scholarly journals A Late Mitotic Regulatory Network Controlling Cyclin Destruction in Saccharomyces cerevisiae

1998 ◽  
Vol 9 (10) ◽  
pp. 2803-2817 ◽  
Author(s):  
Sue L. Jaspersen ◽  
Julia F. Charles ◽  
Rachel L. Tinker-Kulberg ◽  
David O. Morgan
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Regulatory Network ◽  
Growth Defect ◽  
Cyclin Dependent Kinase ◽  
Anaphase Promoting Complex ◽  
Yeast Saccharomyces Cerevisiae ◽  
Mitotic Cyclin ◽  
Low Levels ◽  
Late Mitosis ◽  
Mutant Cells

Exit from mitosis requires the inactivation of mitotic cyclin-dependent kinase–cyclin complexes, primarily by ubiquitin-dependent cyclin proteolysis. Cyclin destruction is regulated by a ubiquitin ligase known as the anaphase-promoting complex (APC). In the budding yeast Saccharomyces cerevisiae, members of a large class of late mitotic mutants, including cdc15,cdc5, cdc14, dbf2, andtem1, arrest in anaphase with a phenotype similar to that of cells expressing nondegradable forms of mitotic cyclins. We addressed the possibility that the products of these genes are components of a regulatory network that governs cyclin proteolysis. We identified a complex array of genetic interactions among these mutants and found that the growth defect in most of the mutants is suppressed by overexpression of SPO12, YAK1, andSIC1 and is exacerbated by overproduction of the mitotic cyclin Clb2. When arrested in late mitosis, the mutants exhibit a defect in cyclin-specific APC activity that is accompanied by high Clb2 levels and low levels of the anaphase inhibitor Pds1. Mutant cells arrested in G1 contain normal APC activity. We conclude that Cdc15, Cdc5, Cdc14, Dbf2, and Tem1 cooperate in the activation of the APC in late mitosis but are not required for maintenance of that activity in G1.

scholarly journals The Anaphase-promoting Complex Promotes Actomyosin-Ring Disassembly during Cytokinesis in Yeast

2009 ◽  
Vol 20 (4) ◽  
pp. 1201-1212 ◽  
Author(s):  
Gregory H. Tully ◽  
Ryuichi Nishihama ◽  
John R. Pringle ◽  
David O. Morgan
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ubiquitin Ligase ◽  
Myosin Light Chain ◽  
Anaphase Promoting Complex ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Division Site ◽  
Specific Proteins ◽  
Mutant Cells ◽  
In Cells

The anaphase-promoting complex (APC) is a ubiquitin ligase that controls progression through mitosis by targeting specific proteins for degradation. It is unclear whether the APC also contributes to the control of cytokinesis, the process that divides the cell after mitosis. We addressed this question in the yeast Saccharomyces cerevisiae by studying the effects of APC mutations on the actomyosin ring, a structure containing actin, myosin, and several other proteins that forms at the division site and is important for cytokinesis. In wild-type cells, actomyosin-ring constituents are removed progressively from the ring during contraction and disassembled completely thereafter. In cells lacking the APC activator Cdh1, the actomyosin ring contracts at a normal rate, but ring constituents are not disassembled normally during or after contraction. After cytokinesis in mutant cells, aggregates of ring proteins remain at the division site and at additional foci in other parts of the cell. A key target of APCCdh1 is the ring component Iqg1, the destruction of which contributes to actomyosin-ring disassembly. Deletion of CDH1 also exacerbates actomyosin-ring disassembly defects in cells with mutations in the myosin light-chain Mlc2, suggesting that Mlc2 and the APC employ independent mechanisms to promote ring disassembly during cytokinesis.

scholarly journals Study of Cyclin Proteolysis in Anaphase-Promoting Complex (APC) Mutant Cells Reveals the Requirement for APC Function in the Final Steps of the Fission Yeast Septation Initiation Network

2001 ◽  
Vol 21 (19) ◽  
pp. 6681-6694 ◽  
Author(s):  
Louise Chang ◽  
Jennifer L. Morrell ◽  
Anna Feoktistova ◽  
Kathleen L. Gould
Keyword(s):  
Kinase Activity ◽  
Nuclear Division ◽  
Cyclin B ◽  
Cyclin Dependent Kinase ◽  
Anaphase Promoting Complex ◽  
Wild Type ◽  
Mitotic Cyclin ◽  
Septation Initiation Network ◽  
Mutant Cells ◽  
Null Mutants

