation factor (Arf) and Arf-like (Arl)1 G proteins, and also has GAP-independent activities. The absence of Gcs1 imposes cold sensitivity for growth and endosomal transport; here we present evidence that dysregulated Arl1 may cause these impairments. We show that gene deletions affecting the Arl1 or Ypt6 vesicle-tethering pathways prevent Arl1 activation and membrane localization, and restore growth and trafficking in the absence of Gcs1. A mutant version of Gcs1 deficient for both ArfGAP and Arl1GAP activity in vitro still allows growth and endosomal transport, suggesting that the function of Gcs1 that is required for these processes is independent of GAP activity. We propose that, in the absence of this GAP-independent regulation by Gcs1, the resulting dysregulated Arl1 prevents growth and impairs endosomal transport at low temperatures. In cells with dysregulated Arl1, an increased abundance of the Arl1 effector Imh1 restores growth and trafficking, and does so through Arl1 binding. Protein sequestration at the trans-Golgi membrane by dysregulated, active Arl1 may therefore be the mechanism of inhibition.


Assuntos
Complexo de Golgi/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Corpos Multivesiculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Rede trans-Golgi/metabolismo , Fatores de Ribosilação do ADP/metabolismo , Aminoácido N-Acetiltransferase/metabolismo , Temperatura Baixa , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endocitose/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Complexo de Golgi/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , Acetiltransferase N-Terminal C , Ligação Proteica , Transporte Proteico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular/genética , Rede trans-Golgi/genética
3.
J Biol Chem ; 286(7): 5187-96, 2011 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-21135091

RESUMO

Vesicular transport shuttles cargo among intracellular compartments. Several stages of vesicular transport are mediated by the small GTPase Arf, which is controlled in a cycle of GTP binding and hydrolysis by Arf guanine-nucleotide exchange factors and Arf GTPase-activating proteins (ArfGAPs), respectively. In budding yeast the Age2 + Gcs1 ArfGAP pair facilitates post-Golgi transport. We have found the AGE1 gene, encoding another ArfGAP, can in high gene-copy number alleviate the temperature sensitivity of cells carrying mutations affecting the Age2 + Gcs1 ArfGAP pair. Moreover, increased AGE1 gene dosage compensates for the complete absence of the otherwise essential Age2 + Gcs1 ArfGAP pair. Increased dosage of SFH2, encoding a phosphatidylinositol transfer protein, also allows cell growth in the absence of the Age2 + Gcs1 pair, but good growth in this situation requires Age1. The ability of Age1 to overcome the need for Age2 + Gcs1 depends on phospholipase D activity that regulates lipid composition. We show by direct assessment of Age1 ArfGAP activity that Age1 is regulated by lipid composition and can provide ArfGAP function for post-Golgi transport.


Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Complexo de Golgi/metabolismo , Lipídeos de Membrana/metabolismo , Fosfolipase D/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vesículas Transportadoras/metabolismo , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Transporte Biológico/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Ativadoras de GTPase/genética , Dosagem de Genes , Complexo de Golgi/genética , Lipídeos de Membrana/genética , Fosfolipase D/genética , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas de Transferência de Fosfolipídeos/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Vesículas Transportadoras/genética
4.
Mol Cell Biol ; 30(5): 1116-29, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20048053

