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PubMed1991

The cell-cycle-regulated budding yeast gene DBF2, encoding a putative protein kinase, has a homologue that is not under cell-cycle control.

The budding yeast cell-cycle gene, DBF2, encoding a putative protein kinase, was shown to have a homologue, designated DBF20. This gene was cloned, sequenced, and confirmed to be highly homologous to DBF2, with over 80% identity in the 490 most C-terminal amino acid residues. Either gene could be deleted by itself, but deletion of both genes simultaneously was lethal, indicating that they are redundant for at least one vital function in yeast. In contrast to the DBF2 mRNA, which is expressed under cell-cycle control at or near START [Johnston et al., Mol. Cell. Biol. 10 (1990) 1358-1366], the DBF20 mRNA is expressed at a low level and not under cell-cycle control. Assuming there is no translational control, the differential expression of the mRNAs would result in a cell-cycle fluctuation of the relative levels of the gene products, which may constitute a novel form of regulation.

Amino Acid Sequence↗
PubMed2000

Cdc14 activates cdc15 to promote mitotic exit in budding yeast.

Inactivation of mitotic cyclin-dependent kinases (Cdks) is required for cells to exit mitosis [1] [2]. In the budding yeast Saccharomyces cerevisiae, Cdk inactivation is triggered by the phosphatase Cdc14, which is activated by a complex network of regulatory proteins that includes the protein kinase Cdc15 [3] [4] [5] [6]. Here we show that the ability of Cdc15 to promote mitotic exit is inhibited by phosphorylation. Cdc15 is phosphorylated in vivo at multiple Cdk-consensus sites during most of the cell cycle, but is transiently dephosphorylated in late mitosis. Although phosphorylation appears to have no effect on Cdc15 kinase activity, a non-phosphorylatable mutant of Cdc15 is a more potent stimulator of mitotic exit than wild-type Cdc15, indicating that phosphorylation inhibits Cdc15 function in vivo. Interestingly, inhibitory phosphorylation of Cdc15 is removed by the phosphatase Cdc14 in vitro, and overproduction of Cdc14 leads to Cdc15 dephosphorylation in vivo. Thus, Cdc15 serves both as an activator and substrate of Cdc14. Although this scheme raises the possibility that positive feedback promotes Cdc14 activation, we present evidence that such feedback is not essential for Cdc14 activation in vivo. Instead, Cdc15 dephosphorylation may promote some additional function of Cdc15 that is independent of its effects on Cdc14 activation.

Cell Cycle↗
PubMed1999

Bifurcation of the mitotic checkpoint pathway in budding yeast.

The coordination of mitotic events is ensured through the spindle assembly checkpoint. BFA1 is required for this checkpoint in budding yeast because its disruption abolishes the mitotic arrest when spindle assembly is inhibited. Analysis of the genetic interaction of BFA1 with known mitotic checkpoint genes suggest that Bfa1 functions in the same pathway with Bub2 but not with Mad1 or Mad2. Both Bfa1 and Bub2 localize to spindle poles, and overexpression of Bfa1 arrests the cell cycle in anaphase. These findings suggest a bifurcation of the spindle assembly checkpoint: whereas one branch of the pathway, consisting of Mad1-3, Bub1 and 3, and Mps1, may prevent premature disjunction of sister chromosomes, the other, consisting of Bfa1 and Bub2, may function at spindle poles to prevent cytokinesis before the completion of chromosome segregation.

Cell Cycle Proteins↗
PubMed2001

Budding yeast GCN1 binds the GI domain to activate the eIF2alpha kinase GCN2.