ABSTRACT Cytokinesis in eukaryotic cells requires the inactivation of mitotic cyclin-dependent kinase complexes. An apparent exception to this relationship is found in Schizosaccharomyces pombemutants with mutations of the anaphase-promoting complex (APC). These conditional lethal mutants arrest with unsegregated chromosomes because they cannot degrade the securin, Cut2p. Although failing at nuclear division, these mutants septate and divide. Since septation requires Cdc2p inactivation in wild-type S. pombe, it has been suggested that Cdc2p inactivation occurs in these mutants by a mechanism independent of cyclin degradation. In contrast to this prediction, we show that Cdc2p kinase activity fluctuates in APCcut mutants due to Cdc13/cyclin B destruction. In APC-null mutants, however, septation and cutting do not occur and Cdc13p is stable. We conclude that APC cut mutants are hypomorphic with respect to Cdc13p degradation. Indeed, overproduction of nondestructible Cdc13p prevents septation in APC cutmutants and the normal reorganization of septation initiation network components during anaphase.

scholarly journals The Molecular Function of the Yeast Polo-like Kinase Cdc5 in Cdc14 Release during Early Anaphase

2009 ◽  
Vol 20 (16) ◽  
pp. 3671-3679 ◽  
Author(s):  
Fengshan Liang ◽  
Fengzhi Jin ◽  
Hong Liu ◽  
Yanchang Wang
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Phosphatase ◽  
Budding Yeast ◽  
Molecular Function ◽  
Cyclin Dependent Kinase ◽  
Yeast Saccharomyces Cerevisiae ◽  
Protein Levels ◽  
Early Anaphase ◽  
Show Evidence ◽  
Mitotic Cyclin

In the budding yeast Saccharomyces cerevisiae , Cdc14 is sequestered within the nucleolus before anaphase entry through its association with Net1/Cfi1, a nucleolar protein. Protein phosphatase PP2ACdc55 dephosphorylates Net1 and keeps it as a hypophosphorylated form before anaphase. Activation of the Cdc fourteen early anaphase release (FEAR) pathway after anaphase entry induces a brief Cdc14 release from the nucleolus. Some of the components in the FEAR pathway, including Esp1, Slk19, and Spo12, inactivate PP2ACdc55, allowing the phosphorylation of Net1 by mitotic cyclin-dependent kinase (Cdk) (Clb2-Cdk1). However, the function of another FEAR component, the Polo-like kinase Cdc5, remains elusive. Here, we show evidence indicating that Cdc5 promotes Cdc14 release primarily by stimulating the degradation of Swe1, the inhibitory kinase for mitotic Cdk. First, we found that deletion of SWE1 partially suppresses the FEAR defects in cdc5 mutants. In contrast, high levels of Swe1 impair FEAR activation. We also demonstrated that the accumulation of Swe1 in cdc5 mutants is responsible for the decreased Net1 phosphorylation. Therefore, we conclude that the down-regulation of Swe1 protein levels by Cdc5 promotes FEAR activation by relieving the inhibition on Clb2-Cdk1, the kinase for Net1 protein.

Yeast dom34 Mutants Are Defective in Multiple Developmental Pathways and Exhibit Decreased Levels of Polyribosomes

Genetics ◽  
1998 ◽  
Vol 149 (1) ◽  
pp. 45-56
Author(s):  
Luther Davis ◽  
JoAnne Engebrecht
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Heterologous Expression ◽  
Protein Translation ◽  
Developmental Pathways ◽  
G1 Phase ◽  
Growth Defect ◽  
Wild Type ◽  
Drosophila Homolog ◽  
Mutant Cells

Abstract The DOM34 gene of Saccharomyces cerevisiae is similar togenes found in diverse eukaryotes and archaebacteria. Analysis of dom34 strains shows that progression through the G1 phase of the cell cycle is delayed, mutant cells enter meiosis aberrantly, and their ability to form pseudohyphae is significantly diminished. RPS30A, which encodes ribosomal protein S30, was identified in a screen for high-copy suppressors of the dom34Δ growth defect. dom34Δ mutants display an altered polyribosome profile that is rescued by expression of RPS30A. Taken together, these data indicate that Dom34p functions in protein translation to promote G1 progression and differentiation. A Drosophila homolog of Dom34p, pelota, is required for the proper coordination of meiosis and spermatogenesis. Heterologous expression of pelota in dom34Δ mutants restores wild-type growth and differentiation, suggesting conservation of function between the eukaryotic members of the gene family.

