Fus3-regulated Tec1 degradation through SCFCdc4 determines MAPK signaling specificity during mating in yeast

Signaling specificity is fundamental for parallel mitogen-activated protein kinase (MAPK) cascades that control growth and differentiation in response to different stimuli. In Saccharomyces cerevisiae, components of the pheromone-responsive MAPK cascade activate Fus3 and Kss1 MAPKs to induce mating and Kss1 to promote filamentation. Active Fus3 is required to prevent the activation of the filamentation program during pheromone response. How Fus3 prevents the crossactivation is not clear. Here we show that Tec1, a cofactor of Ste12 for the expression of filamentation genes, is rapidly degraded during pheromone response. Fus3 but not Kss1 induces Tec1 ubiquination and degradation through the SCFCdc4 ubiquitin ligase. T273 in a predicted high-affinity Cdc4 binding motif is phosphorylated by Fus3 both in vitro and in vivo. Tec1T273V blocks Tec1 ubiquitination and degradation and allows the induction of filamentation genes in response to pheromone. Thus, Fus3 inhibits filamentous growth during mating by degrading Tec1.     

Homothallic switching of yeast mating type cassettes is initiated by a double-stranded cut in the MAT locus

A double-stranded DNA cut has been observed in the mating type (MAT) locus of the yeast Saccharomyces cerevisiae in cultures undergoing homothallic cassette switching. Cutting is observed in exponentially growing cells of genotype HO HML alpha MAT alpha HMR alpha or HO HMLa MATa HMRa, which switch continuously, but not in a/alpha HO/HO diploid strains, in which homothallic switching is known to be shut off. Stationary phase cultures do not exhibit the cut. Although this site-specific cut occurs in a sequence (Z1) common to the silent HML and HMR cassettes and to MAT, only the Z1 sequence at the MAT locus is cut. The cut at MAT occurs in the absence of the HML and HMR donor cassettes, suggesting that cutting initiates the switching process. An assay for switching on hybrid plasmids containing mata- cassettes has been devised, and deletion mapping has shown that the cut site is required for efficient switching. Thus a double-stranded cut at the MAT locus appears to initiate cassette transposition-substitution and defines MAT as the recipient in this process.     

The role of recombinational repair proteins in mating type switching in fission yeast cells

DNA double-strand breaks (DSBs) occur after exposing cells to ionizing radiation or under the action of various antitumor antibiotics. They can be also generated in the course cell processes, such as meiosis and mating type switching in yeast. The most preferential mechanism for the correction of DNA DSB in yeasts is recombinational repair controlled by RAD52 group genes. The role of recombinational repair in mating type switching of fission yeast cells was examined on the example of genes of this group, rhp51+ and rhp51+. We constructed homothallic strains of genotypes h90 rhp51 and h90 rhp55, and found that mutant cells yielded colonies with the mottled phenotype. In addition, h90 cells with deletions in these genes were shown to segregate heterothallic iodine-negative colonies h- and h+. The genome region, responsible for the switching process in these segregants, was analyzed by DNA hybridization. As shown in this analysis, h+ segregants had the h+N or h90 configuration of the mat region, whereas h-, the h90 configuration. Segregants h+ contained DNA duplication in the mat region. DNA rearrangements were not detected at the mating type locus, but the level of DNA DSB formation was drastically decreased in these segregants. Thus, our results show that genes rhp51+ and rhp55+ are involved not only in the repair of induced DNA DSB, but also in the mechanism of mating type switching in fission yeast.     

The role of recombinational repair proteins in mating type switching in fission yeast cells

