Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 10 de 10
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
J Bacteriol ; 181(6): 1793-800, 1999 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-10074071

RESUMO

In Saccharomyces cerevisiae the FUR4-encoded uracil permease catalyzes the first step of the pyrimidine salvage pathway. The availability of uracil has a negative regulatory effect upon its own transport. Uracil causes a decrease in the level of uracil permease, partly by decreasing the FUR4 mRNA level in a promoter-independent fashion, probably by increasing its instability. Uracil entry also triggers more rapid degradation of the existing permease by promoting high efficiency of ubiquitination of the permease that signals its internalization. A direct binding of intracellular uracil to the permease is possibly involved in this feedback regulation, as the behavior of the permease is similar in mutant cells unable to convert intracellular uracil into UMP. We used cells impaired in the ubiquitination step to show that the addition of uracil produces rapid inhibition of uracil transport. This may be the first response prior to the removal of the permease from the plasma membrane. Similar down-regulation of uracil uptake, involving several processes, was observed under adverse conditions mainly corresponding to a decrease in the cellular content of ribosomes. These results suggest that uracil of exogenous or catabolic origin down-regulates the cognate permease to prevent buildup of excess intracellular uracil-derived nucleotides.


Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Transporte de Nucleotídeos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Uracila/metabolismo , Sequência de Bases , Primers do DNA/genética , Regulação para Baixo/efeitos dos fármacos , Genes Fúngicos , Moduladores de Transporte de Membrana , Proteínas de Membrana Transportadoras/antagonistas & inibidores , Proteínas de Membrana Transportadoras/genética , Plasmídeos/genética , Pirimidinas/farmacologia , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Regulação para Cima , Uracila/farmacologia
2.
Yeast ; 15(1): 73-9, 1999 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-10028187

RESUMO

Six open reading frames (ORFs) of unknown function from the left arm of Saccharomyces cerevisiae chromosome XV were deleted in two genetic backgrounds by disruption cassettes with long flanking homology (LFH) (Wach, 1996), within the frame of the research project EUROFAN. The LFH disruption cassettes, obtained by PCR, were made by introducing the kanMX4 marker module between two fragments homologous to the promoter and terminator regions of a given ORF. Transformants resistant to geneticin (G418) were selected. The LFH disruption cassettes were cloned in a bacterial vector. Each cognate gene was also cloned in a centromeric plasmid. Correct deletion of each gene was verified by four different PCR reactions. Sporulation and tetrad analysis of heterozygous deletants revealed that ORF YOL102c is essential. The non-growing haploid spores gave rise to microcolonies. Basic phenotypic analyses were performed on haploid deletants of both mating types of the five non-essential ORFs, YOL018c, YOL098c, YOL101c, YOL104c and YOL105c. Plate growth tests on different media at 15 degrees C, 30 degrees C or 37 degrees C did not reveal any significant differences between parental and mutant cells. Mating and sporulation efficiencies were not affected in any of the viable disruptants as compared to wild-type cells.


Assuntos
Cromossomos Fúngicos , Deleção de Genes , Genes Fúngicos , Saccharomyces cerevisiae/genética , Marcadores Genéticos , Fases de Leitura Aberta/genética , Fenótipo , Reação em Cadeia da Polimerase/métodos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Transformação Genética
3.
Mol Biol Cell ; 9(10): 2873-89, 1998 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-9763449

RESUMO

The ORF YOL018c (TLG2) of Saccharomyces cerevisiae encodes a protein that belongs to the syntaxin protein family. The proteins of this family, t-SNAREs, are present on target organelles and are thought to participate in the specific interaction between vesicles and acceptor membranes in intracellular membrane trafficking. TLG2 is not an essential gene, and its deletion does not cause defects in the secretory pathway. However, its deletion in cells lacking the vacuolar ATPase subunit Vma2p leads to loss of viability, suggesting that Tlg2p is involved in endocytosis. In tlg2Delta cells, internalization was normal for two endocytic markers, the pheromone alpha-factor and the plasma membrane uracil permease. In contrast, degradation of alpha-factor and uracil permease was delayed in tlg2Delta cells. Internalization of positively charged Nanogold shows that the endocytic pathway is perturbed in the mutant, which accumulates Nanogold in primary endocytic vesicles and shows a greatly reduced complement of early endosomes. These results strongly suggest that Tlg2p is a t-SNARE involved in early endosome biogenesis.


