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PubMed2004

Predicting 22 protein localizations in budding yeast.

According to the recent experiments, proteins in budding yeast can be distinctly classified into 22 subcellular locations. Of these proteins, some bear the multi-locational feature, i.e., occur in more than one location. However, so far all the existing methods in predicting protein subcellular location were developed to deal with only the mono-locational case where a query protein is assumed to belong to one, and only one, subcellular location. To stimulate the development of subcellular location prediction, an augmentation procedure is formulated that will enable the existing methods to tackle the multi-locational problem as well. It has been observed thru a jackknife cross-validation test that the success rate obtained by the augmented GO-FnD-PseAA algorithm [BBRC 320 (2004) 1236] is overwhelmingly higher than those by the other augmented methods. It is anticipated that the augmented GO-FunD-PseAA predictor will become a very useful tool in predicting protein subcellular localization for both basic research and practical application.

Algorithms↗
PubMed2003

Mek1 kinase activity functions downstream of RED1 in the regulation of meiotic double strand break repair in budding yeast.

Three meiosis-specific chromosomal components in budding yeast, Mek1, Red1, and Hop1, are required for recombination, proper segregation of homologs, and the meiotic recombination checkpoint. Mek1 is a protein kinase. Mutations that increase the size of the ATP binding pocket of Mek1 (mek1-as1) sensitize the kinase to specific small molecule inhibitors. Experiments using mek1-as1 demonstrate that the requirement for Mek1 kinase activity coincides with the formation of double strand breaks (DSBs) and that this activity is necessary after DSB formation to prevent repair by DMC1-independent pathways. Contrary to previous reports, Red1 is not a substrate for Mek1. Instead, RED1 is required for wild-type levels of Mek1 kinase activity. In addition, activation of Mek1 requires HOP1, the formation of Red1/Hop1 complexes and a functional Mek1 FHA domain. The requirement for RED1 to produce active kinase can be bypassed by a mek1 mutation that creates a constitutively active Mek1 kinase. We propose that Red1 is phosphorylated by a kinase other than MEK1 and that phosphothreonines on Red1 then interact with the Mek1 FHA domain to recruit the kinase to sites of DSBs where Mek1 is activated to prevent DMC1-independent DSB repair.

Adenosine Triphosphate↗
PubMed1996

At the heart of the budding yeast cell cycle.

It might now seem obvious that the mechanisms regulating cell division would be found to be a highly conserved feature of eukaryotic cells. This was less clear 20 years ago when the pioneering genetic studies of the cell cycle were initiated. This article presents one view as to what lies at the heart of the budding yeast cell cycle. It is written on the premise that most of the key players, such as cyclin-dependent kinases, the anaphase-promoting complex, the origin recognition complex, Cdc6p and Mcm proteins, were performing similar functions in the common ancestor of yeast and man. Ideas about the budding yeast cell cycle might, therefore, have universal significance for other eukaryotic cells.

Anaphase↗
PubMed1997

Small heat shock protein suppression of Vpr-induced cytoskeletal defects in budding yeast.

Expression of the auxiliary human immunodeficiency virus type 1 (HIV-1) protein Vpr causes arrest of primate host cells in G2. Expression of this protein in budding yeast has been previously reported to cause growth arrest and a large-cell phenotype. Investigation of the effect of Vpr expression in budding yeast, reported here, showed that it causes disruption of the actin cytoskeleton. Expression of HSP42, the gene for a small heat shock protein (sHSP), from a high-copy-number plasmid reversed this effect. The sHSPs are induced by exposure of cells to thermal, osmotic, and oxidative stresses and to mitogens. In animal cells, overexpression of sHSPs causes increased resistance to stress and stabilization of actin stress fibers. Yeast cells subjected to mild stress, such as shifting from 23 to 39 degrees C, arrest growth and then resume cell division. Growth arrest is accompanied by transient disorganization of the cytoskeleton. Yeast in which the HSP42 gene was disrupted and which was subjected to moderate thermal stress reorganized the actin cytoskeleton more slowly than did wild-type control cells. These results demonstrate that in yeast, as in metazoan cells, sHSPs promote maintenance of the actin cytoskeleton.

Actin Cytoskeleton↗
PubMed1997

Cdc6p-dependent loading of Mcm proteins onto pre-replicative chromatin in budding yeast.

