RESUMO
Photofield emission (PFE) measurements are employed to examine modifications of the surface electronic structure of the tungsten (1 1 1) facet upon deposition of thin films (1-3 monolayers) of titanium. With the help of DFT simulations, the observed PFE features are interpreted as adsorbate-induced resonance states with energies just below the Fermi level, localized predominantly at the exposed surface atoms. Comparison between the computed surface DOS distributions and the measured PFE spectra is also used to verify various possible arrangements of the Ti adatoms, supporting the DFT-favored model of Ti growth in registry with the W(1 1 1) substrate until a full physical overlayer of the adsorbate is completed.
RESUMO
Photofield emission (PFE) measurements are employed to examine modifications of the surface electronic structure of the tungsten (111) facet upon deposition of thin films (1-3 monolayers) of titanium. With the help of DFT simulations, the observed PFE features are interpreted as adsorbate-induced resonance states with energies just below the Fermi level, localized predominantly at the exposed surface atoms. Comparison between the computed surface DOS distributions and the measured PFE spectra is also used to verify various possible arrangements of the Ti adatoms, supporting the DFT-favored model of Ti growth in registry with the W(111) substrate until a full physical overlayer of the adsorbate is completed.
RESUMO
Previously we have shown that the Saccharomyces cerevisiae CCZ1 (YBR131w) gene encodes a protein involved in protein trafficking. Deletion of this gene leads to fragmentation of the vacuole typical of the class B vps mutants. Genetic and biochemical data indicated that Ccz1p is required for fusion of various transport intermediates with the vacuole. Here we present data indicating that CCZ1 is a close partner of the YPT7 gene, which encodes Rab GTPase and is required for fusion of transport vesicles to vacuole and homotypic vacuole fusion. We isolated extragenic suppressors of CCZ1 deletion. All these suppressors belong to one complementation group and correspond to mutated alleles of the YPT7 gene. The mutated residues are located in two Ypt7p domains responsible for guanine binding. These data suggest that Ccz1p and Ypt7p interact physically. Coimmunoprecipitation experiments provide direct evidence that this indeed is the case. A possible mechanism of Ccz1p action is discussed.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Fatores de Troca do Nucleotídeo Guanina , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Proteínas de Transporte Vesicular , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Alanina/química , Alelos , Sequência de Aminoácidos , Ácido Aspártico/química , Transporte Biológico , Cafeína/farmacologia , Cálcio/metabolismo , Proteínas de Transporte/química , Estimulantes do Sistema Nervoso Central/farmacologia , DNA/metabolismo , Epitopos , Deleção de Genes , Biblioteca Gênica , Teste de Complementação Genética , Guanina/química , Hemaglutininas/química , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Fenótipo , Reação em Cadeia da Polimerase , Testes de Precipitina , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/química , Homologia de Sequência de Aminoácidos , Zinco/metabolismoRESUMO
The role of the yeast vacuole, a functional analogue of the mammalian lysosome, in the turnover of proteins and organelles has been well documented. This review provides an overview of the current knowledge of vesicle mediated vacuolar transport in the yeast Saccharomyces cerevisiae cells. Due to the conservation of the molecular transport machinery S. cerevisiae has become an important model system of vacuolar trafficking because of the facile application of genetics, molecular biology and biochemistry.
Assuntos
Lisossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular , Sequência de Aminoácidos , Transporte Biológico , Membrana Celular/metabolismo , Complexo de Golgi/metabolismo , Metabolismo dos Lipídeos , Proteínas de Membrana/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Proteínas SNARE , Homologia de Sequência de Aminoácidos , Vacúolos/química , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
CCZ1 was previously identified by the sensitivity of ccz1(delta) mutants to high concentrations of Caffeine and the divalent ions Ca(2+ )and Zn(2+). In this paper we show that deletion of CCZ1 leads to aberrant vacuole morphology, similar to the one reported for the family of vacuolar protein sorting (vps) mutants of class B. The ccz1(&Dgr;) cells display severe vacuolar protein sorting defects for both the soluble carboxipeptidase Y and the membrane-bound alkaline phosphatase, which are delivered to the vacuole by distinct routes. Ccz1p is a membranous protein and the vast majority of Ccz1p resides in late endosomes. These results, along with a functional linkage found between the CCZ1 and YPT7 genes, indicate that the site of Ccz1p function is at the last step of fusion of multiple transport intermediates with the vacuole.