Assuntos
Endocitose/fisiologia , Fatores de Troca do Nucleotídeo Guanina , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas de Transporte de Nucleotídeos , Organelas/fisiologia , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiologia , ATPases Vacuolares Próton-Translocadoras , Sequência de Aminoácidos , Cromossomos Fúngicos , Clonagem Molecular , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Deleção de Genes , Cinética , Fator de Acasalamento , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Dados de Sequência Molecular , Fases de Leitura Aberta , Organelas/ultraestrutura , Peptídeos/genética , Peptídeos/metabolismo , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Proteínas Qa-SNARE , Proteínas Recombinantes de Fusão/biossíntese , Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
4.
Mol Microbiol ; 21(5): 1061-73, 1996 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-8885275

RESUMO

The uracil permease of Saccharomyces cerevisiae is a 633 residue polytopic plasma membrane protein. Hydropathy profile analysis indicates that this protein has long hydrophilic N- and C-termini and 10-12 potential transmembrane segments. Previous results based on analysis of hybrid proteins allowed identification of the first transmembrane segment of uracil permease, and provided a preliminary indication of the cytoplasmic orientation of its N-terminus. In this work, other experimental approaches were used to confirm this orientation, and to determine that of the C-terminus. Epitopes in the N- and the C-termini of the protein were protected against trypsin degradation on intact protoplasts, but readily digested on permeabilized protoplasts. Immunofluorescent analysis showed that antibodies to the last 10 amino acids of uracil permease bind to detergent-treated protoplasts, but not to intact ones. Carboxypeptidase digested the C-terminus of uracil permease inserted into the sealed dog-pancreas microsomes. These results establish that both N- and C-termini are cytoplasmic, the permease polypeptide spanning the membrane an even number of times. The orientation of several hydrophilic loops with respect to the membrane was investigated by introducing potential glycosylation sites into these regions. We checked whether the resulting mutant proteins were glycosylated when expressed in the presence of dog-pancreas microsomes. Our data show that two loops of the protein are lumenal. Together with previous results, this work indicates that uracil permease is a 10 membrane-spanning protein, with rather small external loops and three main cytoplasmic regions (the N- and C-termini and a central 60-residue loop).


Assuntos
Membrana Celular/química , Proteínas Fúngicas/química , Proteínas de Membrana/química , Proteínas de Membrana Transportadoras/química , Proteínas de Transporte de Nucleotídeos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Animais , Membrana Celular/enzimologia , Permeabilidade da Membrana Celular , Polaridade Celular , Cães , Técnica Indireta de Fluorescência para Anticorpo , Proteínas Fúngicas/efeitos dos fármacos , Glicosilação , Hexosaminidases/farmacologia , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Microssomos/metabolismo , Dados de Sequência Molecular , Conformação Proteica , Dobramento de Proteína , Tripsina/farmacologia
5.
Int Immunol ; 7(7): 1093-106, 1995 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-8527407

RESUMO

BCMA is a human gene expressed preferentially in mature B lymphocytes as a 1.2 kb mRNA, which encodes a 184 amino acid peptide (BCMAp). The study of BCMA mRNA expression, using human malignant B cell lines characteristic of different stages of B lymphocyte differentiation, demonstrated that the BCMA mRNA is absent in the pro-B lymphocyte stage. It is expressed faintly at the pre-B cell stage and its expression increases with B lymphocyte maturation. Polyclonal antibodies were used to show, by cellular fractionation and immunoprecipitation, that BCMAp is a non-glycosylated integral membrane protein. Furthermore, BCMAp inserts, in vitro, into canine microsomes, as a type I integral membrane protein. Cell surface labeling showed that BCMAp is not expressed in the plasma membrane of mature B lymphocytes. Immunofluorescence studies revealed that BCMAp lies in a cap-like structure near the nucleus, that was identified as the Golgi apparatus by co-localization of BCMAp with CTR433, a marker of the medial cisternae of the Golgi apparatus. Confocal scanning laser microscopy of U266 plasma cells labeled with markers of various Golgi apparatus subcompartments strongly suggests that BCMAp is located in the cis part of the Golgi apparatus. Thus, BCMAp is the first Golgi resident protein with a tissue specificity and whose expression is linked to the stage of differentiation of B lymphocytes. The location of BCMAp in the Golgi apparatus and its high expression in plasmocytes (secreting large amounts of Ig) suggest that BCMAp is implicated in the intracellular traffic of Ig.


