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Signalling towards cell wall synthesis in budding yeast.

The budding yeast Saccharomyces cerevisiae has long proved to be a very useful model in cell biology. Its cell morphology is established and maintained at least in part by the cell wall, a rigid but dynamic structure that affords mechanical protection. Although fungal cell walls represent an unique phenomenon, recent progress in research has shown striking parallels between yeast and mammalian cells in the area of cell morphogenesis and proliferation. Further studies promise to shed common light on the processes of cell morphogenesis including the intersections with proliferation control. This review focuses on the recent progress in this promising area in the yeast Saccharomyces cerevisiae. The process of cell wall synthesis in Saccharomyces cerevisiae was reviewed by several authors recently. Briefly, the cell wall represents a complex structure of cross-linked chitin, beta-(1,6)-d-glucan, beta-(1,3)-D-glucan and mannoproteins. Chitin and beta-(1,3)-D-glucan are synthesized by enzymatic complexes at the cell membrane and extruded into the periplasmic space, mannoproteins are synthesized along the yeast secretory pathway, and the site of beta-(1,6)-D-glucan synthesis is still unknown. The principal motif which interconnects individual cell wall constituents was recently identified by Kollár et al. The mechanisms of cross-linking of the polymers in the wall remain unknown, however. Recently, nevertheless, substantial progress has been achieved in understanding the signalling pathways which target the cell wall construction.

Cell Wall↗

Programmed frameshifting in the synthesis of mammalian antizyme is +1 in mammals, predominantly +1 in fission yeast, but -2 in budding yeast.

The coding sequence for mammalian ornithine decarboxylase antizyme is in two different partially overlapping reading frames with no independent ribosome entry to the second ORF. Immediately before the stop codon of the first ORF, a proportion of ribosomes undergo a quadruplet translocation event to shift to the +1 reading frame of the second and main ORF. The proportion that frameshifts is dependent on the polyamine level and, because the product antizyme is a negative regulator of intracellular polyamine levels, the frameshifting acts to complete an autoregulatory circuit by sensing polyamine levels. An mRNA element just 5' of the shift site and a 3' pseudoknot are important for efficient frameshifting. Previous work has shown that a cassette with the mammalian shift site and associated signals directs efficient shifting in the budding yeast Saccharomyces cerevisiae at the same codon to the correct frame, but that the shift is -2 instead of +1. The product contains an extra amino acid corresponding to the shift site. The present work shows efficient frameshifting also occurs in the fission yeast, Schizosaccharomyces pombe. This frameshifting is 80% +1 and 20% -2. The response of S. pombe translation apparatus to the mammalian antizyme recoding signals is more similar to that of the mammalian system than to that of S. cerevisiae. S. pombe provides a good model system for genetic studies on the mechanism of at least this type of programmed mammalian frameshifting.

Amino Acid Sequence↗

Simple centromere, complex kinetochore: linking spindle microtubules and centromeric DNA in budding yeast.

Although the budding yeast centromere is extremely short (125 bp) compared to those of other eukaryotes, the kinetochore that assembles on this DNA displays a rich molecular complexity. Here, we describe recent advances in our understanding of kinetochore function in budding yeast and present a model describing the attachment that is formed between spindle microtubules and centromeric DNA. This analysis may provide general principles for kinetochore function and regulation.

DNA, Fungal↗

The hyper unequal sister chromatid recombination in an sgs1 mutant of budding yeast requires MSH2.

Budding yeast SGS1 and the human Bloom's syndrome (BS) gene, BLM, are homologues of the Escherichia coli recQ. Cells derived from BS patients are characterized by a dramatic increase in sister chromatid exchange (SCE). We previously reported that budding yeast cells deficient in SGS1 showed an increase in the frequency of recombination between unequal sister chromatids recombination (USCR). In this study, we examined the factors influencing the elevated SCR frequency in sgs1 disruptants. The increase in SCR frequency in sgs1 mutants was greatly reduced by disrupting the RAD52 or MSH2 gene, which is involved in mismatch repair. However, a plasmid carrying MSH2, having a missense mutation defective in mismatch repair complemented the reduced USCR in msh2 sgs1 mutants, suggesting that the function of Msh2 in mismatch repair is dispensable for USCR.

