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PubMed1990

An inducible expression vector for both fission and budding yeast.

We have developed a vector system for inducible gene expression in both fission yeast (Schizosaccharomyces pombe) and budding yeast (Saccharomyces cerevisiae). The autonomously replicating expression vector contains multiple glucocorticoid response elements, rendering a linked promoter inducible 20-70-fold by glucocorticoid hormones in the presence of the mammalian glucocorticoid receptor. A polylinker with several unique cloning sites allows insertion of cDNAs of interest. Glucocorticoids are gratuitous signalling molecules in yeast, exerting little or no effect on the expression of genes other than those fused to the regulated promoter.

Base Sequence↗
PubMed1997

Centromere position in budding yeast: evidence for anaphase A.

Although general features of chromosome movement during the cell cycle are conserved among all eukaryotic cells, particular aspects vary between organisms. Understanding the basis for these variations should provide significant insight into the mechanism of chromosome movement. In this context, establishing the types of chromosome movement in the budding yeast Saccharomyces cerevisiae is important since the complexes that mediate chromosome movement (microtubule organizing centers, spindles, and kinetochores) appear much simpler in this organism than in many other eukaryotic cells. We have used fluorescence in situ hybridization to begin an analysis of chromosome movement in budding yeast. Our results demonstrate that the position of yeast centromeres changes as a function of the cell cycle in a manner similar to other eukaryotes. Centromeres are skewed to the side of the nucleus containing the spindle pole in G1; away from the poles in mid-M and clustered near the poles in anaphase and telophase. The change in position of the centromeres relative to the spindle poles supports the existence of anaphase A in budding yeast. In addition, an anaphase A-like activity independent of anaphase B was demonstrated by following the change in centromere position in telophase-arrested cells upon depolymerization and subsequent repolymerization of microtubules. The roles of anaphase A activity and G1 centromere positioning in the segregation of budding yeast chromosomes are discussed. The fluorescence in situ hybridization methodology and experimental strategies described in this study provide powerful new tools to analyze mutants defective in specific kinesin-like molecules, spindle components, and centromere factors, thereby elucidating the mechanism of chromosome movement.

Anaphase↗
PubMed1999

Regulation of G2/M progression by the STE mitogen-activated protein kinase pathway in budding yeast filamentous growth.

Inoculation of diploid budding yeast onto nitrogen-poor agar media stimulates a MAPK pathway to promote filamentous growth. Characteristics of filamentous cells include a specific pattern of gene expression, elongated cell shape, polar budding pattern, persistent attachment to the mother cell, and a distinct cell cycle characterized by cell size control at G2/M. Although a requirement for MAPK signaling in filamentous gene expression is well established, the role of this pathway in the regulation of morphogenesis and the cell cycle remains obscure. We find that ectopic activation of the MAPK signal pathway induces a cell cycle shift to G2/M coordinately with other changes characteristic of filamentous growth. These effects are abrogated by overexpression of the yeast mitotic cyclins Clb1 and Clb2. In turn, yeast deficient for Clb2 or carrying cdc28-1N, an allele of CDK defective for mitotic functions, display enhanced filamentous differentiation and supersensitivity to the MAPK signal. Importantly, activation of Swe1-mediated inhibitory phosphorylation of Thr-18 and/or Tyr-19 of Cdc28 is not required for the MAPK pathway to affect the G2/M delay. Mutants expressing a nonphosphorylatable mutant Cdc28 or deficient for Swe1 exhibit low-nitrogen-dependent filamentous growth and are further induced by an ectopic MAPK signal. We infer that the MAPK pathway promotes filamentous growth by a novel mechanism that inhibits mitotic cyclin/CDK complexes and thereby modulates cell shape, budding pattern, and cell-cell connections.

Cell Cycle↗
PubMed2001

Assembly and regulation of the cytokinetic apparatus in budding yeast.

The identification of an actomyosin-based contractile ring in budding yeast has recently established this organism as a general model for studying cytokinesis. Work over the past three years has provided important new insights into the conserved mechanisms underlying the assembly and regulation of the cytokinetic structures. This review covers the recent progress in studying cytokinesis in budding yeast.

Actomyosin↗
PubMed2003

Spatial and temporal dynamics of budding yeast mitochondria lacking the division component Fis1p.

