Regulation of mating in the budding yeast Saccharomyces cerevisiae by the zinc cluster proteins Sut1 and Sut2

The zinc cluster proteins Sut1 and Sut2 play a role in sterol uptake and filamentous growth in the budding yeast Saccharomyces cerevisiae. In this study, we show that they are also involved in mating. Cells that lack both SUT1 and SUT2 were defective in mating. The expression of the genes NCE102 and PRR2 was increased in the sut1 sut2 double deletion mutant suggesting that Sut1 and Sut2 both repress the expression of NCE102 and PRR2. Consistent with these data, overexpression of either SUT1 or SUT2 led to lower expression of NCE102 and PRR2. Furthermore, expression levels of NCE102, PRR2 and RHO5, another target gene of Sut1 and Sut2, decreased in response to pheromone. Prr2 has been identified as a mating inhibitor before. Here we show that overexpression of NCE102 and RHO5 also reduced mating. Our results suggest that Sut1 and Sut2 positively regulate mating by repressing the expression of the mating inhibitors NCE102, PRR2 and RHO5 in response to pheromone.     

Systematic identification of cell size regulators in budding yeast

Cell size is determined by a complex interplay between growth and division, involving multiple cellular pathways. To identify systematically processes affecting size control in G1 in budding yeast, we imaged and analyzed the cell cycle of millions of individual cells representing 591 mutants implicated in size control. Quantitative metric distinguished mutants affecting the mechanism of size control from the majority of mutants that have a perturbed size due to indirect effects modulating cell growth. Overall, we identified 17 negative and dozens positive size control regulators, with the negative regulators forming a small network centered on elements of mitotic exit network. Some elements of the translation machinery affected size control with a notable distinction between the deletions of parts of small and large ribosomal subunit: parts of small ribosomal subunit tended to regulate size control, while parts of the large subunit affected cell growth. Analysis of small cells revealed additional size control mechanism that functions in G2/M, complementing the primary size control in G1. Our study provides new insights about size control mechanisms in budding yeast.     

蛋白质的磷酸化作用和泛肽化降解作用与芽殖酵母细胞周期的调控

在芽殖酵母（Ｓａｃｃｈａｒｏｍｙｃｅｓｃｅｒｅｖｉｓｉａｅ）细胞中，Ｇ１期的三种ｃｙｃｌｉｎｓ和Ｓ、Ｍ期的五种ｃｙｃｌｉｎｓ之周期性的合成和分解调节着Ｃｄｃ２８的活性，驱动细胞周期的正常运转。除了ＣＤＫ的磷酸化作用外，蛋白质的泛肽化降解作用间接或直接调控细胞周期：ＣＤＣ３４泛肽化途径通过降解Ｃｄｃ２８的专一抑制子而起始ＤＮＡ复制；ＡＰＣ泛肽化途径通过降解Ｍ期后期的抑制子和Ｍ期ｃｙｃｌｉｎｓ，使姐妹染色体分离和Ｍ期终止。     

Isolation and characterization of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by the mating hormone a-factor

Summary Nine independent mutants which are supersensitive (ssl ^–) to G1 arrest by the mating hormone a-factor were isolated by screening mutagenized Saccharomyces cerevisiae MAT cells on solid medium for increased growth inhibition with a-factor. These mutants carried lesions in two complementation groups, ssl1 and ssl2. Mutations at the ssl1 locus were mating type specific: MAT ssl1 ^– cells were supersensitive to -factor but MAT ssl1 ^– were not supersensitive to -factor. In contrast, mutations at the ssl2. locus conferred supersensitivity to the mating hormone of the opposite mating type on both MAT, and MATa cells. The -cell specific capacity to inactivate externally added a-factor was shown to be lacking in MAT ssl1 ^– mutants whereas MAT ssl2. cells were able to inactivate a-factor. Complementation analysis showed that ssl2 and sst2, a mutation originally isolated as conferring supersensitivity to -factor to MATa cells, are lesions in the same gene. The ssl1 gene was mapped 30.5 centi-Morgans distal to ilv5 on chromosome XII.     

Cell cycle commitment in budding yeast emerges from the cooperation of multiple bistable switches

The start-transition (START) in the G1 phase marks the point in the cell cycle at which a yeast cell initiates a new round of cell division. Once made, this decision is irreversible and the cell is committed to progressing through the entire cell cycle, irrespective of arrest signals such as pheromone. How commitment emerges from the underlying molecular interaction network is poorly understood. Here, we perform a dynamical systems analysis of an established cell cycle model, which has never been analysed from a commitment perspective. We show that the irreversibility of the START transition and subsequent commitment can be consistently explained in terms of the interplay of multiple bistable molecular switches. By applying an existing mathematical model to a novel problem and by expanding the model in a self-consistent manner, we achieve several goals: we bring together a large number of experimental findings into a coherent theoretical framework; we increase the scope and the applicability of the original model; we give a systems level explanation of how the START transition and the cell cycle commitment arise from the dynamical features of the underlying molecular interaction network; and we make clear, experimentally testable predictions.     