The Cdc6 protein is essential for the assembly of pre-replicative complexes (pre-RCs) at origins of DNA replication in the budding yeast Saccharomyces cerevisiae. This reaction is blocked in vivo by the cyclin-dependent kinase Cdc28p, together with its regulatory subunits, the B type cyclins that are present throughout S, G2, and M phases. Because the destruction of B type cyclins and the consequent inactivation of the kinase are essential for exit from mitosis, pre-RC formation can only occur after passage through mitosis. Therefore, pre-RC formation has been proposed to be essential for coupling S phase and mitosis and for limiting DNA replication to once per cell cycle. The Mcm2-7 family of proteins has been implicated in limiting replication to once per cell cycle from experiments with Xenopus egg extracts. Here we show that the Mcm proteins of budding yeast are abundant and are quantitatively found in a chromatin-enriched fraction specifically during the G1 phase of the cell cycle. This chromatin binding depends on the de novo synthesis of Cdc6p, providing evidence that a conserved biochemical pathway plays a critical role in coordinating DNA replication with mitosis in both yeast and higher eukaryotes. Cdc6p and the origin recognition complex can be selectively removed from this chromatin-enriched fraction without removing the Mcm proteins. From these results, we propose that Cdc6p (and the origin recognition complex) nucleates the binding of Mcm proteins to chromatin, but once bound, the Mcm proteins appear to interact tightly with some other component of chromatin.

Cell Cycle↗
PubMed2006

Downregulation of PP2A(Cdc55) phosphatase by separase initiates mitotic exit in budding yeast.

After anaphase, the high mitotic cyclin-dependent kinase (Cdk) activity is downregulated to promote exit from mitosis. To this end, in the budding yeast S. cerevisiae, the Cdk counteracting phosphatase Cdc14 is activated. In metaphase, Cdc14 is kept inactive in the nucleolus by its inhibitor Net1. During anaphase, Cdk- and Polo-dependent phosphorylation of Net1 is thought to release active Cdc14. How Net1 is phosphorylated specifically in anaphase, when mitotic kinase activity starts to decline, has remained unexplained. Here, we show that PP2A(Cdc55) phosphatase keeps Net1 underphosphorylated in metaphase. The sister chromatid-separating protease separase, activated at anaphase onset, interacts with and downregulates PP2A(Cdc55), thereby facilitating Cdk-dependent Net1 phosphorylation. PP2A(Cdc55) downregulation also promotes phosphorylation of Bfa1, contributing to activation of the "mitotic exit network" that sustains Cdc14 as Cdk activity declines. These findings allow us to present a new quantitative model for mitotic exit in budding yeast.

Cell Cycle Proteins↗
PubMed1995

A cell cycle checkpoint monitors cell morphogenesis in budding yeast.

Checkpoint controls are regulatory pathways that inhibit cell cycle progression in cells that have not faithfully completed a prior step in the cell cycle. In the budding yeast Saccharomyces cerevisiae, DNA replication and spindle assembly are monitored by checkpoint controls that prevent nuclear division in cells that have failed to complete these processes. During the normal cell cycle, bud formation is temporally coincident with DNA replication and spindle assembly, and the nucleus divides along the mother-bud axis in mitosis. In this report, we show that inhibition of bud formation also causes a dramatic delay in nuclear division. This allows cells to recover from a transient disruption of cell polarity without becoming binucleate. The delay occurs after DNA replication and spindle assembly, and results from delayed activation of the master cell cycle regulatory kinase, Cdc28. Cdc28 activation is inhibited by phosphorylation of Cdc28 on tyrosine 19, and by delayed accumulation of the B-type cyclins Clb1 and Clb2. These results suggest the existence of a novel checkpoint that monitors cell morphogenesis in budding yeast.

CDC28 Protein Kinase, S cerevisiae↗
PubMed1989

Small nuclear RNAs from budding yeasts: phylogenetic comparisons reveal extensive size variation.

Homologues of each of the five metazoan snRNAs required for pre-mRNA splicing have recently been identified in the budding yeast Saccharomyces cerevisiae on the basis of shared structural elements and evidence of similar roles during splicing. However, the spliceosomal snRNAs in this yeast are up to six times larger than their mammalian counterparts, suggesting that they may perform additional, perhaps species-specific, functions in the pre-mRNA processing pathway. We have undertaken a survey of 23 other budding yeasts to determine whether increased snRNA size is unique to Sacch. cerevisiae and, if not, to look for common structural motifs among homologous snRNAs. Our studies reveal that the spliceosomal snRNAs exhibit a surprising degree of size variation among these species. Furthermore, partial sequence analysis has identified a specific domain in the U6 snRNA which accounts for the observed size polymorphisms.