The mitochondrial compartment of budding yeast (Saccharomyces cerevisiae) is a highly dynamic net-like structure of tubules that constantly undergo fusion and fission. The outer membrane protein Fis1p plays a crucial role in mitochondrial fission. Here we report on the temporal and spatial dynamics of this organelle in wild-type cells and in fis1Delta mutants. Mitochondria of fis1Delta mutants adapt their mitochondrial network to a change in carbon source. We find that the frequencies of apparent matrix separation and fusion events decrease in both wild-type cells and in mutants lacking Fis1p upon glucose repression. Matrix separation could be caused by matrix constriction and does not necessarily require fission of the inner or outer membrane. Double-labelling experiments demonstrated that some of these matrix separations in fis1 mutants are due to genuine tubule fissions, whereas others do not involve fission of the outer membrane. The rates of matrix separation in fis1Delta mutants almost approach those of the wildtype, demonstrating that, although apparently involved in outer membrane fission, Fis1p is not crucial for the separation of the mitochondrial matrix. In mutants lacking the GTPase Dnm1p no complete tubule fissions were recorded, although dnm1Delta mutants display matrix separations as well. The data suggest that different molecular machineries are responsible for the separation of the matrix and the fission of the outer membrane in budding yeast.

Carbon↗
PubMed2001

Time course analysis of precocious separation of sister centromeres in budding yeast: continuously separated or frequently reassociated?

BACKGROUND: Sister kinetochores are bioriented toward the spindle poles in eukaryotic metaphase before chromosome segregation. In the budding yeast Saccharomyces cerevisiae, sister centromeres/kinetochores are separated in the early spindle, while the sister arms remain associated. Biorientation is thought to be established in this organism with precocious separation of sister centromeres in early stages of the cell cycle. It is not, however, settled whether this pre-anaphase separation is continuous or only transient and whether the transient separation has any physiological significance. RESULTS: Time-lapse observation of the behaviour of budding yeast centromeres in living cells was performed using GFP alone or in combination with CFP marking. Sixty-three per cent of the cell population showed permanent separation of centromeres for a long period of time from the small-budded stage to the onset of anaphase in the single-colour GFP-CEN construct. The remaining cell population (6 of 16) showed brief apparent reassociation of centromere signals before anaphase, but the frequency of the association was very low. In a time-lapse observation of the double-colour marked cells by GFP-CEN and CFP-SPB (the spindle pole body), the continuous separation of sister centromeres in the short medial spindle was firmly established. CONCLUSIONS: In the budding yeast, once sister centromeres separate, they rarely reassociate in pre-anaphase. Sister centromere cohesion at this stage appears to be irrelevant for normal chromosome segregation. Whether abundant cohesin in the centromere regions has any role in anaphase remains to be determined.

Anaphase↗
PubMed1996

Bud10p directs axial cell polarization in budding yeast and resembles a transmembrane receptor.

BACKGROUND: The budding yeast Saccharomyces cerevisiae can bud in two spatially programmed patterns: axial or bipolar. In the axial budding pattern, cells polarize and divide adjacent to the previous site of cell separation, in response to a cell-division remnant, which includes Bud3p, Bud4p and septin proteins. This paper investigates the role of an additional component of the cell-division remnant, Bud10p, in axial budding. RESULTS: The sequence of Bud10p predicts a protein that contains a single trans-membrane domain but lacks similarity to known proteins. Subcellular fractionations confirm that Bud10p is associated with membranes. Bud10p accumulates as a patch at the bud site prior to bud formation, and then persists at the mother-bud neck as the bud grows. Towards the end of the cell cycle, the localization of Bud10p refines to a tight double ring which splits at cytokinesis into two single rings, one in each progeny cell. Each single ring remains until a new concentration of Bud10p forms at the developing axial bud site, immediately adjacent to the old ring. Certain aspects of Bud10p localization are dependent upon BUD3, suggesting a close functional interaction between Bud10p and Bud3p. CONCLUSIONS: Bud10p is the first example of a transmembrane protein that controls cell polarization during budding. Because Bud10p contains a large extracellular domain, it is possible that Bud10p functions in a manner analogous to an extracellular matrix receptor. Clusters of Bud10p at the mother-bud neck formed in response to Bud3p (and possibly to an extracellular cue, such as a component of the cell wall), might facilitate the docking of downstream components that direct polarization of the cytoskeleton.