DNA Helicases↗

Cleavage of model replication forks by fission yeast Mus81-Eme1 and budding yeast Mus81-Mms4.

The blockage of replication forks can result in the disassembly of the replicative apparatus and reversal of the fork to form a DNA junction that must be processed in order for replication to restart and sister chromatids to segregate at mitosis. Fission yeast Mus81-Eme1 and budding yeast Mus81-Mms4 are endonucleases that have been implicated in the processing of aberrant DNA junctions formed at stalled replication forks. Here we have investigated the activity of purified Mus81-Eme1 and Mus81-Mms4 on substrates that resemble DNA junctions that are expected to form when a replication fork reverses. Both enzymes cleave Holliday junctions and substrates that resemble normal replication forks poorly or not at all. However, forks where the equivalents of either both the leading and lagging strands or just the lagging strand are juxtaposed at the junction point, or where either the leading or lagging strand has been unwound to produce a fork with a single-stranded tail, are cleaved well. Cleavage sites map predominantly between 3 and 6 bp 5' of the junction point. For most substrates the leading strand template is cleaved. The sole exception is a fork with a 5' single-stranded tail, which is cleaved in the lagging strand template.

DNA↗

Ctf3p, the Mis6 budding yeast homolog, interacts with Mcm22p and Mcm16p at the yeast outer kinetochore.

The budding yeast kinetochore is composed of an inner and outer protein complex, which binds to centromere (CEN) DNA and attaches to microtubules. We performed a genetic synthetic dosage lethality screen to identify novel kinetochore proteins in a collection of chromosome transmission fidelity mutants. Our screen identified several new kinetochore-related proteins including YLR381Wp/Ctf3p, which is a member of a conserved family of centromere-binding proteins. Ctf3p interacts with Mcm22p, Mcm16p, and the outer kinetochore protein Ctf19p. We used chromatin immunoprecipitation to demonstrate that Ctf3p, Mcm22p, and Mcm16p bind to CEN DNA in a Ctf19p-dependent manner. In addition, Ctf3p, Mcm22p, and Mcm16p have a localization pattern similar to other kinetochore proteins. The fission yeast Ctf3p homolog, Mis6, is required for loading of a CENP-A centromere specific histone, Cnp1, onto centromere DNA. We find however that Ctf3p is not required for loading of the budding yeast CENP-A homolog, Cse4p, onto CEN DNA. In contrast, Ctf3p and Ctf19p fail to bind properly to the centromere in a cse4-1 mutant strain. We conclude that the requirements for CENP-A loading onto centromere DNA differ in fission versus budding yeast.

Amino Acid Sequence↗

Cdc28-Clb mitotic kinase negatively regulates bud site assembly in the budding yeast.

In the budding yeast Saccharomyces cerevisiae, a prospective mother normally commences the formation of a daughter (the bud) only in the G(1) phase of the cell division cycle. This suggests a strict temporal regulation of the processes that initiate the formation of a new bud. Using cortical localization of bud site components Spa2 and Bni1 as an indicator of bud site assembly, we show that cells assemble a bud site following inactivation of the Cdc28-Clb mitotic kinase but prior to START. Interestingly, an untimely inactivation of the mitotic kinase is sufficient to drive cells to assemble a new bud site inappropriately in G(2) or M phases. The induction of Cdc28/Clb kinase activity in G(1), on the other hand, dramatically reduces a cell's ability to construct an incipient bud site. Our findings strongly suggest that the Cdc28-Clb kinase plays a critical role in the mechanism that restricts the timing of bud formation to the G(1) phase of the cell cycle.