DNA double-strand breaks (DSBs) occur after exposing cells to ionizing radiation or under the action of various antitumor antibiotics. They can be also generated in the course cell processes, such as meiosis and mating type switching in yeast. The most preferential mechanism for the correction of DNA DSB in yeasts is recombinational repair controlled by RAD52 group genes. The role of recombinational repair in mating type switching of fission yeast cells was examined on the example of genes of this group, rhp51 ⁺ and rhp55 ⁺. We constructed homothallic strains of genotypes h ⁹⁰ rhp51 and h ⁹⁰ rhp55, and found that mutant cells yielded colonies with the mottled phenotype. In addition, h ⁹⁰ cells with deletions in these genes were shown to segregate heterothallic iodine-negative colonies h ^? and h ⁺. The genome region, responsible for the switching process in these segregants, was analyzed by DNA hybridization. As shown in this analysis, h ⁺ segregants had the h ^+N or h ⁹⁰ configuration of the mat region, whereas h ^?, the h ⁹⁰ configuration. Segregants h ^+N contained DNA duplication in the mat region. DNA rearrangements were not detected at the mating type locus, but the level of DNA DSB formation was drastically decreased in these segregants. Thus, our results show that genes rhp51 ⁺ and rhp55 ⁺ are involved not only in the repair of induced DNA DSB, but also in the mechanism of mating type switching in fission yeast.     

Cell type-specific chromatin organization of the region that governs directionality of yeast mating type switching.

Switching of mating type in Saccharomyces cerevisiae is directional; MAT alpha cells recombine to transfer information from HMRa while MATa cells switch using the silent cassette at HML alpha. Genetic analysis recently has defined a 700 bp recombination enhancer approximately 29 kb from the left end of chromosome III that is necessary for directionality. The chromatin structure of this region differs strikingly in a- and alpha-cells. Mat alpha2p organizes a 3.7 kb chromatin domain that opposes interaction of trans-acting proteins with the enhancer. In a-cells lacking the alpha2 repressor, two footprinted regions flank an approximately 100 bp section having a unique DNA structure. This structural signature probably reflects interactions of proteins that result in directional mating type switching.     

The DNA repair protein yKu80 regulates the function of recombination enhancer during yeast mating type switching

Recombination enhancer (RE) is essential for regulating donor preference during yeast mating type switching. In this study, by using minichromosome affinity purification (MAP) and mass spectrometry, we found that yeast Ku80p is associated with RE in MATa cells. Chromatin immunoprecipitation assays confirmed its occupancy in vivo. Deletion of YKU80 results in altered chromatin structure in the RE region and more importantly causes a dramatic decrease of HML usage in MATa cells. We also detect directional movement of yKu80p from the RE towards HML during switching. These results indicate a novel function of yeast Ku80p in regulating mating type switching.     

Rearrangements at the mating type locus in fission yeast

Summary Crosses involving the partially defective mating type mutant B102 (functional in conjugation, defective in meiosis) have confirmed the notion that, in Schizosaccharomyces pombe, certain mating type mutations can arise by transposition. A copy of the mat2 ^P segment (specifying + mating type) is transposed and inserted into the mat1 ^M segment (usually specifying mating type). The mat1 ^M segment affected by the insertion loses its former function entirely. The function is, however, fully regained upon excision of the transposed and inserted mat2 ^P segment.At either position, the mat2 ^P segments can undergo inactivations to different states of residual activity. These events can occur about as frequent as other mutations of the mating type locus (ca. 10^–4 per cell division). In certain diploid strains, such inactivations were significantly correlated with recombination. Spontaneous reversions to full activity were also observed.     

Relation between the efficiency of homothallic switching of yeast mating type genes and the distribution of cell types.

Homothallic switching of yeast mating type genes occurs as often as each cell division, so that a colony derived from a single haploid spore soon contains an equal number of MATa and MAT alpha cells. Cells of opposite mating types conjugate, and eventually the colony contains only nonmating MATa/MAT alpha diploids. Mutations that reduce the efficiency of homothallic MAT conversions yield colonies that still contain many haploid cells of the original spore mating type plus a few recently generated cells of the opposite mating type. These (a greater than alpha)- or (alpha greater than a)-mating colonies also contain some nonmating diploid cells. As an alternative to microscopic pedigree analysis to determine the frequency of mating type conversions in a variety of mutant homothallic strains, we analyzed the proportions of MATa, MAT alpha, and MATa/MAT alpha cells in a colony by examining the mating phenotypes of subclones. We developed a mathematical model that described the proportion of cell types in a slow-switching colony. This model predicted that the proportion of nonmating cells would continually increase with the size (age) of a colony derived from a single cell. This prediction was confirmed by determining the proportion of cell types in colonies of an HO swi1 strain that was grown for different numbers of cell divisions. Data from subcloning (a greater than alpha) and (alpha greater than a) colonies from a variety of slow-switching mutations and chromosomal rearrangements were used to calculate the frequency of MAT conversions in these strains.     

