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At least 199 records · Page 11Linked to original sources
PubMed1992

Sexual agglutination in budding yeasts: structure, function, and regulation of adhesion glycoproteins.

The sexual agglutinins of the budding yeasts are cell adhesion proteins that promote aggregation of cells during mating. In each yeast species, complementary agglutinins are expressed by cells of opposite mating type that interact to mediate aggregation. Saccharomyces cerevisiae alpha-agglutinin and its analogs from other yeasts are single-subunit glycoproteins that contain N-linked and O-linked oligosaccharides. The N-glycosidase-sensitive carbohydrate is not necessary for activity. The proposed binding domain of alpha-agglutinin has features characteristic of the immunoglobulin fold structures of cell adhesion proteins of higher eukaryotes. The C-terminal region of alpha-agglutinin plays a role in anchoring the glycoprotein to the cell surface. The S. cerevisiae alpha-agglutinin and its analogs from other species contain multiple subunits; one or more binding subunits, which interact with the opposite agglutinin, are disulfide bonded to a core subunit, which mediates cell wall anchorage. The core subunits are composed of 80 to 95% O-linked carbohydrate. The binding subunits have less carbohydrate, and both carbohydrate and peptide play roles in binding. The alpha-agglutinin and alpha-agglutinin genes from S. cerevisiae have been cloned and shown to be regulated by the mating-type locus, MAT, and by pheromone induction. The agglutinins are necessary for mating under conditions that do not promote cell-cell contact. The role of the agglutinins therefore is to promote close interactions between cells of opposite mating type and possibly to facilitate the response to phermone, thus increasing the efficiency of mating. We speculate that they mediate enhanced response to sex pheromones by providing a synapse at the point of cell-cell contact, at which both pheromone secretion and cell fusion occur.

Agglutinins↗
PubMedDate not supplied

Extraction, purification and characterization of ADH1 from the budding yeast Kluyveromyces marxianus.

The enzyme ADH1 has been extracted and purified from the budding yeast Kluyveromyces marxianus, and its enzymatic activity has been compared, with the ADH1 extracted and purified in the same way from the well known yeast Saccharomyces cerevisiae. K. marxianus ADH1 has an optimal temperature higher than the S. cerevisiae enzyme (45-50 degrees vs 35 degrees C), a better stability to pH variations in the oxidative reaction (pH optimum 7.5), a lower Michaelis constant for acetaldehyde, and a good catalytic activity both for fermentative and oxidative reactions. In fact, while in Saccharomyces the constants ratio (velocity constant fermentation/velocity constant oxidation) is about 20,000, in Kluyveromyces the same ratio is only 15. Even if these two Genera are quite related (they belong to the same subfamily) it seems that their ADH1s possess different catalytic properties.

Alcohol Dehydrogenase↗
PubMed1996

Budding yeast SKP1 encodes an evolutionarily conserved kinetochore protein required for cell cycle progression.

The budding yeast SKP1 gene, identified as a dosage suppressor of a known kinetochore protein mutant, encodes an intrinsic 22.3 kDa subunit of CBF3, a multiprotein complex that binds centromere DNA in vitro. Temperature-sensitive mutations in SKP1 define two distinct phenotypic classes. skp1-4 mutants arrest predominantly as large budded cells with a G2 DNA content and short mitotic spindle, consistent with a role in kinetochore function. skp1-3 mutants, however, arrest predominantly as multiply budded cells with a G1 DNA content, suggesting an additional role during the G1/S phase. Identification of Skp1p homologs from C. elegans, A. thaliana, and H. sapiens indicates that SKP1 is evolutionarily highly conserved. Skp1p therefore represents an intrinsic kinetochore protein conserved throughout eukaryotic evolution and may be directly involved in linking kinetochore function with the cell cycle-regulatory machinery.

Alleles↗
PubMed1995

NAP1 acts with Clb1 to perform mitotic functions and to suppress polar bud growth in budding yeast.

NAP1 is a 60-kD protein that interacts specifically with mitotic cyclins in budding yeast and frogs. We have examined the ability of the yeast mitotic cyclin Clb2 to function in cells that lack NAP1. Our results demonstrate that Clb2 is unable to carry out its full range of functions without NAP1, even though Clb2/p34CDC28-associated kinase activity rises to normal levels. In the absence of NAP1, Clb2 is unable to efficiently induce mitotic events, and cells undergo a prolonged delay at the short spindle stage with normal levels of Clb2/p34CDC28 kinase activity. NAP1 is also required for the ability of Clb2 to induce the switch from polar to isotropic bud growth. As a result, polar bud growth continues during mitosis, giving rise to highly elongated cells. Our experiments also suggest that NAP1 is required for the ability of the Clb2/p34CDC28 kinase complex to amplify its own production, and that NAP1 plays a role in regulation of microtubule dynamics during mitosis. Together, these results demonstrate that NAP1 is required for the normal function of the activated Clb2/p34CDC28 kinase complex, and provide a step towards understanding how cyclin-dependent kinase complexes induce specific events during the cell cycle.