CDC28 Protein Kinase, S cerevisiae↗

Structural heterogeneity in populations of the budding yeast Saccharomyces cerevisiae.

Bud scar analysis integrated with mathematical analysis of DNA and protein distributions obtained by flow microfluorometry have been used to analyze the cell cycle of the budding yeast Saccharomyces cerevisiae. In populations of this yeast growing exponentially in batch at 30 degrees C on different carbon and nitrogen sources with duplication times between 75 and 314 min, the budded period is always shorter (approximately 5 to 10 min) than the sum of the S + G2 + M + G1* phases (determined by the Fried analysis of DNA distributions), and parent cells always show a prereplicative unbudded period. The analysis of protein distributions obtained by flow microfluorometry indicates that the protein level per cell required for bud emergence increases at each new generation of parent cells, as observed previously for cell volume. A wide heterogeneity of cell populations derives from this pattern of budding, since older (and less frequent) parent cells have shorter generation times and produce larger (and with shorter cycle times) daughter cells. A possible molecular mechanism for the observed increase with genealogical age of the critical protein level required for bud emergence is discussed.

DNA, Fungal↗

Kinetics of spindle pole body separation in budding yeast.

In the budding yeast Saccharomyces cerevisiae, the spindle pole body (SPB) serves as the microtubule-organizing center and is the functional analog of the centrosome of higher organisms. By expressing a fusion of a yeast SPB-associated protein to the Aequorea victoria green fluorescent protein, the movement of the SPBs in living yeast cells undergoing mitosis was observed by fluorescence microscopy. The ability to visualize SPBs in vivo has revealed previously unidentified mitotic events. During anaphase, the mitotic spindle has four sequential activities: alignment at the mother-daughter junction, fast elongation, translocation into the bud, and slow elongation. These results indicate that distinct forces act upon the spindle at different times during anaphase.

Base Sequence↗

Improved flow cytometric analysis of the budding yeast cell cycle.

The budding yeast, Saccharomyces cerevisiae has been a remarkably useful model system for the study of eukaryotic cell cycle regulation. Flow cytometric analysis of DNA content in budding yeast has become a standard tool for the analysis of cell cycle progression. However, popular protocols utilizing the DNA binding dye, propidium iodide, suffer from a number of drawbacks that confound accurate analysis by flow cytometry. Here we show the utility of the DNA binding dye, SYTOX Green, in the cell cycle analysis of yeast. Samples analyzed using SYTOX Green exhibited better coefficients of variation, improved linearity between DNA content and fluorescence, and decreased peak drift associated with changes in dye concentration, growth conditions or cell size.

Cell Cycle↗

Characterization of the Net1 cell cycle-dependent regulator of the Cdc14 phosphatase from budding yeast.

In the budding yeast Saccharomyces cerevisiae, the multifunctional protein Net1 is implicated in regulating the cell cycle function of the Cdc14 protein phosphatase. Genetic and cell biological data suggest that during interphase and early mitosis Net1 holds Cdc14 within the nucleolus where its activity is suppressed. Upon its transient release from Net1 at late anaphase, active Cdc14 promotes exit from mitosis by dephosphorylating targets in the nucleus and cytoplasm. In this paper we present evidence supporting the proposed role of Net1 in regulating Cdc14 and exit from mitosis. We show that the NH(2)-terminal fragment Net1(1-600) directly binds Cdc14 in vitro and is a highly specific competitive inhibitor of its activity (K(i) = 3 nm) with five different substrates including the physiologic targets Swi5 and Sic1. An analysis of truncation mutants indicates that the Cdc14 binding site is located within a segment of Net1 containing residues 1-341. We propose that Net1 inhibits by occluding the active site of Cdc14 because it acts as a competitive inhibitor, binds to a site located within the catalytic domain (residues 1-374), binds with reduced affinity to a Cdc14 C283S mutant in which an active site Cys is replaced, and is displaced by tungstate, a transition state analog known to bind in the catalytic site of protein-tyrosine phosphatases.