RESUMO

Asf1 is a conserved histone H3/H4 chaperone that can assemble and disassemble nucleosomes and promote histone acetylation. Set2 is an H3 K36 methyltransferase. The functions of these proteins intersect in the context of transcription elongation by RNA polymerase II: both contribute to the establishment of repressive chromatin structures that inhibit spurious intragenic transcription. Here we characterize further interactions between budding yeast (Saccharomyces cerevisiae) Asf1 and Set2 using assays of intragenic transcription, H3/H4 posttranslational modification, coding region cross-linking of Asf1 and Set2, and cooccurrence of Asf1 and Set2 in protein complexes. We find that at some genes Asf1 and Set2 control chromatin metabolism as components of separate pathways. However, the existence of a low-abundance complex containing both proteins suggests that Asf1 and Set2 can more directly collaborate in chromatin regulation. Consistent with this possibility, we show that Asf1 stimulates Set2 occupancy of the coding region of a highly transcribed gene by a mechanism that depends on Asf1 binding to H3/H4. This function of Asf1 promotes the switch from di- to trimethylation of H3 K36 at that gene. These results support the view that Set2 function in chromatin metabolism can intimately involve histone chaperone Asf1.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Histonas/metabolismo , Metiltransferases/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular/genética , Cromatina/genética , Cromatina/metabolismo , Dano ao DNA , DNA Fúngico/genética , DNA Fúngico/metabolismo , Deleção de Genes , Redes Reguladoras de Genes , Genes Fúngicos , Histonas/química , Metilação , Metiltransferases/genética , Modelos Biológicos , Chaperonas Moleculares/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transcrição Gênica
5.
Mol Genet Genomics ; 282(5): 487-502, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19727824

RESUMO

Transcription by RNA polymerase II is impeded by the nucleosomal organization of DNA; these negative effects are modulated at several stages of nucleosomal DNA transcription by FACT, a heterodimeric transcription factor. At promoters, FACT facilitates the binding of TATA-binding factor, while during transcription elongation FACT mediates the necessary destabilization of nucleosomes and subsequent restoration of nucleosome structure in the wake of the transcription elongation complex. Altered FACT activity can impair the fidelity of transcription initiation and affect transcription patterns. Using reporter genes we have identified new mutant versions of the Spt16 subunit of yeast FACT with dominant negative effects on the fidelity of transcription initiation. Two of these spt16 mutant alleles also affect cell integrity. Cells relying on these spt16 mutant alleles display sorbitol-remediated temperature sensitivity, altered sensitivity to detergent, and abnormal morphologies, and are further inhibited by the ssd1-d mutation. The overexpression of components of protein kinase C (Pkc1) signaling diminishes this spt16 ssd1-d temperature sensitivity, whereas gene deletions eliminating components of Pkc1 signaling further impair these spt16 mutant cells. Thus, the FACT subunit Spt16 and Pkc1 signaling have an overlapping essential function, with an unexpected role for FACT in the maintenance of cell integrity.


Assuntos
Mutação/genética , Subunidades Proteicas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Fatores de Elongação da Transcrição/genética , Alelos , Parede Celular/efeitos dos fármacos , Parede Celular/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Genes Dominantes/genética , Genes Fúngicos/genética , Genes Reporter , Teste de Complementação Genética , Hidroxiureia/farmacologia , Fenótipo , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Supressão Genética/efeitos dos fármacos , Temperatura , Transcrição Gênica/efeitos dos fármacos , Fatores de Elongação da Transcrição/metabolismo , beta-Galactosidase/metabolismo
6.
Traffic ; 10(9): 1362-75, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19602196

RESUMO

The ArfGAP Glo3 is required for coat protein I vesicle generation in the Golgi-endoplasmic reticulum (ER) shuttle. The best-understood role of Glo3 is the stimulation of the GTPase activity of Arf1. In this study, we characterized functional domains of the ArfGAP Glo3 and identified an interaction interface for coatomer, SNAREs and cargo in the central region of Glo3 (BoCCS region). The GAP domain together with the BoCCS region is necessary and sufficient for all vital Glo3 functions. Expression of a truncated Glo3 lacking the GAP domain results in a dominant negative growth phenotype in glo3Delta cells at 37 degrees C. This phenotype was alleviated by mutating either the BoCCS region or the Glo3 regulatory motif (GRM), or by overexpression of ER-Golgi SNAREs or the ArfGAP Gcs1. The GRM is not essential for Glo3 function; it may act as an intrinsic sensor coupling GAP activity to SNARE binding to avoid dead-end complex formation at the Golgi membrane. Our data suggest that membrane-interaction modules and cargo-sensing regions have evolved independently in ArfGAP1s versus ArfGAP2/3s.