Blotting, Northern↗
PubMed2001

Functional correlation between the nuclear localization of Fht1p and its flocculation and heat tolerance activities in budding yeast Saccharomyces cerevisiae.

Fht1p is involved in the flocculation and heat tolerance machinery of budding yeast Saccharomyces cerevisiae. Despite knowledge of its involvement in those phenotypes, a precise mechanism has yet to be discovered. To this end, we monitored the relationship between subcellular localization of Fht1p and its flocculation or heat tolerance function using newly developed expression vectors with a recombinant green fluorescent protein (GFP; S65T/S147P) of Aequorea victoria added at both the N- and C-terminus of Fht1p. The main fluorescent signal of the GFP tagged with either a wild-type Fht1p or mutants which preserve their flocculation function was detected in the nucleus, whereas signals of functionless mutants were dispersed to the cytoplasm.

Cell Nucleus↗
PubMed2001

The budding yeast Msh4 protein functions in chromosome synapsis and the regulation of crossover distribution.

The budding yeast MSH4 gene encodes a MutS homolog produced specifically in meiotic cells. Msh4 is not required for meiotic mismatch repair or gene conversion, but it is required for wild-type levels of crossing over. Here, we show that a msh4 null mutation substantially decreases crossover interference. With respect to the defect in interference and the level of crossing over, msh4 is similar to the zip1 mutant, which lacks a structural component of the synaptonemal complex (SC). Furthermore, epistasis tests indicate that msh4 and zip1 affect the same subset of meiotic crossovers. In the msh4 mutant, SC formation is delayed compared to wild type, and full synapsis is achieved in only about half of all nuclei. The simultaneous defects in synapsis and interference observed in msh4 (and also zip1 and ndj1/tam1) suggest a role for the SC in mediating interference. The Msh4 protein localizes to discrete foci on meiotic chromosomes and colocalizes with Zip2, a protein involved in the initiation of chromosome synapsis. Both Zip2 and Zip1 are required for the normal localization of Msh4 to chromosomes, raising the possibility that the zip1 and zip2 defects in crossing over are indirect, resulting from the failure to localize Msh4 properly.

Carrier Proteins↗
PubMed2003

TOR regulates the subcellular localization of Ime1, a transcriptional activator of meiotic development in budding yeast.

The transcriptional activator Ime1 is a key regulator of meiosis and sporulation in budding yeast. Ime1 is controlled at different levels by nutrients and cell-type signals. Previously, we have proposed that G(1) cyclins would transmit nutritional signals to the Ime1 pathway by preventing the accumulation of Ime1 within the nucleus. We show here that nutritional signals regulate the subcellular localization of Ime1 through the TOR pathway. The inactivation of TOR with rapamycin promotes the nuclear accumulation and stabilization of Ime1, with consequent induction of early meiotic genes. On the contrary, the activation of TOR by glutamine induces the relocalization of Ime1 to the cytoplasm. Thus, TOR may sense optimal nitrogen- and carbon-limiting conditions to modulate Ime1 function. Besides TOR, ammonia induces an independent mechanism that prevents the accumulation of Ime1 in the nucleus. Both TOR and ammonia regulate Ime1 localization in the absence of Cdk1 activity and therefore use mechanisms different from those exerted by G(1) cyclins. Integration of independent mechanisms into a single early controlling step, such as the nuclear accumulation of Ime1, may help explain why yeast cells execute the meiotic program only when the appropriate internal and external conditions are met together.

Antibiotics, Antineoplastic↗
PubMed2004

Effect of ethanol on cell growth of budding yeast: genes that are important for cell growth in the presence of ethanol.

The budding yeast Saccharomyces cerevisiae has been used in the fermentation of various kinds of alcoholic beverages. But the effect of ethanol on the cell growth of this yeast is poorly understood. This study shows that the addition of ethanol causes a cell-cycle delay associated with a transient dispersion of F-actin cytoskeleton, resulting in an increase in cell size. We found that the tyrosine kinase Swe1, the negative regulator of Cdc28-Clb kinase, is related to the regulation of cell growth in the presence of ethanol. Indeed, the increase in cell size due to ethanol was partially abolished in the SWE1-deleted cells, and the amount of Swe1 protein increased transiently in the presence of ethanol. These results indicated that Swe1 is involved in cell size control in the presence of ethanol, and that a signal produced by ethanol causes a transient up-regulation of Swe1. Further we investigated comprehensively the ethanol-sensitive strains in the complete set of 4847 non-essential gene deletions and identified at least 256 genes that are important for cell growth in the presence of ethanol.