Binding Sites↗

Kinase activity-dependent nuclear export opposes stress-induced nuclear accumulation and retention of Hog1 mitogen-activated protein kinase in the budding yeast Saccharomyces cerevisiae.

Budding yeast adjusts to increases in external osmolarity via a specific mitogen-activated protein kinase signal pathway, the high-osmolarity glycerol response (HOG) pathway. Studies with a functional Hog1-green fluorescent protein (GFP) fusion reveal that even under nonstress conditions the mitogen-activated protein kinase Hog1 cycles between cytoplasmic and nuclear compartments. The basal distribution of the protein seems independent of its activator, Pbs2, and independent of its phosphorylation status. Upon osmotic challenge, the Hog1-GFP fusion becomes rapidly concentrated in the nucleus from which it is reexported after return to an iso-osmotic environment or after adaptation to high osmolarity. The preconditions and kinetics of increased nuclear localization correlate with those found for the dual phosphorylation of Hog1-GFP. The duration of Hog1 nuclear residence is modulated by the presence of the general stress activators Msn2 and Msn4. Reexport of Hog1 to the cytoplasm does not require de novo protein synthesis but depends on Hog1 kinase activity. Thus, at least three different mechanisms contribute to the intracellular distribution pattern of Hog1: phosphorylation-dependent nuclear accumulation, retention by nuclear targets, and a kinase-induced export.

Base Sequence↗

Actin and septin ultrastructures at the budding yeast cell cortex.

Budding yeast has been a powerful model organism for studies of the roles of actin in endocytosis and septins in cell division and in signaling. However, the depth of mechanistic understanding that can be obtained from such studies has been severely hindered by a lack of ultrastructural information about how actin and septins are organized at the cell cortex. To address this problem, we developed rapid-freeze and deep-etch techniques to image the yeast cell cortex in spheroplasted cells at high resolution. The cortical actin cytoskeleton assembles into conical or mound-like structures composed of short, cross-linked filaments. The Arp2/3 complex localizes near the apex of these structures, suggesting that actin patch assembly may be initiated from the apex. Mutants in cortical actin patch components with defined defects in endocytosis disrupted different stages of cortical actin patch assembly. Based on these results, we propose a model for actin function during endocytosis. In addition to actin structures, we found that septin-containing filaments assemble into two kinds of higher order structures at the cell cortex: rings and ordered gauzes. These images provide the first high-resolution views of septin organization in cells.

Actin-Related Protein 2↗

Activation of silent replication origins at autonomously replicating sequence elements near the HML locus in budding yeast.

In the budding yeast, Saccharomyces cerevisiae, replicators can function outside the chromosome as autonomously replicating sequence (ARS) elements; however, within chromosome III, certain ARSs near the transcriptionally silent HML locus show no replication origin activity. Two of these ARSs comprise the transcriptional silencers E (ARS301) and I (ARS302). Another, ARS303, resides between HML and the CHA1 gene, and its function is not known. Here we further localized and characterized ARS303 and in the process discovered a new ARS, ARS320. Both ARS303 and ARS320 are competent as chromosomal replication origins since origin activity was seen when they were inserted at a different position in chromosome III. However, at their native locations, where the two ARSs are in a cluster with ARS302, the I silencer, no replication origin activity was detected regardless of yeast mating type, special growth conditions that induce the transcriptionally repressed CHA1 gene, trans-acting mutations that abrogate transcriptional silencing at HML (sir3, orc5), or cis-acting mutations that delete the E and I silencers containing ARS elements. These results suggest that, for the HML ARS cluster (ARS303, ARS320, and ARS302), inactivity of origins is independent of local transcriptional silencing, even though origins and silencers share key cis- and trans-acting components. Surprisingly, deletion of active replication origins located 25 kb (ORI305) and 59 kb (ORI306) away led to detection of replication origin function at the HML ARS cluster, as well as at ARS301, the E silencer. Thus, replication origin silencing at HML ARSs is mediated by active replication origins residing at long distances from HML in the chromosome. The distal active origins are known to fire early in S phase, and we propose that their inactivation delays replication fork arrival at HML, providing additional time for HML ARSs to fire as origins.