When starved for a single amino acid, the budding yeast Saccharomyces cerevisiae activates the eukaryotic initiation factor 2alpha (eIF2alpha) kinase GCN2 in a GCN1-dependent manner. Phosphorylated eIF2alpha inhibits general translation but selectively derepresses the synthesis of the transcription factor GCN4, which leads to coordinated induction of genes involved in biosynthesis of various amino acids, a phenomenon called general control response. We recently demonstrated that this response requires binding of GCN1 to the GI domain occurring at the N terminus of GCN2 (Kubota, H., Sakaki, Y., and Ito, T. (2000) J. Biol. Chem. 275, 20243-20246). Here we provide the first evidence for the involvement of GCN1-GCN2 interaction in activation of GCN2 per se. We identified a C-terminal segment of GCN1 sufficient to bind the GI domain and used a novel dual bait two-hybrid method to identify mutations rendering GCN1 incapable of interacting with GCN2. The yeast bearing such an allele, gcn1-F2291L, fails to display derepression of GCN4 translation and hence general control response, as does a GI domain mutant, gcn2-Y74A, defective in association with GCN1. Furthermore, we demonstrated that phosphorylation of eIF2alpha is impaired in both mutants. Since GCN2 is the sole eIF2alpha kinase in yeast, these findings indicate a critical role of GCN1-GCN2 interaction in activation of the kinase in vivo.

Amino Acid Sequence↗
PubMed2003

Budding yeast PAK kinases regulate mitotic exit by two different mechanisms.

We report the characterization of the dominant-negative CLA4t allele of the budding yeast CLA4 gene, encoding a member of the p21-activated kinase (PAK) family of protein kinases, which, together with its homologue STE20, plays an essential role in promoting budding and cytokinesis. Overproduction of the Cla4t protein likely inhibits both endogenous Cla4 and Ste20 and causes a delay in the onset of anaphase that correlates with inactivation of Cdc20/anaphase-promoting complex (APC)-dependent proteolysis of both the cyclinB Clb2 and securin. Although the precise mechanism of APC inhibition by Cla4t remains to be elucidated, our results suggest that Cla4 and Ste20 may regulate the first wave of cyclinB proteolysis mediated by Cdc20/APC, which has been shown to be crucial for activation of the mitotic exit network (MEN). We show that the Cdk1-inhibitory kinase Swe1 is required for the Cla4t-dependent delay in cell cycle progression, suggesting that it might be required to prevent full Cdc20/APC and MEN activation. In addition, inhibition of PAK kinases by Cla4t prevents mitotic exit also by a Swe1-independent mechanism impinging directly on the MEN activator Tem1.

Anaphase↗
PubMed1993

A note on modelling the dynamics of budding yeast populations using branching processes.

A multitype branching process is proposed as a model for the behaviour of populations of the budding yeast Saccharomyces Cerevisiae. Using the idea of branching processes counted by random characteristics, we are able to obtain explicit expressions describing different aspects of the asymptotic composition of such populations. The main purpose of this note is to show that the branching process approach is an alternative to deterministic population models based on differential equation methods.

Mathematics↗
PubMed2006

The spatial arrangement of ORC binding modules determines the functionality of replication origins in budding yeast.

In the quest to define autonomously replicating sequences (ARSs) in eukaryotic cells, an ARS consensus sequence (ACS) has emerged for budding yeast. This ACS is recognized by the replication initiator, the origin recognition complex (ORC). However, not every match to the ACS constitutes a replication origin. Here, we investigated the requirements for ORC binding to origins that carry multiple, redundant ACSs, such as ARS603. Previous studies raised the possibility that these ACSs function as individual ORC binding sites. Detailed mutational analysis of the two ACSs in ARS603 revealed that they function in concert and give rise to an initiation pattern compatible with a single bipartite ORC binding site. Consistent with this notion, deletion of one base pair between the ACS matches abolished ORC binding at ARS603. Importantly, loss of ORC binding in vitro correlated with the loss of ARS activity in vivo. Our results argue that replication origins in yeast are in general comprised of bipartite ORC binding sites that cannot function in random alignment but must conform to a configuration that permits ORC binding. These requirements help to explain why only a limited number of ACS matches in the yeast genome qualify as ORC binding sites.

Base Sequence↗
PubMed1983

The cell cycle of the budding yeast Sterigmatomyces halophilus: culture fractionation by zonal centrifugation and the accumulation of DNA, RNA and protein.