Assuntos
Linfócitos B/química , Complexo de Golgi/química , Proteínas de Membrana/química , Receptores do Fator de Necrose Tumoral , Antígeno de Maturação de Linfócitos B , Compartimento Celular , Imunofluorescência , Complexo de Golgi/imunologia , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Microscopia Confocal , Microssomos/química , Conformação Proteica , RNA Mensageiro/química , Células Tumorais Cultivadas
6.
J Biol Chem ; 269(44): 27478-85, 1994 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-7961662

RESUMO

The yeast SSS1 gene has been isolated as an extragenic high copy suppressor of sec61, a mutant displaying defects in protein translocation into the endoplasmic reticulum (ER). We found that SSS1 is an essential gene required for transfer of secretory precursors through the ER membrane. Here we demonstrate that the SSS1 product (Sss1p) is firmly bound to the ER membrane and exposes its amino-terminal half on the cytosolic side. Only detergent, or an alkali treatment, is effective at extracting Sss1p from the membrane. Coimmunoprecipitation experiments revealed that Sss1p and Sec61p participate in the same multisubunit complex. Cross-linking followed by immunoprecipitation specifically yielded an additional polypeptide of molecular mass 73 kDa. Moreover, Sss1p and Sec61p show mutually stabilizing interactions: Sss1p is destabilized in a sec61 mutant context, and mutated Sec61p is stabilized by Sss1p overproduction. These observations account for the isolation of SSS1 as a dosage-dependent suppressor of sec61. Since the polytopic integral membrane protein Sec61p is adjacent to translocating precursors and to ribosomes, and given the comparable translocation deficiencies of sss1 or sec61 mutants, we propose that Sss1p belongs to the "Sec61 subcomplex" that constitutes the pore of the membrane-bound translocation apparatus.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae , Transporte Biológico , Genes Fúngicos , Substâncias Macromoleculares , Proteínas de Membrana Transportadoras , Precursores de Proteínas/metabolismo , Canais de Translocação SEC , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
7.
EMBO J ; 12(11): 4083-93, 1993 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-8223425

RESUMO

The SEC61, SEC62 and SEC63 yeast gene products are membrane components of the apparatus that catalyses protein translocation into the endoplasmic reticulum (ER). In the hope of uncovering additional components of the translocation apparatus, we sought yeast genes whose overexpression would restore partial thermoresistance in a sec61 translocation-deficient mutant. The first extragenic Sec sixty-one suppressor, SSS1, is an essential single copy gene whose overexpression restores translocation in the sec61 mutant. Another extragenic suppressor was identified as TDH3, which encodes the major isozyme of the most abundant yeast protein, glyceraldehyde-3-phosphate dehydrogenase. TDH3 overexpression could exert an indirect effect by competitively inhibiting protein synthesis, thereby allowing the impaired translocation apparatus to cope with a reduced flow of newly synthesized secretory proteins. Depletion of the Sss1 protein rapidly results in accumulation of multiple secretory or membrane proteins devoid of post-translational modifications; the normally secreted alpha-factor accumulates on the cytosolic side of ER membranes. Thus, the SSS1 gene is required for continued translocation of secretory preproteins beyond their early association to ER membranes. Consistent with its essential role in protein translocation, the Sss1 protein localizes to the ER and homologues were detected in higher eukaryotes.


