scholarly journals The dihydrosphingosine-1-phosphate phosphatases of Saccharomyces cerevisiae are important regulators of cell proliferation and heat stress responses

1999 ◽  
Vol 342 (3) ◽  
pp. 667-675 ◽  
Author(s):  
Cungui MAO ◽  
Julie D. SABA ◽  
Lina M. OBEID
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Heat Stress ◽  
Cell Growth ◽  
Stress Responses ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Yeast Cells ◽  
Sphingolipid Metabolism ◽  
Mrna Levels

We have identified YSR2 and YSR3 of Saccharomyces cerevisiae as genes encoding dihydrosphingosine-1-phosphate phophatases which are involved in regulation of sphingolipid metabolism [Mao, Wadleigh, Jenkins, Hannun and Obeid (1997) J. Biol. Chem. 272, 28690-28694]. In this study, we explored the physiological roles that these enzymes may have in S. cerevisiae.Deletion of either YSR2, YSR3 or both did not affect viability or growth rate of yeast cells. However, overexpression of YSR2 significantly prolonged the doubling time of cell growth, whereas overexpression of YSR3 affected cell growth only slightly. Cell cycle analysis suggested that overexpression of either YSR2 or, to a lesser extent, YSR3 caused cell cycle arrest at the G1 phase. Disruption of YSR2, but not YSR3, conferred increased thermotolerance. On the other hand, overexpression of either YSR2 or YSR3 diminished thermotolerance. Using labelled dihydrosphingosine and dihydrosphingosine-1-P (DHS-1-P), we found that overexpression of YSR2 significantly increased ceramide formation, whereas deletion of YSR2, YSR3, or both, accumulated DHS-1-P, and deletion of YSR2 decreased ceramide formation. Together, these results show that the phenotypes of YSR2 are associated with changes in endogenous levels of the different sphingolipids. Green fluorescent protein tagging showed that in the exponentially growing cells, YSR2 and YSR3 had the same cellular localization to endoplasmic reticulum. However, YSR2 and YSR3 differ in mRNA levels: YSR2 had significantly higher mRNA levels than YSR3. This discrepancy might result in the functional differences that these proteins exhibited. In addition, this study implicates sphingolipids and their metabolism in the regulation of growth and heat stress responses of the yeast S. cerevisiae.

scholarly journals Monitoring Stress-Related Genes during the Process of Biomass Propagation of Saccharomyces cerevisiae Strains Used for Wine Making

2005 ◽  
Vol 71 (11) ◽  
pp. 6831-6837 ◽  
Author(s):  
Roberto Pérez-Torrado ◽  
Jose M. Bruno-Bárcena ◽  
Emilia Matallana
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Stress Response ◽  
Biomass Production ◽  
Stress Responses ◽  
Environmental Changes ◽  
Wine Yeast ◽  
Yeast Cells ◽  
Mrna Levels ◽  
Yeast Biomass ◽  
Stress Related Genes

ABSTRACT Physiological capabilities and fermentation performance of Saccharomyces cerevisiae strains to be employed during industrial wine fermentations are critical for the quality of the final product. During the process of biomass propagation, yeast cells are dynamically exposed to a mixed and interrelated group of known stresses such as osmotic, oxidative, thermic, and/or starvation. These stressing conditions can dramatically affect the parameters of the fermentation process and the technological abilities of the yeast, e.g., the biomass yield and its fermentative capacity. Although a good knowledge exists of the behavior of S. cerevisiae under laboratory conditions, insufficient knowledge is available about yeast stress responses under the specific media and growth conditions during industrial processes. We performed growth experiments using bench-top fermentors and employed a molecular marker approach (changes in expression levels of five stress-related genes) to investigate how the cells respond to environmental changes during the process of yeast biomass production. The data show that in addition to the general stress response pathway, using the HSP12 gene as a marker, other specific stress response pathways were induced, as indicated by the changes detected in the mRNA levels of two stress-related genes, GPD1 and TRX2. These results suggest that the cells were affected by osmotic and oxidative stresses, demonstrating that these are the major causes of the stress response throughout the process of wine yeast biomass production.

scholarly journals Calmodulin concentrates at regions of cell growth in Saccharomyces cerevisiae.