Sterigmatomyces halophilus is an unusual budding yeast in which daughter cells are formed, remote from the mother cell, on fine projections called sterigmata. Some fundamental properties of the cell cycle have been explored by separating cells from an exponentially growing culture into size, and thus age, classes by density-gradient centrifugation. Rate separations on high capacity, high resolution, equivolumetric gradients of sucrose, or, alternatively, isopycnic separations on gradients of Urografin revealed consistent and reproducible patterns of accumulation of DNA, RNA and protein through the cell cycle. Total DNA accumulation was stepwise, synthesis occurring late in the cycle, whilst protein accumulated continuously with no evidence for the discontinuities reported in some other lower eukaryotes. Total RNA accumulation, measured either colorimetrically or by long-term incorporation of radioactively-labelled uracil was transiently elevated early in the cycle and then accumulated continuously. A mathematical analysis of the volume distributions of the cells in fractions from the gradients showed that there is a hyperbolic relationship between cell age and size but that, to a first approximation, measurements of cell size (and density) are direct measures of age. The results are discussed with reference to (1) the unusually high buoyant density of this yeast, (2) the resolution of zonal cell separation methods and (3) macromolecular accumulation in the cell cycles of other eukaryotic micro-organisms.

Cell Cycle↗
PubMed1992

Active genes in budding yeast display enhanced in vivo accessibility to foreign DNA methylases: a novel in vivo probe for chromatin structure of yeast.

Unlike higher eukaryotes, where an inverse correlation has been generally observed between gene expression and methylation of CpG sites, the budding yeast Saccharomyces cerevisiae lacks DNA methylation. Gene regulatory mechanisms can function independently of DNA methylation in yeast, and yeast strains expressing foreign DNA methylases that modify adenine and CpG residues have been found to be viable. We have used such strains to determine whether the transcriptional status of genes can influence the level of their DNA methylation in vivo. Several genes were tested, for example, GAL1, -7, and -10, PHO5, HMRa and HML alpha, and STE2 and STE3. Surprisingly, we found that all the genes displayed severalfold more methylation in the expressed state as compared to the repressed state. This procedure serves as a novel in vivo probe for the chromatin structure of yeast and potentially for higher eukaryotes.

Blotting, Southern↗
PubMed1999

Altered states: programmed proteolysis and the budding yeast cell cycle.

The recent identification of an essential RING-H2 finger protein in the SCF E3 ubiquitin ligase complex of budding yeast has uncovered a family of related E3 enzymes, including the other main cell cycle E3 complex, the anaphase promoting complex (APC). Recent insights into APC-dependent proteolysis include a novel protease activity that dissolves cohesion between sister chromatids at anaphase, and a crucial phosphatase, Cdc14, whose release from the nucleolus eliminates cyclin-dependent kinase activity and thereby drives exit from mitosis.

Anaphase-Promoting Complex-Cyclosome↗
PubMed1998

Activated transcription independent of the RNA polymerase II holoenzyme in budding yeast.

We investigated whether the multisubunit holoenzyme complex of RNA polymerase II (Pol II) and mediator is universally required for transcription in budding yeast. DeltaCTD Pol II lacking the carboxy-terminal domain of the large subunit cannot assemble with mediator but can still transcribe the CUP1 gene. CUP1 transcripts made by DeltaCTD Pol II initiated correctly and some extended past the normal poly(A) site yielding a novel dicistronic mRNA. Most CUP1 transcripts made by DeltaCTD Pol II were degraded but could be stabilized by deletion of the XRN1 gene. Unlike other genes, transcription of CUP1 and HSP82 also persisted after inactivation of the CTD kinase Kin28 or the mediator subunit Srb4. The upstream-activating sequence (UAS) of the CUP1 promoter was sufficient to drive Cu2+ inducible transcription without Srb4 and heat shock inducible transcription without the CTD. We conclude that the Pol II holoenzyme is not essential for all UAS-dependent activated transcription in yeast.

Carrier Proteins↗
PubMed1993

Evidence for two cell division cycle (CDC) genes that govern yeast bud emergence in the pathogenic fungus Wangiella dermatitidis.