Assuntos
Retículo Endoplasmático/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Proteínas de Membrana/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Transporte Biológico/genética , Compartimento Celular , Clonagem Molecular , Sequência Conservada , Imunofluorescência , Proteínas Fúngicas/isolamento & purificação , Regulação Fúngica da Expressão Gênica , Genes Supressores , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras , Dados de Sequência Molecular , RNA Mensageiro/genética , Canais de Translocação SEC , Homologia de Sequência de Aminoácidos
8.
Biochem Biophys Res Commun ; 150(3): 979-86, 1988 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-2449211

RESUMO

The illumination of Escherichia coli cells with UVA light, 320 nm less than or equal to lambda less than or equal to 380 nm, triggers a transient growth and division delay. The built-in 4-thiouridine chromophore which absorbs light at 340 nm leads to the quantitative 8-13 crosslinking of a number of tRNA species corresponding to 50% of the bulk tRNA molecules. Determination of the tRNA acylation level by the various aminoacids shows that only the tRNA species acylated by Phe and Pro are strikingly affected in vivo. Both acylation levels decrease to less than 10% of their initial value during the illumination period, remain stable all along the growth lag and increase concomitantly with cell mass when growth resumes. Hence tRNA(Phe) and tRNA(Pro) are the UVA light molecular targets triggering growth delay and related effects of biological significance such as cell volume reduction, photoprotection and protection against UV mutagenesis (antiphotomutagenesis).


Assuntos
Escherichia coli/efeitos da radiação , RNA Bacteriano/efeitos da radiação , Aminoacil-RNA de Transferência/efeitos da radiação , Raios Ultravioleta , Acilação , Reagentes de Ligações Cruzadas , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Conformação de Ácido Nucleico , Fenilalanina/análogos & derivados , Fenilalanina/farmacologia , Aminoacil-RNA de Transferência/metabolismo , Tiouridina
9.
Biochem Biophys Res Commun ; 141(2): 847-54, 1986 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-2432896

RESUMO

Monkey kidney cells (CV-1) cultivated in the presence of 0.1 mM 4-thiouridine (S4U) and subsequently illuminated at 365 nm exhibit a marked RNA synthesis inhibition. Maximal effect (approximately 40%) was obtained for a 4 h S4U incubation and a 45 KJ/m2 dose. Under these conditions up to 20% of total cellular RNA is retained at the interphase during phenol-chloroform extraction. The fraction of RNA crosslinked to proteins amounts to 50% of the 3H-uridine labeled RNA synthesized during S4U incorporation and less than 10% for the control samples. This strongly suggests that S4U incorporated within the RNA chains acts as a photoaffinity probe. The data above provide the basis of a method for studying in vivo RNA-protein interactions under non destructive conditions.


Assuntos
Reagentes de Ligações Cruzadas , Nucleoproteínas , RNA/metabolismo , Tiouridina/metabolismo , Animais , Células Cultivadas , Chlorocebus aethiops , Fotoquímica , RNA/biossíntese
10.
J Gen Microbiol ; 121(1): 203-12, 1980 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-7252480

RESUMO

A phage isolated from lysates of phage CP-54 grown on Bacillus cereus 569 and selected on the basis of its ability to infect Bacillus thuringiensis var. berliner 1715 (serotype I) was designated CP-54Ber. Phages CP-54Ber and CP-54 were similar in size, morphology, cryosensitivity and stabilization by dimethyl sulphoxide. They showed significant differences with regard to inactivation by specific antiserum, adsorption to the berliner strains and host range. Phage CP-54Ber was able to mediate generalized transduction in the host strain berliner 1715 with frequencies ranging between 1 x 10(-5) and 1 x 10(-6). Cotransduction of markers was demonstrated. Cross-transduction occurred between strains belonging to serotype I whereas it was more difficult to observe when lysates were prepared on strains from other serotypes.


Assuntos
Bacillus thuringiensis/genética , Bacteriófagos/genética , Transdução Genética , Bacteriófagos/ultraestrutura , DNA Viral , Ligação Genética , Microscopia Eletrônica , Mutação
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...