Base Sequence↗

The tomato nia gene promoter functions in fission yeast but not in budding yeast.

A fragment comprising 1 kb of the 5' region and the 81 first nucleotides of the coding region of the tomato nitrate reductase nia gene was placed in translational fusion with the lacZ reporter gene. This construct was introduced in budding and in fission yeast using a derivative of the Saccharomyces cerevisiae/Schizosaccharomyces pombe autonomously replicating vector pUZL. Beta-galactosidase activity was detected in S. pombe but not in S. cerevisiae. Primer extension experiments show that in fission yeast transcripts are initiated at the same starting point as in tomato, indicating for the first time that a plant promoter can be correctly recognized in fission yeast.

Base Sequence↗

Comparative analysis of regulatory transcription factors in Schizosaccharomyces pombe and budding yeasts.

Regulatory transcription factors (rTFs), which bind specific DNA sequences in the regulatory regions of genes and subsequently activate or repress transcription, play a central role in programming genomic expression. The number of rTFs in a species might therefore reflect its functional complexity. For simple organisms like yeast, a relatively small number of rTFs might be expected that is fairly constant between yeast species. We show that the budding yeast, Saccharomyces cerevisiae, contains 201 rTfs, which is one of the largest rTF numbers found in yeast species for which genome sequences are available. This is a much higher number than the 129 rTFs found in the fission yeast, Schizosaccharomyces pombe, which is currently the yeast with the lowest number of rTFs. Comparative analysis of several different budding yeast species shows that most of the 'extra' rTFs found in S. cerevisiae were probably acquired as a result of a whole genome duplication (WGD) event that occurred in an ancestor of a subset of budding yeast species. However, we also show that budding yeast species that have not been affected by the WGD contain a greater number of rTFs than S. pombe (mean = 145). Thus, two or more mechanisms have led to the 60% increase in rTFs in S. cerevisiae compared to S. pombe. This difference may correlate with a more extensive functional divergence in budding yeasts compared to fission yeasts. The relatively small number of rTFs in S. pombe make this organism an attractive model for global studies of mechanisms that programme gene expression.

Evolution, Molecular↗

The spindle cycle in budding yeast.

The mitotic spindle of the budding yeast Saccharomyces cerevisiae will probably be the first such organelle to be understood in molecular detail. Here we describe the mitotic spindle cycle of budding yeast using electron-microscope-derived structures and dynamic live-cell imaging. Recent work has revealed that many general aspects of mitosis are conserved, making budding yeast an excellent model for the study of mitosis.

Anaphase↗

Mathematical model of the morphogenesis checkpoint in budding yeast.

The morphogenesis checkpoint in budding yeast delays progression through the cell cycle in response to stimuli that prevent bud formation. Central to the checkpoint mechanism is Swe1 kinase: normally inactive, its activation halts cell cycle progression in G2. We propose a molecular network for Swe1 control, based on published observations of budding yeast and analogous control signals in fission yeast. The proposed Swe1 network is merged with a model of cyclin-dependent kinase regulation, converted into a set of differential equations and studied by numerical simulation. The simulations accurately reproduce the phenotypes of a dozen checkpoint mutants. Among other predictions, the model attributes a new role to Hsl1, a kinase known to play a role in Swe1 degradation: Hsl1 must also be indirectly responsible for potent inhibition of Swe1 activity. The model supports the idea that the morphogenesis checkpoint, like other checkpoints, raises the cell size threshold for progression from one phase of the cell cycle to the next.

Algorithms↗