Amino Acid Sequence↗
PubMed1991

Homologous mRNA 3' end formation in fission and budding yeast.

Sequences resembling polyadenylation signals of higher eukaryotes are present downstream of the Schizosaccharomyces pombe ura4+ and cdc10+ coding regions and function in HeLa cells. However, these and other mammalian polyadenylation signals are inactive in S. pombe. Instead, we find that polyadenylation signals of the CYC1 gene of budding yeast Saccharomyces cerevisiae function accurately and efficiently in fission yeast. Furthermore, a 38 bp deletion which renders this RNA processing signal non-functional in S. cerevisiae has the equivalent effect in S. pombe. We demonstrate that synthetic pre-mRNAs encoding polyadenylation sites of S. pombe genes are accurately cleaved and polyadenylated in whole cell extracts of S. cerevisiae. Finally, as is the case in S. cerevisiae, DNA sequences encoding regions proximal to the S. pombe mRNA 3' ends are found to be extremely AT rich; however, no general sequence motif can be found. We conclude that although fission yeast has many genetic features in common with higher eukaryotes, mRNA 3' end formation is significantly different and appears to be formed by an RNA processing mechanism homologous to that of budding yeast. Since fission and budding yeast are evolutionarily divergent, this lower eukaryotic mechanism of mRNA 3' end formation may be generally conserved.

Base Sequence↗
PubMed1995

The CLN gene family: central regulators of cell cycle Start in budding yeast.

The Start transition in the budding yeast cell cycle is the point of most physiological regulation of cell cycle commitment. This transition is controlled by the CLN1,2,3 gene family. We review what is known about the regulation, inter-regulation and function of these genes in controlling the Start transition.

Cell Cycle↗
PubMed2006

Ploidy controls the success of mutators and nature of mutations during budding yeast evolution.

BACKGROUND: We used the budding yeast Saccharomyces cerevisiae to ask how elevated mutation rates affect the evolution of asexual eukaryotic populations. Mismatch repair defective and nonmutator strains were competed during adaptation to four laboratory environments (rich medium, low glucose, high salt, and a nonfermentable carbon source). RESULTS: In diploids, mutators have an advantage over nonmutators in all conditions, and mutators that win competitions are on average fitter than nonmutator winners. In contrast, haploid mutators have no advantage when competed against haploid nonmutators, and haploid mutator winners are less fit than nonmutator winners. The diploid mutator winners were all superior to their ancestors both in the condition they had adapted to, and in two of the other conditions. This phenotype was due to a mutation or class of mutations that confers a large growth advantage during the respiratory phase of yeast cultures that precedes stationary phase. This generalist mutation(s) was not selected in diploid nonmutator strains or in haploid strains, which adapt primarily by fixing specialist (condition-specific) mutations. In diploid mutators, such mutations also occur, and the majority accumulates after the fixation of the generalist mutation. CONCLUSIONS: We conclude that the advantage of mutators depends on ploidy and that diploid mutators can give rise to beneficial mutations that are inaccessible to nonmutators and haploid mutators.

Adaptation, Physiological↗
PubMed2000

Conservation of eukaryotic sterol homeostasis: new insights from studies in budding yeast.

The model eukaryote Saccharomyces cerevisiae (budding yeast) has provided significant insight into sterol homeostasis. The study of sterol metabolism in a genetically amenable model organism such as yeast is likely to have an even greater impact and relevance to human disease with the advent of the complete human genome sequence. In addition to definition of the sterol biosynthetic pathway, almost to completion, the remarkable conservation of other components of sterol homeostasis are described in this review.

Animals↗
PubMed1990

Regulation of proliferation by the budding yeast Saccharomyces cerevisiae.