The yeast UME6 gene product is required for transcriptional repression mediated by the CAR1 URS1 repressor binding site.

URS1 is known to be a repressor binding site in Saccharomyces cerevisiae that negatively regulates expression of many genes including CAR1 (arginase), several required for sporulation, mating type switching, inositol metabolism, and oxidative carbon metabolism. In addition to the proteins previously shown to directly bind to the URS1 site, we show here that the UME6 gene product is required for URS1 to mediate repression of gene expression in the absence of inducer. We also show that mutations in the CAR80 (CARGRI) gene are allelic to those in UME6.     

Physical monitoring of mating type switching in Saccharomyces cerevisiae.

The kinetics of mating type switching in Saccharomyces cerevisiae can be followed at the DNA level by using a galactose-inducible HO (GAL-HO) gene to initiate the event in synchronously growing cells. From the time that HO endonuclease cleaves MAT a until the detection of MAT alpha DNA took 60 min. When unbudded G1-phase cells were induced, switched to the opposite mating type in "pairs." In the presence of the DNA synthesis inhibitor hydroxyurea, HO-induced cleavage occurred but cells failed to complete switching. In these blocked cells, the HO-cut ends of MATa remained stable for at least 3 h. Upon removal of hydroxyurea, the cells completed the switch in approximately 1 h. The same kinetics of MAT switching were also seen in asynchronous cultures and when synchronously growing cells were induced at different times of the cell cycle. Thus, the only restriction that confined normal homothallic switching to the G1 phase of the cell cycle was the expression of HO endonuclease. Further evidence that galactose-induced cells can switch in the G2 phase of the cell cycle was the observation that these cells did not always switch in pairs. This suggests that two chromatids, both cleaved with HO endonuclease, can interact independently with the donors HML alpha and HMRa.     

Rum1, an inhibitor of cyclin-dependent kinase in fission yeast, is negatively regulated by mitogen-activated protein kinase-mediated phosphorylation at Ser and Thr residues.

The p25(rum1) is an inhibitor of Cdc2 kinase expressed in fission yeast and plays an important role in cell-cycle control. As its amino-acid sequence suggests that p25(rum1) has putative phosphorylation sites for mitogen-activated protein kinase (MAPK), we investigated the ability of MAPK to phosphorylate p25(rum1). Direct in vitro kinase assay using GST-fusion proteins of wild-type as well as various mutants of p25(rum1) demonstrated that MAPK phosphorylates the N-terminal portion of p25(rum1) and residues Thr13 and Ser19 are major phosphorylation sites for MAPK. In addition, phosphorylation of p25(rum1) by MAPK revealed markedly reduced Cdc2 kinase inhibitor ability of the protein. Together with the fact that replacement of both Thr13 and Ser19 with Glu, which mimics the phosphorylated state of these residues, also significantly reduces the activity of p25(rum1) as a Cdc2 inhibitor, it was suggested that the phosphorylation of Thr13 and Ser19 negatively regulates the function of p25(rum1). Further evidence indicates that phosphorylation of Thr13 and Ser19 may retain a negative effect on the function of p25(rum1) even in vivo. Therefore, MAPK may regulate the function of p25(rum1) via phosphorylation of its Thr and Ser residues and thus participate in cell cycle control in fission yeast.     

Genes involved in mating type expression of fission yeast

Summary Mutations of Schizosaccharomyces pombe are described, which change homothallism to heterothallism without affecting the mating type locus itself. The genetic block was effective at the level of agglutinated cells in two instances, or before that in two other cases. Mutations of particular interest affected meiosis as well, when homozygous diploid strains were constructed. In these strains azygotic meiosis was inducible by the addition of h ⁺ cells, even in the absence of cellular fusion.This work was supported by NIH grant GM 13234 initially, and by DFG (SFB 46).