Strains Mc2 and Mc3 are morphological mutants of the melanized, pathogenic fungus Wangiella dermatitidis. These strains possess temperature-sensitive (ts) mutations designated mcm2 and mcm3, respectively. At the restrictive temperature (37 degrees C), uninucleate yeast cells of strains Mc2 and Mc3 cease budding and initiate an isotropic mode of cellular development, which is reflected in the formation of a multicellular and multinucleate morphology. Because W. dermatitidis either lacks or has an undiscovered sexual cycle, parasexual methods of analysis were used to confirm that mcm2 and mcm3 define separate bud emergence control genes in the wild-type strain. Spheroplasts of albino auxotrophs derived from strains Mc2 and Mc3 were fused and then regenerated on minimal medium. The resulting fusion products grew as darkly pigmented, prototrophic colonies. When incubated at 37 degrees C, all fusion products exhibited polarized growth predominantly as uninucleate, budding yeasts and less frequently as pseudohyphae and moniliform hyphae. Subsequent analysis of cultures derived from albino, ts segregants, which were induced from fusion products by using methyl benzimidazole-2-yl-carbamate, revealed three types of cell populations. Two resembled those expressed by strain Mc2 or Mc3. The third consisted of a cell population unlike the former, suggesting the presence of both ts mutations in all cells. These results imply that yeast development in the fusion products resulted from intergenic complementation of mcm2 and mcm3, i.e., they are nonallelic. Because mcm2 and mcm3 are equivalent to certain cdc lesions in the yeast Saccharomyces cerevisiae, we have renamed the analogous genes defined by the mutations in W. dermatitidis as CDC1 and CDC2. To our knowledge, these are the first CDC genes identified in a dematiaceous fungus.

Alleles↗
PubMed2003

The fission yeast spo14+ gene encoding a functional homologue of budding yeast Sec12 is required for the development of forespore membranes.

The Schizosaccharomyces pombe spo14-B221 mutant was originally isolated as a sporulation-deficient mutant. However, the spo14(+) gene is essential for cell viability and growth. spo14(+) is identical to the previously characterized stl1(+) gene encoding a putative homologue of Saccharomyces cerevisiae Sec12, which is essential for protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus. In the spo14 mutant cells, ER-like membranes were accumulated beneath the plasma membrane and the ER/Golgi shuttling protein Rer1 remained in the ER. Sec12 is a guanine nucleotide exchange factor for the Sar1 GTPase. Overproduction of psr1(+) coding for an S. pombe Sar1 homologue suppressed both the sporulation defect of spo14-B221 and cold-sensitive growth of newly isolated spo14-6 and spo14-7 mutants. These results indicate that Spo14 is involved in early steps of the protein secretory pathway. The spo14-B221 allele carries a single nucleotide change in the branch point consensus of the fifth intron, which reduces the abundance of the spo14 mRNA. During meiosis II, the forespore membrane was initiated near spindle pole bodies; however, subsequent extension of the membrane was arrested before its closure into a sac. We conclude that Spo14 is responsible for the assembly of the forespore membrane by supplying membrane vesicles.

Amino Acid Sequence↗
PubMed2003

Genetic evidence for a role of phospholipase C at the budding yeast kinetochore.

Chromosome segregation during mitosis requires kinetochores, specialized organelles that mediate chromosome attachment to spindle microtubules. We have shown previously that in budding yeast, Plc1p (phosphoinositide-specific phospholipase C) localizes to centromeric loci, associates with the kinetochore proteins Ndc10p and Cep3p, and affects the function of kinetochores. Deletion of PLC1 results in nocodazole sensitivity, mitotic delay, and a higher frequency of chromosome loss. We report here that despite the nocodazole sensitivity of plc1Delta cells, Plc1p is not required for the spindle checkpoint. However, plc1Delta cells require a functional BUB1/BUB3-dependent spindle checkpoint for viability. PLC1 displays strong genetic interactions with genes encoding components of the inner kinetochore, including NDC10, SKP1, MIF2, CEP1, CEP3, and CTF13. Furthermore, plc1Delta cells display alterations in chromatin structure in the core centromere. Chromatin immunoprecipitation experiments indicate that Plc1p localizes to centromeric loci independently of microtubules, and accumulates at the centromeres during G(2)/M stage of cell cycle. These results are consistent with the view that Plc1p affects kinetochore function, possibly by modulating the structure of centromeric chromatin.

Cell Nucleus↗
PubMed2006

Morphologically-structured models of growing budding yeast populations.