Mutations in the budding yeast Saccharomyces cerevisiae define regulatory activities both for the mitotic cell cycle and for resumption of proliferation from the quiescent stationary-phase state. In each case, the regulation of proliferation occurs in the prereplicative interval that precedes the initiation of DNA replication. This regulation is particularly responsive to the nutrient environment and the biosynthetic capacity of the cell. Mutations in components of the cAMP-mediated effector pathway and in components of the biosynthetic machinery itself affect regulation of proliferation within the mitotic cell cycle. In the extreme case of nutrient starvation, cells cease proliferation and enter stationary phase. Mutations in newly defined genes prevent stationary-phase cells from reentering the mitotic cell cycle, but have no effect on proliferating cells. Thus stationary phase represents a unique developmental state, with requirements to resume proliferation that differ from those for the maintenance of proliferation in the mitotic cell cycle.

Cell Cycle↗
PubMed2005

Crm1-mediated nuclear export of Cdc14 is required for the completion of cytokinesis in budding yeast.

The mitotic exit network (MEN) controls the exit from mitosis in budding yeast. The proline-directed phosphatase, Cdc14p, is a key component of MEN and promotes mitotic exit by activating the degradation of Clb2p and by reversing Cdk-mediated mitotic phosphorylation. Cdc14p is sequestered in the nucleolus during much of the cell cycle and is released in anaphase from the nucleolus to the nucleoplasm and cytoplasm to perform its functions. Release of Cdc14p from the nucleolus during anaphase is well understood. In contrast, less is known about the mechanism by which Cdc14p is released from the nucleus to the cytoplasm. Here we show that Cdc14p contains a leucine-rich nuclear export signal (NES) that interacts with Crm1p physically. Mutations in the NES of Cdc14p allow Clb2p degradation and mitotic exit, but cause abnormal morphology and cytokinesis defects at non-permissive temperatures. Cdc14p localizes to the bud neck, among other cytoplasmic structures, following its release from the nucleolus in late anaphase. This bud neck localization of Cdc14p is disrupted by mutations in its NES and by the leptomycin B-mediated inhibition of Crm1p. Our results suggest a requirement for Crm1p-dependent nuclear export of Cdc14p in coordinating mitotic exit and cytokinesis in budding yeast.

Active Transport, Cell Nucleus↗
PubMed1995

A yeast transcription factor bypassing the requirement for SBF and DSC1/MBF in budding yeast has homology to bacterial signal transduction proteins.

The transcription factors SBF and DSC1/MBF bind SCB and MCB promoter elements, respectively, and are essential for the cell cycle progression of Saccharomyces cerevisiae through the control of G1 cyclin gene expression. We isolated a gene (BRY1; Bacterial Response regulator in Yeast) able to activate either MCB or SCB promoter elements on a reporter plasmid which, when overexpressed, can bypass the normally essential requirement for SBF and DSC1/MBF by the stimulation of CLN1 and CLN2 expression. In the case of CLN2 at least, this expression depends upon the MCB and SCB promoter elements. In wild-type yeast, the disruption of BRY1 has no apparent phenotype, but under conditions where the activities of SBF and DSC1/MBF are reduced, BRY1 becomes essential. Our data imply the existence of a third pathway affecting cyclin expression. BRY1 is the same gene as SKN7 which has significant sequence homology to the receiver domains found in response regulator proteins from the bacterial two-component signal transduction pathways. SKN7 is thought to affect cell wall structure, and when highly overexpressed we find that BRY1/SKN7 is lethal perhaps because of perturbations in cell wall biosynthesis. The lethality is partially rescued by genes from the protein kinase C pathway, but genetic data imply that BRY1/SKN7 and protein kinase C are not in the same pathway. Our results suggest that Bry1/Skn7 can influence the expression of MCB- and SCB-driven gene expression in budding yeast, perhaps including genes involved in cell wall metabolism, via a two-component signal transduction pathway which activates Bry1/Skn7 in response to an unidentified signal.

Cyclins↗
PubMed2006

The budding yeast endocytic pathway.

Recent live-cell imaging studies, coupled with powerful genetic, biochemical and pharmacological tests of function, have expanded our understanding of the molecular events that underlie clathrin/actin mediated-endocytosis in budding yeast. Many features of this pathway are evolutionarily conserved (Engqvist-Goldstein and Drubin, 2003; Kaksonen et al, 2006). Therefore, insights into the intricate molecular choreography of endocytic events in budding yeast will provide a basis for elucidating such mechanisms in more complex organisms. This poster depicts our current understanding of the dynamics of endocytosis in budding yeast.