scholarly journals Genetic and Biochemical Evaluation of the Importance of Cdc6 in Regulating Mitotic Exit

2003 ◽  
Vol 14 (11) ◽  
pp. 4592-4604 ◽  
Author(s):  
Vincent Archambault ◽  
Caihong X. Li ◽  
Alan J. Tackett ◽  
Ralph Wäsch ◽  
Brian T. Chait ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mitotic Exit ◽  
Terminal Region ◽  
Cyclin Dependent Kinase ◽  
Replication Control ◽  
Mitotic Cyclin ◽  
Biochemical Evaluation ◽  
Inhibitory Domain ◽  
Late Mitosis ◽  
Control Protein

We evaluated the hypothesis that the N-terminal region of the replication control protein Cdc6 acts as an inhibitor of cyclin-dependent kinase (Cdk) activity, promoting mitotic exit. Cdc6 accumulation is restricted to the period from mid-cell cycle until the succeeding G1, due to proteolytic control that requires the Cdc6 N-terminal region. During late mitosis, Cdc6 is present at levels comparable with Sic1 and binds specifically to the mitotic cyclin Clb2. Moderate overexpression of Cdc6 promotes viability of CLB2Δdb strains, which otherwise arrest at mitotic exit, and rescue is dependent on the N-terminal putative Cdk-inhibitory domain. These observations support the potential for Cdc6 to inhibit Clb2-Cdk, thus promoting mitotic exit. Consistent with this idea, we observed a cytokinesis defect in cdh1Δ sic1Δ cdc6Δ2–49 triple mutants. However, we were able to construct viable strains, in three different backgrounds, containing neither SIC1 nor the Cdc6 Cdk-inhibitory domain, in contradiction to previous work. We conclude, therefore, that although both Cdc6 and Sic1 have the potential to facilitate mitotic exit by inhibiting Clb2-Cdk, mitotic exit nevertheless does not require any identified stoichiometric inhibitor of Cdk activity.

scholarly journals Fermentación de la almendra de copoazú (Theobroma grandiflorum [Willd. ex Spreng.] Schum.): evaluación y optimización del proceso

2010 ◽  
Vol 11 (2) ◽  
pp. 107 ◽  
Author(s):  
Jenifer Criollo ◽  
Dagoberto Criollo ◽  
Angélica Sandoval Aldana
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Process Optimization ◽  
Fermentation Process ◽  
Raw Material ◽  
Sensory Panel ◽  
Yeast Saccharomyces Cerevisiae ◽  
Theobroma Grandiflorum ◽  
Cutting Test ◽  
Maximum Range ◽  
Low Levels

<p>La almendra de copoazú como producto promisorio para la industria de cosméticos, chocolate, bebidas, licores y conservas, se evaluó el proceso de fermentación variando el tiempo de remoción de la masa (24 y 48 horas) y la pulpa inicial (30 y 100%). Se tuvieron en cuenta las condiciones de los productores en el acceso a equipos de despulpado. Se cuantificó la temperatura de la masa en tres puntos (superior, medio e inferior), acidez, pH, humedad, prueba de corte y análisis sensorial. Se encontró bajo desarrollo de la temperatura de fermentación en los tratamientos con 100% de pulpa y se registraron las máximas temperaturas entre 35 y 36°C que indican deficiencias en el proceso; no se alcanzó los 40°C requeridos para la muerte del embrión. El 30% de pulpa inicial y la remoción cada 24 horas por 9 días, fueron las mejores condiciones encontradas. La optimización con 0,1% de levadura (Saccharomyces cerevisiae) aumentó la temperatura de fermentación hasta 44°C, los granos fermentados hasta 56,14% y el mayor desarrollo de sabores frutales con intensidad de 4, mostrando un mejor proceso de fermentación. El panel sensorial mostró que los licores de copoazú tienen notas frutales destacadas y bajos valores de otros sabores evaluados. Los resultados son semejantes a los cacaos criollos, conocidos en el mundo como materia prima de licores finos y de aroma.</p><p> </p><p><strong>Fermentation of the copoazu kernel (Theobroma grandiflorum [Willd. ex Spreng.] Schum.): Assessmente and process optimization</strong></p><p>The fermentation of copoazu kernels (a promising product for the cosmetics industry, chocolate, beverages, liquors and preserves) was evaluated varying the time of mass removal (24 and 48 hours) and the initial pulp (30 and 100%). This study took into account the degree of access the producers had to pulping equipment. We quantified temperature of the mass at three points (top, middle and bottom), acidity, pH, moisture, cutting test and sensory analysis. The observed temperatures during fermentation in the treatments with 100% pulp reached a maximum range between 35 and 36°C which indicated deficiencies in the process as the 40°C required for the death of the seed was not attained. Thirty percent initial pulp with removal every 24 hours for 9 days yielded the best results. Optimization with 0.1% yeast (Saccharomyces cerevisiae) increased the fermentation temperature to 44°C, augmented fermented beans to 56.14% and saw a development of fruit flavors with an intensity of 4, demonstrating a better fermentation process. The sensory panel showed that copoazu liquors have outstanding fruity notes and low levels of other evaluated flavors. The results are similar to the criollo cacao, known worldwide as a raw material for fine liquors and fragrances.</p>