It has been well recognized that many key aspects of cell cycle regulation are encoded into the size distributions of growing budding yeast populations due to the tight coupling between cell growth and cell division present in this organism. Several attempts have been made to model the cell size distribution of growing yeast populations in order to obtain insight on the underlying control mechanisms, but most were based on the age structure of asymmetrically dividing populations. Here we propose a new framework that couples a morphologically-structured representation of the population with population balance theory to formulate a dynamic model for the size distribution of growing yeast populations. An advantage of the presented framework is that it allows derivation of simpler models that are directly identifiable from experiments. We show how such models can be derived from the general framework and demonstrate their utility in analyzing yeast population data. Finally, by employing a recently proposed numerical scheme, we proceed to integrate numerically the full distributed model to provide predictions of dynamics of the cell size structure of growing yeast populations.

Cell Cycle↗
PubMed2002

Hsp90 enables Ctf13p/Skp1p to nucleate the budding yeast kinetochore.

Binding of CBF3, a protein complex consisting of Ndc10p, Cep3p, Ctf13p, and Skp1p, to the centromere DNA nucleates kinetochore formation in budding yeast. Here, we investigate how the Ctf13p/Skp1p complex becomes competent to form the CBF3-centromere DNA complex. As revealed by mass spectrometry, Ctf13p and Skp1p carry two and four phosphate groups, respectively. Complete dephosphorylation of Ctf13p and Skp1p does not interfere with the formation of CBF3-centromere DNA complexes in vitro. Furthermore, deletion of corresponding phosphorylation sites results in viable cells. Thus, in contrast to the current view, phosphorylation of Ctf13p and Skp1p is not essential for the formation of CBF3-centromere DNA complexes. Instead, the formation of active Ctf13p/Skp1p requires Hsp90. Several lines of evidence support this conclusion: activation of heterologous Ctf13p/Skp1p by reticulocyte lysate is inhibited by geldanamycin and Hsp90 depletion. skp1 mutants exhibit growth defects on media containing geldanamycin. A skp1 mutation together with Hsp90 mutations exhibits synthetic lethality. An Hsp90 mutant contains decreased levels of active Ctf13p/Skp1p.

Amino Acid Sequence↗
PubMed2004

The budding yeast mei5 and sae3 proteins act together with dmc1 during meiotic recombination.

Here we provide evidence that the Mei5 and Sae3 proteins of budding yeast act together with Dmc1, a meiosis-specific, RecA-like recombinase. The mei5 and sae3 mutations reduce sporulation, spore viability, and crossing over to the same extent as dmc1. In all three mutants, these defects are largely suppressed by overproduction of Rad51. In addition, mei5 and sae3, like dmc1, suppress the cell-cycle arrest phenotype of the hop2 mutant. The Mei5, Sae3, and Dmc1 proteins colocalize to foci on meiotic chromosomes, and their localization is mutually dependent. The localization of Rad51 to chromosomes is not affected in either mei5 or sae3. Taken together, these observations suggest that the Mei5 and Sae3 proteins are accessory factors specific to Dmc1. We speculate that Mei5 and Sae3 are necessary for efficient formation of Dmc1-containing nucleoprotein filaments in vivo.

Amino Acid Sequence↗
PubMed1994

A prenylation motif is required for plasma membrane localization and biochemical function of casein kinase I in budding yeast.

The subcellular distribution of three casein kinase I (CK1) homologs, encoded by the YCK1, YCK2, and HRR25 genes, has been determined in budding yeast through a combination of subcellular fractionation and immunofluorescence methods. Both Yck proteins are tightly associated with the plasma membrane or underlying cytoskeleton and require both high-salt and nonionic detergent for extraction. Association is mediated primarily by the prenylation motif found at the C terminus of both Yck proteins. In contrast, the third CK1 homolog, Hrr25p, is found predominantly in the nucleus and only partially in the plasma membrane. Despite partial colocalization with the Yck proteins, Hrr25p is unable to rescue the yck1 delta yck2 delta phenotype. However, a chimeric kinase containing the N-terminal kinase domain of Hrr25p and the C-terminal region of Yck2p contains full Yck activity in vivo. These data suggest that members of the casein kinase I family have distinct but partially overlapping distributions in the cell that are mediated by their unique C-terminal regions.

Base Sequence↗