1992 ◽  
Vol 118 (3) ◽  
pp. 619-629 ◽  
Author(s):  
S E Brockerhoff ◽  
T N Davis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cell Growth ◽  
Polyclonal Antibodies ◽  
Neck Region ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Asymmetric Distribution ◽  
Double Labeling ◽  
Bud Emergence

Calmodulin was localized in Saccharomyces cerevisiae by indirect immunofluorescence using affinity-purified polyclonal antibodies. Calmodulin displays an asymmetric distribution that changes during the cell cycle. In unbudded cells, calmodulin concentrates at the presumptive site of bud formation approximately 10 min before bud emergence. In small budded cells, calmodulin accumulates throughout the bud. As the bud grows, calmodulin concentrates at the tip, then disperses, and finally concentrates in the neck region before cytokinesis. An identical staining pattern is observed when wild-type calmodulin is replaced with mutant forms of calmodulin impaired in binding Ca2+. Thus, the localization of calmodulin does not depend on its ability to bind Ca2+ with a high affinity. Double labeling of yeast cells with affinity-purified anti-calmodulin antibody and rhodamine-conjugated phalloidin indicates that calmodulin and actin concentrate in overlapping regions during the cell cycle. Furthermore, disrupting calmodulin function using a temperature-sensitive calmodulin mutant delocalizes actin, and act1-4 mutants contain a random calmodulin distribution. Thus, calmodulin and actin distributions are interdependent. Finally, calmodulin localizes to the shmoo tip in cells treated with alpha-factor. This distribution, at sites of cell growth, implicates calmodulin in polarized cell growth in yeast.

scholarly journals Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

1999 ◽  
Vol 339 (2) ◽  
pp. 299-307 ◽  
Author(s):  
Arthur L. KRUCKEBERG ◽  
Ling YE ◽  
Jan A. BERDEN ◽  
Karel van DAM
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Glucose Transport ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Hexose Transporter ◽  
High Concentrations ◽  
Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

scholarly journals CLN3 expression is sufficient to restore G1-to-S-phase progression in Saccharomyces cerevisiae mutants defective in translation initiation factor eIF4E

1999 ◽  
Vol 340 (1) ◽  
pp. 135-141 ◽  
Author(s):  
Parisa DANAIE ◽  
Michael ALTMANN ◽  
Michael N. HALL ◽  
Hans TRACHSEL ◽  
Stephen B. HELLIWELL
Keyword(s):  
Cell Cycle ◽  
S Phase ◽  
Translation Initiation Factor ◽  
Yeast Cells ◽  
Initiation Factor ◽  
G1 Phase ◽  
Mrna Levels ◽  
Wild Type ◽  
Phase Progression ◽  
Type Gene

The essential cap-binding protein (eIF4E) of Saccharomycescerevisiae is encoded by the CDC33 (wild-type) gene, originally isolated as a mutant, cdc33-1, which arrests growth in the G1 phase of the cell cycle at 37 °C. We show that other cdc33 mutants also arrest in G1. One of the first events required for G1-to-S-phase progression is the increased expression of cyclin 3. Constructs carrying the 5ʹ-untranslated region of CLN3 fused to lacZ exhibit weak reporter activity, which is significantly decreased in a cdc33-1 mutant, implying that CLN3 mRNA is an inefficiently translated mRNA that is sensitive to perturbations in the translation machinery. A cdc33-1 strain expressing either stable Cln3p (Cln3-1p) or a hybrid UBI4 5ʹ-CLN3 mRNA, whose translation displays decreased dependence on eIF4E, arrested randomly in the cell cycle. In these cells CLN2 mRNA levels remained high, indicating that Cln3p activity is maintained. Induction of a hybrid UBI4 5ʹ-CLN3 message in a cdc33-1 mutant previously arrested in G1 also caused entry into a new cell cycle. We conclude that eIF4E activity in the G1-phase is critical in allowing sufficient Cln3p activity to enable yeast cells to enter a new cell cycle.

Codon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression

1987 ◽  
Vol 7 (8) ◽  
pp. 2914-2924
Author(s):  
A Hoekema ◽  
R A Kastelein ◽  
M Vasser ◽  
H A de Boer
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Steady State ◽  
Codon Usage ◽  
Experimental Approach ◽  
Mrna Translation ◽  
Yeast Cells ◽  
Expression Level ◽  
Start Codon ◽  
Mrna Levels

The coding sequences of genes in the yeast Saccharomyces cerevisiae show a preference for 25 of the 61 possible coding triplets. The degree of this biased codon usage in each gene is positively correlated to its expression level. Highly expressed genes use these 25 major codons almost exclusively. As an experimental approach to studying biased codon usage and its possible role in modulating gene expression, systematic codon replacements were carried out in the highly expressed PGK1 gene. The expression of phosphoglycerate kinase (PGK) was studied both on a high-copy-number plasmid and as a single copy gene integrated into the chromosome. Replacing an increasing number (up to 39% of all codons) of major codons with synonymous minor ones at the 5' end of the coding sequence caused a dramatic decline of the expression level. The PGK protein levels dropped 10-fold. The steady-state mRNA levels also declined, but to a lesser extent (threefold). Our data indicate that this reduction in mRNA levels was due to destabilization caused by impaired translation elongation at the minor codons. By preventing translation of the PGK mRNAs by the introduction of a stop codon 3' and adjacent to the start codon, the steady-state mRNA levels decreased dramatically. We conclude that efficient mRNA translation is required for maintaining mRNA stability in S. cerevisiae. These findings have important implications for the study of the expression of heterologous genes in yeast cells.