scholarly journals Two types of TATA elements for the CYC1 gene of the yeast Saccharomyces cerevisiae.

1991 ◽  
Vol 11 (2) ◽  
pp. 666-676 ◽  
Author(s):  
W Z Li ◽  
F Sherman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Analysis ◽  
Start Codon ◽  
Nucleotide Position ◽  
Yeast Saccharomyces Cerevisiae ◽  
The Third ◽  
Direct Initiation ◽  
Different Types ◽  
Low Levels ◽  
Alpha Type

Functional TATA elements in the 5' untranslated region of the CYC1 gene in the yeast Saccharomyces cerevisiae have been defined by transcriptional analysis of site-directed mutations. Five sites previously suggested to contain functional TATA elements were altered individually and in all possible combinations. The results indicated that only two elements are required for transcription at the normal level and the normal start sites. The two functional TATA elements are located at sites -178 and -123, where the A of the ATG start codon is assigned nucleotide position +1. They direct initiation within windows encompassing -70 to -46 and -46 to -28, respectively. Only when both of the upstream TATA sites were rendered nonfunctional were the third and fourth downstream TATA-like sequences activated, as indicated by the presence of low levels of transcription starting at -28. The two upstream functional TATA elements differed in sequence. The sequence of the most 5' one at site 1, denoted beta-type, was ATATATATAT, whereas that of the second one at site 2, denoted alpha-type, was TATATAAAA. The following rearrangements of the beta-type and alpha-type elements at two sites (1 and 2) were examined: site1 beta-site2 alpha; site 1 alpha-site 2 beta; site1 alpha-site2 alpha; and site1 beta-site2 beta. When different types were at different sites (site1 beta-site2 alpha and site1 alpha-site2 beta), both were used equally. In contrast, when the same type was present at both sites (site1 alpha-site2 alpha and site1 beta-site2 beta), only the upstream element was used. We suggest that the two TATA elements are recognized by different factors of the transcription apparatus.

scholarly journals The HXT2 gene of Saccharomyces cerevisiae is required for high-affinity glucose transport.

1990 ◽  
Vol 10 (11) ◽  
pp. 5903-5913 ◽  
Author(s):  
A L Kruckeberg ◽  
L F Bisson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glucose Transport ◽  
Large Family ◽  
Transmembrane Domains ◽  
Growth Defect ◽  
Multicopy Plasmid ◽  
Sugar Transporter ◽  
Yeast Saccharomyces Cerevisiae ◽  
High Affinity ◽  
Transporter Family

The HXT2 gene of the yeast Saccharomyces cerevisiae was identified on the basis of its ability to complement the defect in glucose transport of a snf3 mutant when present on the multicopy plasmid pSC2. Analysis of the DNA sequence of HXT2 revealed an open reading frame of 541 codons, capable of encoding a protein of Mr 59,840. The predicted protein displayed high sequence and structural homology to a large family of procaryotic and eucaryotic sugar transporters. These proteins have 12 highly hydrophobic regions that could form transmembrane domains; the spacing of these putative transmembrane domains is also highly conserved. Several amino acid motifs characteristic of this sugar transporter family are also present in the HXT2 protein. An hxt2 null mutant strain lacked a significant component of high-affinity glucose transport when under derepressing (low-glucose) conditions. However, the hxt2 null mutation did not incur a major growth defect on glucose-containing media. Genetic and biochemical analyses suggest that wild-type levels of high-affinity glucose transport require the products of both the HXT2 and SNF3 genes; these genes are not linked. Low-stringency Southern blot analysis revealed a number of other sequences that cross-hybridize with HXT2, suggesting that S. cerevisiae possesses a large family of sugar transporter genes.