Assuntos
Proteína Coatomer/metabolismo , Proteínas Ativadoras de GTPase/fisiologia , Proteínas SNARE/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Vesículas Transportadoras/fisiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Complexo I de Proteína do Envoltório/metabolismo , Proteínas de Ligação a DNA/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Complexo de Golgi/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas SNARE/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Vesículas Transportadoras/metabolismo
7.
Mol Biol Cell ; 17(4): 1845-58, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16452633

RESUMO

Gcs1 is an Arf GTPase-activating protein (Arf-GAP) that mediates Golgi-ER and post-Golgi vesicle transport in yeast. Here we show that the Snc1,2 v-SNAREs, which mediate endocytosis and exocytosis, interact physically and genetically with Gcs1. Moreover, Gcs1 and the Snc v-SNAREs colocalize to subcellular structures that correspond to the trans-Golgi and endosomal compartments. Studies performed in vitro demonstrate that the Snc-Gcs1 interaction results in the efficient binding of recombinant Arf1Delta17N-Q71L to the v-SNARE and the recruitment of purified coatomer. In contrast, the presence of Snc had no effect on Gcs1 Arf-GAP activity in vitro, suggesting that v-SNARE binding does not attenuate Arf1 function. Disruption of both the SNC and GCS1 genes results in synthetic lethality, whereas overexpression of either SNC gene inhibits the growth of a distinct subset of COPI mutants. We show that GFP-Snc1 recycling to the trans-Golgi is impaired in gcs1Delta cells and these COPI mutants. Together, these results suggest that Gcs1 facilitates the incorporation of the Snc v-SNAREs into COPI recycling vesicles and subsequent endosome-Golgi sorting in yeast.


Assuntos
Proteínas Fúngicas/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Complexo de Golgi/metabolismo , Proteínas R-SNARE/metabolismo , Leveduras/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Endocitose , Endossomos , Proteínas Fúngicas/análise , Proteínas Fúngicas/genética , Proteínas Ativadoras de GTPase/genética , Genes Fúngicos , Genes Letais , Imunoprecipitação , Mapeamento de Interação de Proteínas , Transporte Proteico , Proteínas R-SNARE/análise , Proteínas R-SNARE/genética , Deleção de Sequência , Técnicas do Sistema de Duplo-Híbrido , Leveduras/química
8.
Proc Natl Acad Sci U S A ; 102(36): 12777-82, 2005 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-16126894

RESUMO

The budding yeast Saccharomyces cerevisiae contains a family of Arf (ADP-ribosylation factor) GTPase activating protein (GAP) proteins with the Gcs1 + Age2 ArfGAP pair providing essential overlapping function for the movement of transport vesicles from the trans-Golgi network. We have generated a temperature-sensitive but stable version of the Gcs1 protein that is impaired only for trans-Golgi transport and find that deleterious effects of this enfeebled Gcs1-4 mutant protein are relieved by increased gene dosage of the gcs1-4 mutant gene itself or by the SFH2 gene (also called CSR1), encoding a phosphatidylinositol transfer protein (PITP). This effect was not seen for the SEC14 gene, encoding the founding member of the yeast PITP protein family, even though the Gcs1 and Age2 ArfGAPs are known to be downstream effectors of Sec14-mediated activity for trans-Golgi transport. Sfh2-mediated suppression of inadequate Gcs1-4 function depended on phospholipase D, whereas inadequate Gcs1-4 activity was relieved by increasing levels of diacylglycerol (DAG). Recombinant Gcs1 protein was found to bind certain phospholipids but not DAG. Our findings favor a model of Gcs1 localization through binding to specific phospholipids and activation of ArfGAP activity by DAG-mediated membrane curvature as the transport vesicle is formed. Thus, ArfGAPs are subject to both temporal and spatial regulation that is facilitated by Sfh2-mediated modulation of the lipid environment.