Actins↗
PubMed1997

Filamentous growth of the budding yeast Saccharomyces cerevisiae induced by overexpression of the WHi2 gene.

The WHI2 gene of the budding yeast Saccharomyces cerevisiae is required for the arrest of cell proliferation upon nutrient exhaustion: whi2 mutants carry on dividing and in the absence of growth become abnormally small. It is reported here that overexpression of Whi2 from the GAL1 promoter results in filamentous growth - cells fail to complete cytokinesis, the budding pattern changes from axial to polar, cells become elongated and cell size increases threefold. In many ways, these filaments resemble the pseudohyphae which result from nitrogen-limited growth and the filaments seen during the invasive growth of haploids. However, Whi2-induced filament formation is reduced, but not blocked, by mutations in STE7, STE12 or STE20 which do block pseudohypha formation. Furthermore, pseudohypha formation can still occur in a diploid in which both copies of the WHI2 gene have been deleted. Thus Whi2-induced filament formation and pseudohypha formation must come about through the action of different pathways. Despite this, a mutation in the STE11 gene, which is required for pseudohypha formation, does block Whi2-induced filament formation. Concanavalin A pulse-chase experiments show that new cell wall material is incorporated only into the tips of the apical cells. An extragenic suppressor of the Whi2 allele also results in filamentous growth. These results suggest that Whi2 negatively regulates a function required for the budding mode of cell proliferation including cytokinesis. This function is defined wholly or in part by the fsw1 allele.

Cell Cycle Proteins↗
PubMed1996

A new efficient gene disruption cassette for repeated use in budding yeast.

The dominant kanr marker gene plays an important role in gene disruption experiments in budding yeast, as this marker can be used in a variety of yeast strains lacking the conventional yeast markers. We have developed a loxP-kanMX-loxP gene disruption cassette, which combines the advantages of the heterologous kanr marker with those from the Cre-lox P recombination system. This disruption cassette integrates with high efficiency via homologous integration at the correct genomic locus (routinely 70%). Upon expression of the Cre recombinase the kanMX module is excised by an efficient recombination between the loxP sites, leaving behind a single loxP site at the chromosomal locus. This system allows repeated use of the kanr marker gene and will be of great advantage for the functional analysis of gene families.

Base Sequence↗
PubMed2006

Budding yeast Hed1 down-regulates the mitotic recombination machinery when meiotic recombination is impaired.

In budding yeast, there are two RecA homologs: Rad51 and Dmc1. While Rad51 is involved in both mitotic and meiotic recombination, Dmc1 participates specifically in meiotic recombination. Here, we describe a meiosis-specific protein (Hed1) with a novel Rad51 regulatory function. Several observations indicate that Hed1 attenuates Rad51 activity when Dmc1 is absent. First, although double-strand breaks are normally poorly repaired in the dmc1 mutant, repair becomes efficient when Hed1 is absent, and this effect depends on Rad51. Second, Rad51 and Hed1 colocalize as foci on meiotic chromosomes, and chromosomal localization of Hed1 depends on Rad51. Third, production of Hed1 in vegetative cells inhibits Rad51-dependent recombination events. Fourth, the Hed1 protein shows an interaction with Rad51 in the yeast two-hybrid protein system. We propose that Hed1 provides a mechanism to ensure the coordinated action of Rad51 and Dmc1 during meiosis, by down-regulating Rad51 activity when Dmc1 is unavailable.

Chromosomes, Fungal↗
PubMed2005

Budding yeast PDS5 plays an important role in meiosis and is required for sister chromatid cohesion.

Budding yeast PDS5 is an essential gene in mitosis and is required for chromosome condensation and sister chromatid cohesion. Here we report that PDS also is required in meiosis. Pds5p localizes on chromosomes at all stages during meiotic cycle, except anaphase I. PDS5 plays an important role at first meiotic prophase. Failure in function of PDS5 causes premature separation of chromosomes. The loading of Pds5p onto chromosome requires the function of REC8, but the association of Rec8p with chromosome is independent of PDS5. Mutant analysis and live cell imaging indicate that PDS5 play a role in meiosis II as well.

Anaphase↗
PubMed2004

A role for the MutL homologue MLH2 in controlling heteroduplex formation and in regulating between two different crossover pathways in budding yeast.