scholarly journals The effect of calnexin deletion on the expression level of PDI in Saccharomyces cerevisiae under heat stress conditions

2008 ◽  
Vol 13 (1) ◽  
Author(s):  
Huili Zhang ◽  
Jianwei He ◽  
Yanyan Ji ◽  
Akio Kato ◽  
Youtao Song
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Rate ◽  
Heat Stress ◽  
Protein Expression ◽  
Molecular Chaperone ◽  
Mrna Level ◽  
Stress Conditions ◽  
Mrna Levels ◽  
Wild Type ◽  
Wild Type Yeast

AbstractWe cultured calnexin-disrupted and wild-type Saccharomyces cerevisiae strains under conditions of heat stress. The growth rate of the calnexin-disrupted yeast was almost the same as that of the wild-type yeast under those conditions. However, the induced mRNA level of the molecular chaperone PDI in the ER was clearly higher in calnexin-disrupted S. cerevisiae relative to the wild type at 37°C, despite being almost the same in the two strains under normal conditions. The western blotting analysis for PDI protein expression in the ER yielded results that show a parallel in their mRNA levels in the two strains. We suggest that PDI may interact with calnexin under heat stress conditions, and that the induction of PDI in the ER can recover part of the function of calnexin in calnexin-disrupted yeast, and result in the same growth rate as in wild-type yeast.

Regulation of STA1 gene expression by MAT during the life cycle of Saccharomyces cerevisiae

1989 ◽  
Vol 9 (9) ◽  
pp. 3992-3998
Author(s):  
A M Dranginis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mating Type ◽  
Yeast Cells ◽  
Specific Gene ◽  
Activity Levels ◽  
Sporulation Medium ◽  
Mrna Levels ◽  
Yeast Saccharomyces Cerevisiae ◽  
Type Locus ◽  
Diploid Cells

STA1 encodes a secreted glucoamylase of the yeast Saccharomyces cerevisiae var. diastaticus. Glucoamylase secretion is controlled by the mating type locus MAT; a and alpha haploid yeast cells secrete high levels of the enzyme, but a/alpha diploid cells produce undetectable amounts. It has been suggested that STA1 is regulated by MATa2 (I. Yamashita, Y. Takano, and S. Fukui, J. Bacteriol. 164:769-773, 1985), which is a MAT transcript of previously unknown function. In contrast, this work shows that deletion of the entire MATa2 gene had no effect on STA1 regulation but that deletion of MATa1 sequences completely abolished mating-type control. In all cases, glucoamylase activity levels reflected STA1 mRNA levels. It appears that STA1 is a haploid-specific gene that is regulated by MATa1 and a product of the MAT alpha locus and that this regulation occurs at the level of RNA accumulation. STA1 expression was also shown to be glucose repressible. STA1 mRNA was induced in diploids during sporulation along with SGA, a closely linked gene that encodes an intracellular sporulation-specific glucoamylase of S. cerevisiae. A diploid strain with a MATa1 deletion showed normal induction of STA1 in sporulation medium, but SGA expression was abolished. Therefore, these two homologous and closely linked glucoamylase genes are induced by different mechanisms during sporulation. STA1 induction may be a response to the starvation conditions necessary for sporulation, while SGA induction is governed by the pathway by which MAT regulates sporulation. The strain containing a complete deletion of MATa2 grew, mated, and sporulated normally.

GPD1, which encodes glycerol-3-phosphate dehydrogenase, is essential for growth under osmotic stress in Saccharomyces cerevisiae, and its expression is regulated by the high-osmolarity glycerol response pathway

1994 ◽  
Vol 14 (6) ◽  
pp. 4135-4144
Author(s):  
J Albertyn ◽  
S Hohmann ◽  
J M Thevelein ◽  
B A Prior
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Osmotic Stress ◽  
Yeast Cells ◽  
Mrna Levels ◽  
Hog Pathway ◽  
Hypertonic Medium ◽  
High Osmolarity ◽  
High Osmolarity Glycerol ◽  
Enhanced Production ◽  
Glycerol 3 Phosphate Dehydrogenase