Assuntos
Fatores de Ribosilação do ADP/metabolismo , Membrana Celular/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Fatores de Ribosilação do ADP/genética , Membrana Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Diglicerídeos/química , Diglicerídeos/farmacologia , Ativação Enzimática , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/genética , Complexo de Golgi/efeitos dos fármacos , Mutação/genética , Fosfolipase D/genética , Fosfolipase D/metabolismo , Proteínas de Transferência de Fosfolipídeos/classificação , Proteínas de Transferência de Fosfolipídeos/genética , Transporte Proteico/efeitos dos fármacos , Piridoxal/análogos & derivados , Piridoxal/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/classificação , Proteínas de Saccharomyces cerevisiae/genética , Temperatura
9.
Nucleic Acids Res ; 32(19): 5894-906, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15520471

RESUMO

The abundant nuclear complex termed FACT affects several DNA transactions in a chromatin context, including transcription, replication, and repair. Earlier studies of yeast FACT, which indicated the apparent dispensability of conserved sequences at the N terminus of the FACT subunit Cdc68/Spt16, prompted genetic and biochemical studies reported here that suggest the domain organization for Spt16 and the other FACT subunit Pob3, the yeast homolog of the metazoan SSRP1 protein. Our findings suggest that each FACT subunit is a multidomain protein, and that FACT integrity depends on Pob3 interactions with the Spt16 Mid domain. The conserved Spt16 N-terminal domain (NTD) is shown to be without essential function during normal growth, but becomes important under conditions of replication stress. Genetic interactions suggest that some functions carried out by the Spt16 NTD may be partially redundant within FACT.


Assuntos
Proteínas de Ciclo Celular/química , Chaperonas Moleculares/química , Fatores de Transcrição/química , Sequência de Aminoácidos , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Sequência Conservada , Replicação do DNA , Histonas/metabolismo , Mutação , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Elongação da Transcrição
10.
Biochem Cell Biol ; 82(4): 419-27, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15284894

RESUMO

The chromatin configuration of DNA inhibits access by enzymes such as RNA polymerase II. This inhibition is alleviated by FACT, a conserved transcription elongation factor that has been found to reconfigure nucleosomes to allow transit along the DNA by RNA polymerase II, thus facilitating transcription. FACT also reorganizes nucleosomes after the passage of RNA polymerase II, as indicated by the effects of certain FACT mutations. The larger of the two subunits of FACT is Spt16/Cdc68, while the smaller is termed SSRP1 (vertebrates) or Pob3 (budding yeast). The HMG-box domain at the C terminus of SSRP1 is absent from Pob3; the function of this domain for yeast FACT is supplied by the small HMG-box protein Nhp6. In yeast, this "detachable" HMG domain is a general chromatin component, unlike FACT, which is found only in transcribed regions and associated with RNA polymerase II. The several domains of the larger FACT subunit are also likely to have different functions. Genetic studies suggest that FACT mediates nucleosome reorganization along several pathways, and reinforce the notion that protein unfolding and (or) refolding is involved in FACT activity for transcription.


Assuntos
Cromatina/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/fisiologia , Proteínas de Grupo de Alta Mobilidade/química , Proteínas de Grupo de Alta Mobilidade/fisiologia , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/fisiologia , Alelos , Animais , Ciclo Celular , Cromatina/metabolismo , DNA/química , Proteínas de Ligação a DNA/genética , Genes Supressores , Proteínas de Grupo de Alta Mobilidade/genética , Modelos Biológicos , Mutação , Nucleossomos/metabolismo , Desnaturação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Saccharomycetales/fisiologia , Relação Estrutura-Atividade , Temperatura , Fatores de Elongação da Transcrição/genética
11.
Mol Biol Cell ; 15(9): 4064-72, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15254269

RESUMO

The small GTPase Arf and coatomer (COPI) are required for the generation of retrograde transport vesicles. Arf activity is regulated by guanine exchange factors (ArfGEF) and GTPase-activating proteins (ArfGAPs). The ArfGAPs Gcs1 and Glo3 provide essential overlapping function for retrograde vesicular transport from the Golgi to the endoplasmic reticulum. We have identified Glo3 as a component of COPI vesicles. Furthermore, we find that a mutant version of the Glo3 protein exerts a negative effect on retrograde transport, even in the presence of the ArfGAP Gcs1. Finally, we present evidence supporting a role for ArfGAP protein in the generation of COPI retrograde transport vesicles.