Actins↗
PubMed2000

A myosin light chain mediates the localization of the budding yeast IQGAP-like protein during contractile ring formation.

Cytokinesis in animal cells is accomplished through constriction of an actomyosin ring [1] [2] [3], which must assemble at the correct time and place in order to ensure proper division of genetic material and organelles. Budding yeast is a useful model system for determining the biochemical pathway of contractile ring assembly. The budding yeast IQGAP-like protein, Cyk1/Iqg1p, has multiple roles in the assembly and contraction of the actomyosin ring [4] [5] [6]. Previously, the IQ motifs of Cyk1/Iqg1p were shown to be required for the localization of this protein at the bud neck [6]. We have investigated the binding partner of the IQ motifs, which are predicted to interact with calmodulin-like proteins. Mlc1p was originally identified as a light chain for a type V myosin, Myo2p; however, a cytokinesis defect associated with disruption of the MLC1 gene suggested that the essential function of Mlc1p may involve interactions with other proteins [7]. We show that Mlc1p binds the IQ motifs of Cyk1/Iqg1p and present evidence that this interaction recruits Cyk1/Iqg1p to the bud neck. Immunofluorescence staining shows that Mlc1p is localized to sites of polarized cell growth as well as the bud neck before and independently of Cyk1p. These results demonstrate that Mlc1p is important for the assembly of the actomyosin ring in budding yeast and that this function is mediated through interaction with Cyk1/Iqg1p.

Fungal Proteins↗
PubMed1999

Functional conservation and cell cycle localization of the Nhp2 core component of H + ACA snoRNPs in fission and budding yeasts.

We report the identification of a novel nucleolar protein from fission yeast, p17(nhp2), which is homologous to the recently identified Nhp2p core component of H+ACA snoRNPs in Saccharomyces cerevisiae. We show that the fission yeast p17(nhp2) localizes to the nucleolus in live S. cerevisiae or Schizosaccharomyces pombe cells and is functionally conserved since the fission yeast gene can complement a deletion of the NHP2 gene in budding yeast. Analysis of p17(nhp2) during the mitotic cell cycles of living fission and budding yeast cells shows that this protein, and by implication H+ACA snoRNPs, remains localized with nucleolar material during mitosis, although the gross organization of partitioning of p17(nhp2) during anaphase is different in a comparison of the two yeasts. During anaphase in S. pombe p17(nhp2) trails segregating chromatin, while in S. cerevisiae the protein segregates alongside bulk chromatin. The pattern of segregation comparing haploid and diploid S. cerevisiae cells suggests that p17(nhp2) is closely associated with the rDNA during nuclear division.

Amino Acid Sequence↗
PubMed2003

Conservation of mechanisms controlling entry into mitosis: budding yeast wee1 delays entry into mitosis and is required for cell size control.

BACKGROUND: In fission yeast, the Wee1 kinase delays entry into mitosis until a critical cell size has been reached; however, a similar role for Wee1-related kinases has not been reported in other organisms. SWE1, the budding yeast homolog of wee1, is thought to function in a morphogenesis checkpoint that delays entry into mitosis in response to defects in bud morphogenesis. RESULTS: In contrast to previous studies, we found that budding yeast swe1 Delta cells undergo premature entry into mitosis, leading to birth of abnormally small cells. Additional experiments suggest that conditions that activate the morphogenesis checkpoint may actually be activating a G2/M cell size checkpoint. For example, actin depolymerization is thought to activate the morphogenesis checkpoint by inhibiting bud morphogenesis. However, actin depolymerization also inhibits bud growth, suggesting that it could activate a cell size checkpoint. Consistent with this possibility, we found that actin depolymerization fails to induce a G2/M delay once daughter buds pass a critical size. Other conditions that activate the morphogenesis checkpoint block bud formation, which could also activate a size checkpoint if cell size at G2/M is monitored in the daughter bud. Previous work reported that Swe1 is degraded during G2, which was proposed to account for failure of large-budded cells to arrest in response to actin depolymerization. However, we found that Swe1 is present throughout G2 and undergoes hyperphosphorylation as cells enter mitosis, as found in other organisms. CONCLUSIONS: Our results suggest that the mechanisms known to coordinate entry into mitosis in other organisms have been conserved in budding yeast.

Blotting, Western↗