Assuntos
Proteínas de Transporte de Cátions , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Trocadores de Sódio-Hidrogênio , Vacúolos/metabolismo , Proteínas de Transporte Vesicular , Proteínas rab de Ligação ao GTP/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/genética , ATPases Transportadoras de Cálcio/metabolismo , Proteínas de Transporte/análise , Proteínas de Transporte/metabolismo , Compartimento Celular/fisiologia , Endocitose/fisiologia , Proteínas Fúngicas/análise , Deleção de Genes , Dosagem de Genes , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Chaperonas Moleculares , Mutagênese/fisiologia , Fenótipo , ATPases Transportadoras de Cálcio da Membrana Plasmática , Plasmídeos , Receptores de Superfície Celular/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Vacúolos/química , Zinco/metabolismo , Proteínas rab de Ligação ao GTP/genéticaRESUMO
A PCR-based method for targeted gene deletion by kanMX4 module was used to construct complete deletion mutants of six individual open reading frames from chromosome II: YBR128c, YBR131w, YBR133c, YBR137w, YBR138c and YBR142w. The ORFs were deleted in two diploid strains, FY1679 and W303. Sporulation and tetrad analysis revealed that only one ORF, YBR142w, encoding a putative DEAD-box RNA helicase, is an essential gene. A systematic phenotypic analysis of the deleted mutants was carried out. Homozygous diploids ybr128cDelta/ybr128cDelta and ybr131wDelta/ybr131wDelta did not sporulate. The ybr131cDelta mutant whether haploid or homozygous diploid, in addition displayed an increased sensitivity to Caffeine, Calcium and Zinc, and to emphasize this phenotype we named the gene CCZ1. ORF YBR133c was independently reported by others as Histone Synthetic Lethal (HSL7) (Ma et al., 1996). We found that the aberrant morphology characteristic for ybr133cDelta (hsl7Delta) cells was observed in W303 but not in FY1679 genetic background. Furthermore, we observed that deletion of YBR133c had a pleiotropic effect under a wide range of conditions, including increased sensitivity to calcium, caffeine, calcofluor white, vanadate and verapamil. The effects of the deletion were reinforced in W303 background. We found no phenotypic effects of the two remaining deletions, ybr137wDelta and ybr138cDelta.
Assuntos
Cromossomos Fúngicos/genética , Genes Fúngicos , Saccharomyces cerevisiae/genética , Antifúngicos/farmacologia , Cafeína/farmacologia , Cátions/farmacologia , Clonagem Molecular , Primers do DNA , Deleção de Genes , Genes Essenciais , Fenótipo , Reação em Cadeia da Polimerase , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/fisiologia , Esporos Fúngicos/fisiologia , Transformação Genética , Verapamil/farmacologiaRESUMO
We have deleted the yeast gene YJR025c and shown that this leads to an auxotrophy for nicotinic acid. The deduced protein sequence of the gene product is homologous to the human 3-hydroxyanthranilic acid dioxygenase (EC 1.13.11.6) which is part of the kynurenine pathway for the degradation of tryptophan and the biosynthesis of nicotinic acid. In cell-free extracts the 3-hydroxyanthranilic acid dioxygenase activity is proportional to the copy number of the YJR025c gene. As YJR025c encodes the yeast 3-hydroxyanthranilic acid dioxygenase, we have named this gene BNA1 for biosynthesis of nicotinic acid.
