ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
Subject(s)
Carrier Proteins/genetics , Carrier Proteins/metabolism , Guanine Nucleotide Exchange Factors , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/metabolism , Vacuoles/metabolism , Vesicular Transport Proteins , rab GTP-Binding Proteins/genetics , rab GTP-Binding Proteins/metabolism , Alanine/chemistry , Alleles , Amino Acid Sequence , Aspartic Acid/chemistry , Biological Transport , Caffeine/pharmacology , Calcium/metabolism , Carrier Proteins/chemistry , Central Nervous System Stimulants/pharmacology , DNA/metabolism , Epitopes , Gene Deletion , Gene Library , Genetic Complementation Test , Guanine/chemistry , Hemagglutinins/chemistry , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Phenotype , Polymerase Chain Reaction , Precipitin Tests , Protein Binding , Saccharomyces cerevisiae Proteins/chemistry , Sequence Homology, Amino Acid , Zinc/metabolismABSTRACT
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.
Subject(s)
Lysosomes/metabolism , Saccharomyces cerevisiae/metabolism , Vesicular Transport Proteins , Amino Acid Sequence , Biological Transport , Cell Membrane/metabolism , Golgi Apparatus/metabolism , Lipid Metabolism , Membrane Proteins/metabolism , Models, Biological , Molecular Sequence Data , SNARE Proteins , Sequence Homology, Amino Acid , Vacuoles/chemistry , rab GTP-Binding Proteins/metabolismABSTRACT
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.
Subject(s)
Cation Transport Proteins , Fungal Proteins/genetics , Fungal Proteins/metabolism , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/metabolism , Sodium-Hydrogen Exchangers , Vacuoles/metabolism , Vesicular Transport Proteins , rab GTP-Binding Proteins/metabolism , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Calcium/metabolism , Calcium-Transporting ATPases/genetics , Calcium-Transporting ATPases/metabolism , Carrier Proteins/analysis , Carrier Proteins/metabolism , Cell Compartmentation/physiology , Endocytosis/physiology , Fungal Proteins/analysis , Gene Deletion , Gene Dosage , Membrane Proteins/genetics , Membrane Proteins/metabolism , Molecular Chaperones , Mutagenesis/physiology , Phenotype , Plasma Membrane Calcium-Transporting ATPases , Plasmids , Receptors, Cell Surface/genetics , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/genetics , Vacuoles/chemistry , Zinc/metabolism , rab GTP-Binding Proteins/geneticsABSTRACT
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.
Subject(s)
Chromosomes, Fungal/genetics , Genes, Fungal , Saccharomyces cerevisiae/genetics , Antifungal Agents/pharmacology , Caffeine/pharmacology , Cations/pharmacology , Cloning, Molecular , DNA Primers , Gene Deletion , Genes, Essential , Phenotype , Polymerase Chain Reaction , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/physiology , Spores, Fungal/physiology , Transformation, Genetic , Verapamil/pharmacologyABSTRACT
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.