Base Sequence↗
PubMed1999

Fission yeast Pob1p, which is homologous to budding yeast Boi proteins and exhibits subcellular localization close to actin patches, is essential for cell elongation and separation.

The fission yeast pob1 gene encodes a protein of 871 amino acids carrying an SH3 domain, a SAM domain, and a PH domain. Gene disruption and construction of a temperature-sensitive pob1 mutant indicated that pob1 is essential for cell growth. Loss of its function leads to quick cessation of cellular elongation. Pob1p is homologous to two functionally redundant Saccharomyces cerevisiae proteins, Boi1p and Boi2p, which are necessary for cell growth and relevant to bud formation. Overexpression of pob1 inhibits cell growth, causing the host cells to become round and swollen. In growing cells, Pob1p locates at cell tips during interphase and translocates near the division plane at cytokinesis. Thus, this protein exhibits intracellular dynamics similar to F-actin patches. However, Pob1p constitutes a layer, rather than patches, at growing cell tips. It generates two split discs flanking the septum at cytokinesis. The pob1-defective cells no longer elongate but swell gradually at the middle, eventually assuming a lemon-like morphology. Analysis using the pob1-ts allele revealed that Pob1p is also essential for cell separation. We speculate that Pob1p is located on growing plasma membrane, possibly through the function of actin patches, and may recruit proteins required for the synthesis of cell wall.

Actins↗
PubMed1999

A Bub2p-dependent spindle checkpoint pathway regulates the Dbf2p kinase in budding yeast.

Exit from mitosis in all eukaroytes requires inactivation of the mitotic kinase. This occurs principally by ubiquitin-mediated proteolysis of the cyclin subunit controlled by the anaphase-promoting complex (APC). However, an abnormal spindle and/or unattached kinetochores activates a conserved spindle checkpoint that blocks APC function. This leads to high mitotic kinase activity and prevents mitotic exit. DBF2 belongs to a group of budding yeast cell cycle genes that when mutated prevent cyclin degradation and block exit from mitosis. DBF2 encodes a protein kinase which is cell cycle regulated, peaking in metaphase-anaphase B/telophase, but its function remains unknown. Here, we show the Dbf2p kinase activity to be a target of the spindle checkpoint. It is controlled specifically by Bub2p, one of the checkpoint components that is conserved in fission yeast and higher eukaroytic cells. Significantly, in budding yeast, Bub2p shows few genetic or biochemical interactions with other members of the spindle checkpoint. Our data now point to the protein kinase Mps1p triggering a new parallel branch of the spindle checkpoint in which Bub2p blocks Dbf2p function.

Anaphase↗
PubMed1999

Microtubule dynamics from mating through the first zygotic division in the budding yeast Saccharomyces cerevisiae.

We have used time-lapse digital imaging microscopy to examine cytoplasmic astral microtubules (Mts) and spindle dynamics during the mating pathway in budding yeast Saccharomyces cerevisiae. Mating begins when two cells of opposite mating type come into proximity. The cells arrest in the G1 phase of the cell cycle and grow a projection towards one another forming a shmoo projection. Imaging of microtubule dynamics with green fluorescent protein (GFP) fusions to dynein or tubulin revealed that the nucleus and spindle pole body (SPB) became oriented and tethered to the shmoo tip by a Mt-dependent search and capture mechanism. Dynamically unstable astral Mts were captured at the shmoo tip forming a bundle of three or four astral Mts. This bundle changed length as the tethered nucleus and SPB oscillated toward and away from the shmoo tip at growth and shortening velocities typical of free plus end astral Mts (approximately 0.5 micrometer/min). Fluorescent fiduciary marks in Mt bundles showed that Mt growth and shortening occurred primarily at the shmoo tip, not the SPB. This indicates that Mt plus end assembly/disassembly was coupled to pushing and pulling of the nucleus. Upon cell fusion, a fluorescent bar of Mts was formed between the two shmoo tip bundles, which slowly shortened (0.23 +/- 0.07 micrometer/min) as the two nuclei and their SPBs came together and fused (karyogamy). Bud emergence occurred adjacent to the fused SPB approximately 30 min after SPB fusion. During the first mitosis, the SPBs separated as the spindle elongated at a constant velocity (0.75 micrometer/min) into the zygotic bud. There was no indication of a temporal delay at the 2-micrometer stage of spindle morphogenesis or a lag in Mt nucleation by replicated SPBs as occurs in vegetative mitosis implying a lack of normal checkpoints. Thus, the shmoo tip appears to be a new model system for studying Mt plus end dynamic attachments and much like higher eukaryotes, the first mitosis after haploid cell fusion in budding yeast may forgo cell cycle checkpoints present in vegetative mitosis.