BACKGROUND AND AIMS: Mismatch repair proteins play important roles during meiotic recombination in the budding yeast Saccharomyces cerevisiae and most eukaryotic organisms studied to date. To study the functions of the mismatch repair protein Mlh2p in meiosis, we constructed mlh2Delta strains and measured rates of crossing over, gene conversion, post-meiotic segregation and spore viability. We also analysed mlh1Delta, mlh3Delta, msh4Delta, msh5Delta, exo1Delta and mus81Delta mutant strains singularly and in various combinations. RESULTS: Loss of MLH2 resulted in a small but significant decrease in spore viability and a significant increase in gene conversion frequencies but had no apparent effect on crossing over. Deletion of MLH2 in mlh3Delta, msh4Delta or msh5Delta strains resulted in significant proportion of the "lost" crossovers found in single deletion strains being regained in some genetic intervals. We and others propose that there are at least two pathways to generate crossovers in yeast (Ross-Macdonald and Roeder, 1994; Zalevsky et al., 1999; Khazanehdari and Borts, 2000; Novak et al., 2001; de los Santos et al., 2003). Most recombination intermediates are processed by the "major", Msh4-dependent pathway, which requires the activity of Mlh1p/Mlh3p/Msh4p/Msh5p as well as a number of other proteins. The minor pathway(s) utilizes Mms4p/Mus81p. We suggest that the absence of Mlh2p allows some crossovers from the MSH4 pathway to traverse the MUS81-dependent pathway.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Pulsed-field gel electrophoresis of budding yeast chromosomes.

Pulsed-field gel electrophoresis (PFGE) can be used to separate the 16 budding yeast chromosomes on the basis of size. Here we describe a detailed, practical protocol that will allow a novice to perform informative PFGE experiments. We first describe the culture of yeast prior to analysis, along with details of embedding cells in agarose before removal of cell walls. We then detail the procedure to remove protein and RNA from chromosomes and how naked chromosomes are loaded into agarose gels before being subjected to electrophoresis. Finally, we describe how the separated chromosomes can be visualized and photographed.

Cell Fractionation↗
PubMed2004

Does chromosome size affect map distance and genetic interference in budding yeast?

The hypothesis that chromosome size affects the rate and distribution of meiotic crossovers in budding yeast was tested. Map distance and interference were measured in the same genetic intervals present on either small (340 and 508 kb) or large (917 and 1085 kb) chromosomes, respectively. No differences were observed.

Chromosome Mapping↗
PubMed2006

Budding yeast Saccharomyces cerevisiae as a model to study oxidative modification of proteins in eukaryotes.

The budding yeast Saccharomyces cerevisiae is a well studied unicellular eukaryotic organism the genome of which has been sequenced. The use of yeast in many commercial systems makes its investigation important not only from basic, but also from practical point of view. Yeast may be grown under both aerobic and anaerobic conditions. The investigation of the response of eukaryotes to different kinds of stresses was pioneered owing to yeast and here we focus mainly on the so-called oxidative stress. It is a result of an imbalance between the formation and decomposition of reactive oxygen species increasing their steady-state concentration. Reactive oxygen species may attack any cellular component. In the present review oxidation of proteins in S. cerevisiae is analyzed. There are two connected approaches to study oxidative protein modification - characterization of the overall process and identification of individual oxidized proteins. Because all aerobic organisms possess special systems which defend them against reactive oxygen species, the involvement of so-called antioxidant enzymes, particularly superoxide dismutase and catalase, in the protection of proteins is also analyzed.

Eukaryotic Cells↗
PubMed1993

Actin structure and function: roles in mitochondrial organization and morphogenesis in budding yeast and identification of the phalloidin-binding site.

To further elucidate the functions of actin in budding yeast and to relate actin structure to specific roles and interactions in vivo, we determined the phenotypes caused by 13 charged-to-alanine mutations isolated previously in the single Saccharomyces cerevisiae actin gene. Defects in actin organization, morphogenesis, budding pattern, chitin deposition, septation, nuclear segregation, and mitochondrial organization were observed. In wild-type cells, mitochondria were found to be aligned along actin cables. Many of the amino acid substitutions that had the most severe effects on mitochondrial organization are located under the myosin "footprint" on the actin monomer, suggesting that actin-myosin interactions might underlie mitochondrial organization in yeast. In addition, one mutant (act1-129; R177A, D179A) produced an actin that assembled into cables and patches that could be visualized by anti-actin immunofluorescence in situ and that assembled into microfilaments of normal appearance in vitro as judged by electron microscopy but which could not be labeled by rhodamine-phalloidin in situ or in vitro. Rhodamine-phalloidin could label actin filaments assembled from all of the other mutant actins, including one (act1-119; R116A, E117A, K118A) that is altered at a residue (E117) that can be chemically cross-linked to phalloidin. The implication of residues R177 and/or D179 in phalloidin binding is in close agreement with a recently reported molecular model in which the phalloidin-binding site is proposed to be at the junction of two or three actin monomers in the filament.

Actin Cytoskeleton↗