scholarly journals Phosphatase 2A Negatively Regulates Mitotic Exit in Saccharomyces cerevisiae

2006 ◽  
Vol 17 (1) ◽  
pp. 80-89 ◽  
Author(s):  
Yanchang Wang ◽  
Tuen-Yung Ng
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitotic Exit ◽  
Regulatory Subunit ◽  
Temperature Sensitive ◽  
Cyclin Dependent Kinase ◽  
Yeast Saccharomyces Cerevisiae ◽  
Catalytic Subunits ◽  
Phosphatase 2A ◽  
Negative Role

In budding yeast Saccharomyces cerevisiae, Cdc5 kinase is a component of mitotic exit network (MEN), which inactivates cyclin-dependent kinase (CDK) after chromosome segregation. cdc5-1 mutants arrest at telophase at the nonpermissive temperature due to the failure of CDK inactivation. To identify more negative regulators of MEN, we carried out a genetic screen for genes that are toxic to cdc5-1 mutants when overexpressed. Genes that encode the B-regulatory subunit (Cdc55) and the three catalytic subunits (Pph21, Pph22, and Pph3) of phosphatase 2A (PP2A) were isolated. In addition to cdc5-1, overexpression of CDC55, PPH21, or PPH22 is also toxic to other temperature-sensitive mutants that display defects in mitotic exit. Consistently, deletion of CDC55 partially suppresses the temperature sensitivity of these mutants. Moreover, in the presence of spindle damage, PP2A mutants display nuclear localized Cdc14, the key player in MEN pathway, indicative of MEN activation. All the evidence suggests the negative role of PP2A in mitotic exit. Finally, our genetic and biochemical data suggest that PP2A regulates the phosphorylation of Tem1, which acts at the very top of MEN pathway.

scholarly journals The HXT1 gene product of Saccharomyces cerevisiae is a new member of the family of hexose transporters.

1991 ◽  
Vol 11 (7) ◽  
pp. 3804-3813 ◽  
Author(s):  
D A Lewis ◽  
L F Bisson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glucose Transport ◽  
Significant Proportion ◽  
Growth Defect ◽  
Lacz Gene ◽  
Multicopy Plasmid ◽  
Hexose Transport ◽  
Yeast Saccharomyces Cerevisiae ◽  
High Affinity ◽  
Membrane Spanning

Two novel genes affecting hexose transport in the yeast Saccharomyces cerevisiae have been identified. The gene HXT1 (hexose transport), isolated from plasmid pSC7, was sequenced and found to encode a hydrophobic protein which is highly homologous to the large family of sugar transporter proteins from eucaryotes and procaryotes. Multicopy expression of the HXT1 gene restored high-affinity glucose transport to the snf3 mutant, which is deficient in a significant proportion of high-affinity glucose transport. HXT1 was unable to complement the snf3 growth defect in low copy number. The HXT1 protein was found to contain 12 putative membrane-spanning domains with a central hydrophilic domain and hydrophilic N- and C-terminal domains. The HXT1 protein is 69% identical to GAL2 and 66% identical to HXT2, and all three proteins were found to have a putative leucine zipper motif at a consensus location in membrane-spanning domain 2. Disruption of the HXT1 gene resulted in loss of a portion of high-affinity glucose and mannose transport, and wild-type levels of transport required both the HXT1 and SNF3 genes. Unexpectedly, expression of beta-galactosidase activity by using a fusion of the lacZ gene to the HXT1 promoter in a multicopy plasmid was maximal during lag and early exponential phases of growth, decreasing approximately 100-fold upon further entry into exponential growth. Deletion analysis of pSC7 revealed the presence of another gene (called ORF2) capable of suppressing the snf3 null mutant phenotype by restoring high-affinity glucose transport and increased low-affinity transport.

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