Cell Fusion↗
PubMed2005

Functional conservation between fission yeast moc1/sds23 and its two orthologs, budding yeast SDS23 and SDS24, and phenotypic differences in their disruptants.

The moc1/sds23 gene was isolated to induce sexual development of a sterile strain due to overexpression of adenylate cyclase in Schizosaccharomyces pombe. Here, we studied the functional conservation between moc1/sds23 and its two orthologs SDS23 and SDS24 in Saccharomyces cerevisiae. We observed that the temperature sensitivity, salt tolerance, cell morphology, and sterility of the Deltamoc1 mutant in S. pombe were recovered by expressing either S. cerevisiae SDS23 or SDS24. We found that deletion of both SDS23 and SDS24 resulted in the production of a large vacuole that was reversed by the expression of S. pombe moc1/sds23. In these ways we found that S. pombe Moc1/Sds23 and S. cerevisiae SDS23p or SDS24p are functional homologs. In addition we found that the Deltasds23 Deltasds24 diploid strain reduces cell separation in forming pseudohyphal-like growth in S. cerevisiae. Thus S. pombe moc1/sds23 and S. cerevisiae SDS23 or SDS24 are interchangeable with each other, but their disruptants are phenotypically dissimilar.

Cell Cycle Proteins↗
PubMed2006

Evidence for ORC-dependent repression of budding yeast genes induced by starvation and other stresses.

The highly conserved origin recognition complex (ORC) is required for repressing genes in the silent mating type loci of budding yeast. Here we report that at a non-permissive temperature, the temperature-sensitive orc2-1 mutation induces the expression of more than 500 genes, the majority of which are also induced during starvation of wild-type cells. Many genes induced by starvation or by the orc2-1 mutation are also induced by inactivation of proteins required for chromatin-mediated repression of transcription. Genes induced by the orc2-1 mutation, starvation, or inactivation of repressor proteins, map near ORC-binding loci significantly more frequently compared to all genes. Genes repressed by starvation map near ORC-binding sites less frequently compared to all genes, which suggests they have been evolutionarily excluded from regions of repressive chromatin near ORC-binding sites. Deletion of sequences containing ORC-binding sites near the DAL2 and DAL4 genes in the DAL gene cluster, which are induced by either the orc2-1 mutation or by starvation, constitutively activates these genes and abolishes their activation by the orc2-1 mutation. Our findings suggest a role for ORC in the repression of a large number of budding yeast genes induced by starvation or other aspects of a deleterious environment.

Apoptosis↗
PubMed2002

IBD2 encodes a novel component of the Bub2p-dependent spindle checkpoint in the budding yeast Saccharomyces cerevisiae.

During mitosis, genomic integrity is maintained by the proper coordination of mitotic events through the spindle checkpoint. The bifurcated spindle checkpoint blocks cell cycle progression at metaphase by monitoring unattached kinetochores and inhibits mitotic exit in response to the incorrect orientation of the mitotic spindle. Bfa1p is a spindle checkpoint regulator of budding yeast in the Bub2p checkpoint pathway for proper mitotic exit. We have isolated a novel Bfa1p interacting protein named Ibd2p in the budding yeast Saccharomyces cerevisiae. We found that IBD2 (Inhibition of Bud Division 2) is not an essential gene but its deletion mutant proceeded through the cell cycle in the presence of microtubule-destabilizing drugs, thereby inducing a sharp decrease in viability. In addition, overexpression of Mps1p caused partial mitotic arrest in ibd2Delta as well as in bub2Delta, suggesting that IBD2 encodes a novel component of the spindle checkpoint downstream of MPS1. Overexpression of Ibd2p induced mitotic arrest with increased levels of Clb2p in wild type and mad2Delta, but not in deletion mutants of BUB2 and BFA1. Pds1p was also stabilized by the overexpression of Ibd2p in wild-type cells. The mitotic arrest defects observed in ibd2Delta in the presence of nocodazole were restored by additional copies of BUB2, BFA1, and CDC5, whereas an extra copy of IBD2 could not rescue the mitotic arrest defects of bub2Delta and bfa1Delta. The mitotic arrest defects of ibd2Delta were not recovered by MAD2, or vice versa. Analysis of the double mutant combinations ibd2Deltamad2Delta, ibd2Deltabub2Delta, and ibd2Deltadyn1Delta showed that IBD2 belongs to the BUB2 epistasis group. Taken together, these data demonstrate that IBD2 encodes a novel component of the BUB2-dependent spindle checkpoint pathway that functions upstream of BUB2 and BFA1.