Assuntos
Fatores de Ribosilação do ADP/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Ribosilação do ADP/genética , Sequência de Bases , Transporte Biológico Ativo , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Ativadoras de GTPase/genética , Genes Fúngicos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia Eletrônica , Mutagênese Sítio-Dirigida , Mutação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular
12.
Microbiol Mol Biol Rev ; 68(2): 187-206, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15187181

RESUMO

The cells of organisms as diverse as bacteria and humans can enter stable, nonproliferating quiescent states. Quiescent cells of eukaryotic and prokaryotic microorganisms can survive for long periods without nutrients. This alternative state of cells is still poorly understood, yet much benefit is to be gained by understanding it both scientifically and with reference to human health. Here, we review our knowledge of one "model" quiescent cell population, in cultures of yeast grown to stationary phase in rich media. We outline the importance of understanding quiescence, summarize the properties of quiescent yeast cells, and clarify some definitions of the state. We propose that the processes by which a cell enters into, maintains viability in, and exits from quiescence are best viewed as an environmentally triggered cycle: the cell quiescence cycle. We synthesize what is known about the mechanisms by which yeast cells enter into quiescence, including the possible roles of the protein kinase A, TOR, protein kinase C, and Snf1p pathways. We also discuss selected mechanisms by which quiescent cells maintain viability, including metabolism, protein modification, and redox homeostasis. Finally, we outline what is known about the process by which cells exit from quiescence when nutrients again become available.


Assuntos
Fase de Repouso do Ciclo Celular/fisiologia , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Sobrevivência Celular , Meios de Cultura , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Modelos Biológicos , Mutação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Transcrição Gênica
13.
Mol Biol Cell ; 13(7): 2193-206, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12134061

RESUMO

Yeast phosphatidylinositol transfer protein (Sec14p) coordinates lipid metabolism with protein-trafficking events. This essential Sec14p requirement for Golgi function is bypassed by mutations in any one of seven genes that control phosphatidylcholine or phosphoinositide metabolism. In addition to these "bypass Sec14p" mutations, Sec14p-independent Golgi function requires phospholipase D activity. The identities of lipids that mediate Sec14p-dependent Golgi function, and the identity of the proteins that respond to Sec14p-mediated regulation of lipid metabolism, remain elusive. We now report genetic evidence to suggest that two ADP ribosylation factor-GTPase-activating proteins (ARFGAPs), Gcs1p and Age2p, may represent these lipid-responsive elements, and that Gcs1p/Age2p act downstream of Sec14p and phospholipase D in both Sec14p-dependent and Sec14p-independent pathways for yeast Golgi function. In support, biochemical data indicate that Gcs1p and Age2p ARFGAP activities are both modulated by lipids implicated in regulation of Sec14p pathway function. These results suggest ARFGAPs are stimulatory factors required for regulation of Golgi function by the Sec14p pathway, and that Sec14p-mediated regulation of lipid metabolism interfaces with the activity of proteins involved in control of the ARF cycle.


Assuntos
Fatores de Ribosilação do ADP/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Fosfolipídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Fatores de Ribosilação do ADP/genética , Sequência de Aminoácidos , Transporte Biológico/fisiologia , Proteínas Sanguíneas/genética , Proteínas de Transporte/genética , Proteínas de Ligação a DNA/genética , Proteínas Ativadoras de GTPase/genética , Proteínas de Membrana/genética , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Fosfolipase D/genética , Fosfolipase D/metabolismo , Proteínas de Transferência de Fosfolipídeos , Fosfoproteínas/genética , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Vacúolos/metabolismo , Vacúolos/ultraestrutura
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