The yeast Saccharomyces cerevisiae responds to osmotic stress, i.e., an increase in osmolarity of the growth medium, by enhanced production and intracellular accumulation of glycerol as a compatible solute. We have cloned a gene encoding the key enzyme of glycerol synthesis, the NADH-dependent cytosolic glycerol-3-phosphate dehydrogenase, and we named it GPD1. gpd1 delta mutants produced very little glycerol, and they were sensitive to osmotic stress. Thus, glycerol production is indeed essential for the growth of yeast cells during reduced water availability. hog1 delta mutants lacking a protein kinase involved in osmostress-induced signal transduction (the high-osmolarity glycerol response [HOG] pathway) failed to increase glycerol-3-phosphate dehydrogenase activity and mRNA levels when osmotic stress was imposed. Thus, expression of GPD1 is regulated through the HOG pathway. However, there may be Hog1-independent mechanisms mediating osmostress-induced glycerol accumulation, since a hog1 delta strain could still enhance its glycerol content, although less than the wild type. hog1 delta mutants are more sensitive to osmotic stress than isogenic gpd1 delta strains, and gpd1 delta hog1 delta double mutants are even more sensitive than either single mutant. Thus, the HOG pathway most probably has additional targets in the mechanism of adaptation to hypertonic medium.

scholarly journals Mediation of the inhibitory effect of thyroid hormone on proliferation of hepatoma cells by transforming growth factor-beta

2006 ◽  
Vol 36 (1) ◽  
pp. 9-21 ◽  
Author(s):  
Chun-Che Yen ◽  
Ya-Hui Huang ◽  
Chu-Yu Liao ◽  
Cheng-Jung Liao ◽  
Wan-Li Cheng ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Growth Factor ◽  
Thyroid Hormone ◽  
Cell Growth ◽  
Growth Inhibition ◽  
Transforming Growth Factor ◽  
Cyclin E ◽  
Hepatoma Cell ◽  
Stable Cell Line ◽  
Mrna Levels

Thyroid hormone (triiodothyronine, T3) regulates growth, development and differentiation. To examine the influence of T3 on hepatoma cell growth, thyroid receptor (TR)α1 or TRβ1 over-expressing HepG2 cell lines were used. Growth of the HepG2-TR stable cell line was inhibited by over 50% following treatment with T3. However, transforming growth factor (TGF)-β neutralizing antibody, but not the control antibody can reverse the cell growth inhibition effect of T3. Flow cytometric analysis indicated that the growth inhibition was apparent at the transition point between the G1 and S phases of the cell cycle. The expression of major cell cycle regulators was used to provide further evidence for the growth inhibition. Cyclin-dependent kinase 2 (cdk2) and cyclin E were down-regulated in HepG2-TR cells. Moreover, p21 protein or mRNA levels were up-regulated by around 5-fold or 7.3-fold respectively following T3 treatment. Furthermore, phospho-retinoblastoma (ppRb) protein was down-regulated by T3. The expression of TGF-β was studied to delineate the repression mechanism. TGF-β was stimulated by T3 and its promoter activity was enhanced six- to eight-fold by T3. Furthermore, both T3 and TGF-β repressed the expression of cdk2, cyclin E and ppRb. On the other hand, TGF-β neutralizing but not control antibody blocked the repression of cdk2, cyclin E and ppRb by T3. These results demonstrated that T3 might play a key role in liver tumor cell proliferation.

scholarly journals The spindle checkpoint protein Mad2 regulates APC/C activity during prometaphase and metaphase of meiosis I in Saccharomyces cerevisiae

2011 ◽  
Vol 22 (16) ◽  
pp. 2848-2861 ◽  
Author(s):  
Dai Tsuchiya ◽  
Claire Gonzalez ◽  
Soni Lacefield
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Chromosome Segregation ◽  
Meiotic Division ◽  
Spindle Checkpoint ◽  
Yeast Cells ◽  
Meiotic Cell ◽  
Checkpoint Protein ◽  
Sister Chromatids ◽  
Meiosis I

In many eukaryotes, disruption of the spindle checkpoint protein Mad2 results in an increase in meiosis I nondisjunction, suggesting that Mad2 has a conserved role in ensuring faithful chromosome segregation in meiosis. To characterize the meiotic function of Mad2, we analyzed individual budding yeast cells undergoing meiosis. We find that Mad2 sets the duration of meiosis I by regulating the activity of APCCdc20. In the absence of Mad2, most cells undergo both meiotic divisions, but securin, a substrate of the APC/C, is degraded prematurely, and prometaphase I/metaphase I is accelerated. Some mad2Δ cells have a misregulation of meiotic cell cycle events and undergo a single aberrant division in which sister chromatids separate. In these cells, both APCCdc20 and APCAma1 are prematurely active, and meiosis I and meiosis II events occur in a single meiotic division. We show that Mad2 indirectly regulates APCAma1 activity by decreasing APCCdc20 activity. We propose that Mad2 is an important meiotic cell cycle regulator that ensures the timely degradation of APC/C substrates and the proper orchestration of the meiotic divisions.

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