Gbetagamma recruits Rho1 to the site of polarized growth during mating in budding yeast

In mating mixtures of Saccharomyces cerevisiae, cells polarize their growth toward their conjugation partners along a pheromone gradient. This chemotropic phenomenon is mediated by structural proteins such as Far1 and Bem1 and by signaling proteins such as Cdc24, Cdc42, and Gbetagamma. The Gbetagamma subunit is thought to provide a positional cue that recruits the polarity establishment proteins, and thereby induces polarization of the actin cytoskeleton. We identified RHO1 in a screen for allele-specific high-copy suppressors of Gbetagamma overexpression, suggesting that Rho1 binds Gbetagamma in vivo. Inactivation of Rho1 GTPase activity augmented the rescue phenotype, suggesting that it is the activated form of Rho1 that binds Gbetagamma. We also found, in a pull-down assay, that Rho1 associates with GST-Ste4 and that Rho1 is localized to the neck and tip of mating projections. Moreover, a mutation in STE4 that disrupts Gbetagamma-Rho1 interaction reduces the projection tip localization of Rho1 and compromises the integrity of pheromone-treated cells deficient in Rho1 activity. In addition to its roles as a positive regulator of 1,3-beta-glucan synthase and of the cell integrity MAP kinase cascade, it was recently shown that Rho1 is necessary for the formation of mating projections. Together, these results suggest that Gbetagamma recruits Rho1 to the site of polarized growth during mating.     

DNA-end capping by the budding yeast transcription factor and subtelomeric binding protein Tbf1

Telomere repeats in budding yeast are maintained at a constant average length and protected ('capped'), in part, by mechanisms involving the TG(1-3) repeat-binding protein Rap1. However, metazoan telomere repeats (T(2)AG(3)) can be maintained in yeast through a Rap1-independent mechanism. Here, we examine the dynamics of capping and telomere formation at an induced DNA double-strand break flanked by varying lengths of T(2)AG(3) repeats. We show that a 60-bp T(2)AG(3) repeat array induces a transient G2/M checkpoint arrest, but is rapidly elongated by telomerase to generate a stable T(2)AG(3)/TG(1-3) hybrid telomere. In contrast, a 230-bp T(2)AG(3) array induces neither G2/M arrest nor telomerase elongation. This capped state requires the T(2)AG(3)-binding protein Tbf1, but is independent of two Tbf1-interacting factors, Vid22 and Ygr071c. Arrays of binding sites for three other subtelomeric or Myb/SANT domain-containing proteins fail to display a similar end-protection effect, indicating that Tbf1 capping is an evolved function. Unexpectedly, we observed strong telomerase association with non-telomeric ends, whose elongation is blocked by a Mec1-dependent mechanism, apparently acting at the level of Cdc13 binding.     

G1 cyclins regulate proliferation of the budding yeast Saccharomyces cerevisiae.

The eukaryotic cell cycle is regulated at two points, the G1-S and G2-M boundaries. The molecular basis for these regulatory activities has recently been elucidated, in large part by the use of molecular and genetic analyses using unicellular yeast. The molecular characterization of cell-cycle regulation has revealed striking functional conservation among evolutionarily diverse cell types. For many eukaryotic cells, regulation of cell proliferation occurs primarily in the G1 interval. The G1 regulatory step, termed START, requires the activation of a highly conserved p34 protein kinase by association with a functionally redundant family of proteins, the G1 cyclins. Here we review studies using the genetically tractable budding yeast Saccharomyces cerevisiae, which have provided insight into the role of G1 cyclins in the regulation of START.     

Multiple stable states and hysteresis in continuous, oscillating cultures of budding yeast

The conditions that precede the onset of autonomous oscillations in continuous yeast cultures were studied in three different types of experiments. It was found that the final state of the culture depended on the protocol used to start up the reactor. Batch cultures, switched to continuous operation at different stages of the batch growth curve, all exhibited similar dynamics-ethanol depletion followed by autonomous oscillations. Small perturbations of the distribution of states in the reactor, achieved by addition of externally grown cells, were able to quench the oscillatory dynamics. Reaching the desired operating point by slow dilution rate changes gave rise to different final states, two oscillatory states and one steady state, depending on the rate of change in dilution rate. The multiplicity of stable states at a single operating point is not explained by any current distributed model and points toward a segregated mechanism of these oscillations.     

Cell-cell recognition and pheromone response of the yeast Saccharomyces globosus

Sexual agglutination and pheromone interaction between cells of two mating types, a and alpha, in the yeast Saccharomyces globosus were studied. S. globosus was shown to produce mating-type-specific factors analogs to a- and alpha-mating pheromones of Saccharomyces cerevisiae and to undergo the sexual agglutination reaction between cells of two mating types. While the sexual agglutination of cells of different species was not observed, mating type a cells of each species were shown to respond to alpha-factors produced by the other species. Thus, the mating response of S. globosus was shown to be identical to what has been observed in two other species of the same genera: S. cerevisiae and Saccharomyces kluyveri.     

Sic1 is phosphorylated by CK2 on Ser201 in budding yeast cells

We have previously identified Ser201 of Sic1, a yeast cyclin-dependent kinase inhibitor, as an in vitro target of protein kinase CK2. Here we present new evidence, by using specific anti-P-Ser201 antibodies and 2-D gel electrophoresis coupled to MALDI mass spectrometry analysis, that Sic1 is phosphorylated in vivo on Ser201 shortly after its de novo synthesis, during late anaphase in glucose-grown cells. This phosphorylation is also detected in Sic1 immunopurified from G1 cells. In agreement with these data we also show that the catalytic alpha' subunit of CK2, whose function is required for cell cycle progression, is detected in Sic1 immunopurified complexes, and that phosphorylation on Ser201 is reduced after CK2 inactivation at the non-permissive temperature in a cka1delta cka2(ts) yeast strain. These data strongly support the notion that CK2 phosphorylates Sic1 in vivo.