Blotting, Northern↗
PubMed1998

The cyclin family of budding yeast: abundant use of a good idea.

Cyclins are highly conserved proteins that activate cyclin-dependent kinases (CDKs) to regulate the cell cycle, transcription and other cellular processes. The completion of the genome sequence of the budding yeast Saccharomyces cerevisiae allows an appraisal of the functions of the entire complement of cyclins in a eukaryotic organism. The cyclin family of budding yeast is reviewed from a functional perspective with an emphasis on what genetic and biochemical experiments have revealed about cyclin-CDK substrates.

Animals↗
PubMed2006

Gamma-tubulin is required for proper recruitment and assembly of Kar9-Bim1 complexes in budding yeast.

Microtubule plus-end-interacting proteins (+TIPs) promote the dynamic interactions between the plus ends (+ends) of astral microtubules and cortical actin that are required for preanaphase spindle positioning. Paradoxically, +TIPs such as the EB1 orthologue Bim1 and Kar9 also associate with spindle pole bodies (SPBs), the centrosome equivalent in budding yeast. Here, we show that deletion of four C-terminal residues of the budding yeast gamma-tubulin Tub4 (tub4-delta dsyl) perturbs Bim1 and Kar9 localization to SPBs and Kar9-dependent spindle positioning. Surprisingly, we find Kar9 localizes to microtubule +ends in tub4-delta dsyl cells, but these microtubules fail to position the spindle when targeted to the bud. Using cofluorescence and coaffinity purification, we show Kar9 complexes in tub4-delta dsyl cells contain reduced levels of Bim1. Astral microtubule dynamics is suppressed in tub4-delta dsyl cells, but it are restored by deletion of Kar9. Moreover, Myo2- and F-actin-dependent dwelling of Kar9 in the bud is observed in tub4-delta dsyl cells, suggesting defective Kar9 complexes tether microtubule +ends to the cortex. Overproduction of Bim1, but not Kar9, restores Kar9-dependent spindle positioning in the tub4-delta dsyl mutant, reduces cortical dwelling, and promotes Bim1-Kar9 interactions. We propose that SPBs, via the tail of Tub4, promote the assembly of functional +TIP complexes before their deployment to microtubule +ends.

Actins↗
PubMed1991

A 240 kd multisubunit protein complex, CBF3, is a major component of the budding yeast centromere.

A key protein component (CBF3) of the budding yeast (S. cerevisiae) centromere/kinetochore has been purified and characterized. CBF3 is a 240 kd multisubunit protein complex that binds specifically to the yeast wild-type centromere DNA (CEN), but not to nonfunctional CEN DNA containing a single base substitution in the critical CDEIII consensus sequence. When purified by affinity chromatography, CBF3 contains three protein components: CBF3A (110 kd), CBF3B (64 kd), and CBF3C (58 kd). Highly purified CBF3 requires the presence of a separate assembly factor or chaperone activity to bind to CEN DNA. Treatment with phosphatase inactivates CBF3, indicating that at least one of the CBF3 subunits must be phosphorylated for DNA binding to occur. A 56 bp region including the 26 bp CDEIII consensus is protected from DNAase I cleavage in the CBF3-CEN DNA complex.

Base Sequence↗
PubMed2004

Cold adaptation in budding yeast.

We have determined the transcriptional response of the budding yeast Saccharomyces cerevisiae to cold. Yeast cells were exposed to 10 degrees C for different lengths of time, and DNA microarrays were used to characterize the changes in transcript abundance. Two distinct groups of transcriptionally modulated genes were identified and defined as the early cold response and the late cold response. A detailed comparison of the cold response with various environmental stress responses revealed a substantial overlap between environmental stress response genes and late cold response genes. In addition, the accumulation of the carbohydrate reserves trehalose and glycogen is induced during late cold response. These observations suggest that the environmental stress response (ESR) occurs during the late cold response. The transcriptional activators Msn2p and Msn4p are involved in the induction of genes common to many stress responses, and we show that they mediate the stress response pattern observed during the late cold response. In contrast, classical markers of the ESR were absent during the early cold response, and the transcriptional response of the early cold response genes was Msn2p/Msn4p independent. This implies that the cold-specific early response is mediated by a different and as yet uncharacterized regulatory mechanism.

Adaptation, Physiological↗