A genetic screen reveals a role for the late G1-specific transcription factor Swi4p in diverse cellular functions including cytokinesis.

The transcription factor Swi4p plays a crucial role in the control of the initiation of the cell cycle in budding yeast. To further understand Swi4p function, we set up a synthetic lethal screen for genes interacting with SWI4. Fourteen conditional mutations which resulted in lethality only in the absence of SWI4 have been isolated. Only two of them were suppressed by ectopic expression of CLN2, indicating that Swi4p is involved in diverse cellular processes in addition to its requirement for CLN1,2 regulation. In most of the mutants a cell cycle phenotype was observed, including defects in G1 progression, budding, the G2/M transition and cytokinesis. In addition, four of the mutations resulted in massive cell lysis at the restrictive temperature, indicating that Swi4p is involved in the maintenance of cell integrity. One of the mutants, rsf1 swi4delta, was characterized in detail and it is defective in cytokinesis at the restrictive temperature. Staining with Calcofluor revealed that the rsf1 swi4delta mutant is impaired in chitin biosynthesis. rsf1 is allelic to the AGM1 gene, coding for N-acetylglucosamine-phosphate mutase, an enzyme involved in the biosynthesis of chitin. A single copy of SWI4 suppressed the cytokinesis defect. The above data suggest that Swi4p has a role in cytokinesis and becomes essential in this process when chitin biosynthesis is compromised. As overexpression or ectopic expression of CLN did not suppress the rsf1 swi4delta mutant phenotype, Swi4p must control some other gene(s) involved in cytokinesis.

Coordinated regulation of gene expression by the cell cycle transcription factor Swi4 and the protein kinase C MAP kinase pathway for yeast cell integrity.

Specific transcription in late G1, mediated by the transcription factors SBF (Swi4p-Swi6p) and MBF (Mbp1p-Swi6p), is crucial for cell cycle progression in budding yeast. In order to better understand the G1/S transition, we initiated a search for conditional mutations synthetic lethal with swi4delta. One of the isolated mutants, rsf8swi4delta, showed a growth defect due to cell lysis. rsf8 is allelic to PKC1, encoding a protein kinase C homologue which controls cell integrity. In the presence of the rsf8/(pkc1-8) mutation, a functional SBF but not MBF is required for viability. Importantly, swi4delta and swi6delta strains are hypersensitive to calcofluor white and SDS, indicating that they possess a weakened cell wall. Overexpression or ectopic expression of CLN did not suppress the pkc1-8swi4delta mutant phenotype, thus SBF must control cell integrity independently, rather than acting through CLN expression. We found that at least six genes involved in cell wall biosynthesis are periodically expressed at the G1/S phase boundary. In all six cases, cell cycle-regulated expression is due mainly to Swi4p. Finally, we found that the PKC1 MAP kinase pathway is a positive regulator of five of these cell wall genes, these genes being novel targets of regulation by this pathway. We suggest that SBF and the PKC1 MAP kinase pathway act in concert to maintain cell integrity during bud formation.

Respiration and low cAMP-dependent protein kinase activity are required for high-level expression of the peroxisomal thiolase gene in Saccharomyces cerevisiae.

Transcription of genes for peroxisomal proteins is repressed by glucose and induced by oleate. At least for the peroxisomal thiolase gene (POT1) there is a third regulatory mechanism, mediated by the transcription factor Adr1p, which is responsible for the high-level expression of the gene in stationary phase. Here we show that a region in the POT1 promoter that extends from positions -238 to -152 mediates this mechanism, and we suggest that Adr1p acts indirectly on POT1. We have also analyzed the role of the cAMP-dependent protein kinase (PKA) in the transcriptional regulation of POT1. PKA exerts a negative control: the high, unregulated PKA activity in a bcy1 mutant maintains POT1 transcription at the repressed level. In a ras2 mutant, which has low PKA activity, glucose repression is not alleviated but in non-repressing conditions POT1 regulation is perturbed and expression prematurely increases during exponential phase. This suggests that the PKA signalling pathway controls the regulation of POT1 in stationary phase. Finally, we have found that Adr1p-dependent expression in stationary phase and induction by oleate are both abolished when respiration is blocked. Utilization of fatty acids as carbon source requires respiration. Our result points to the existence of mechanisms that co-ordinate the level of expression of thiolase and the functional state of the mitochondria.

ADR1 and SNF1 mediate different mechanisms in transcriptional regulation of yeast POT1 gene.

We studied the consequences of adr1 and snf1 mutations on POT1 gene expression in different growth conditions. The results obtained reveal that ADR1 and SNF1 genes affect POT1 transcription in different ways: ADR1 has a minor role in derepression in low concentration of glucose but is essential for activation in stationary phase whereas SNF1 is essential for derepression and activation, although it does not seem to be directly involved in the molecular mechanism of activation in stationary phase.

Phylogenetic analysis of the thiolase family. Implications for the evolutionary origin of peroxisomes.

The thiolase family is a widespread group of proteins present in prokaryotes and three cellular compartments of eukaryotes. This fact makes this family interesting in order to study the evolutionary process of eukaryotes. Using the sequence of peroxisomal thiolase from Saccharomyces cerevisiae recently obtained by us and the other known thiolase sequences, a phylogenetic analysis has been carried out. It shows that all these proteins derived from a primitive enzyme, present in the common ancestor of eubacteria and eukaryotes, which evolved into different specialized thiolases confined to various cell compartments. The evolutionary tree obtained is compatible with the endosymbiotic theory for the origin of peroxisomes.

The POT1 gene for yeast peroxisomal thiolase is subject to three different mechanisms of regulation.

The Saccharomyces cerevisiae POT1 gene is, as are other yeast peroxisomal protein genes, inducible by fatty acids and repressible by glucose. We have now found that it is also induced during the stationary phase of the culture. To investigate these three regulatory circuits, we have studied the mRNA levels of regulatory mutants as well as the changes in chromatin structure upon gene activation. We conclude that the regulation of transcriptional activity in glucose repression, oleate induction, and stationary phase induction follow different molecular mechanisms. We suggest that this multiplicity of regulatory mechanisms may represent a general rule for the yeast peroxisomal protein genes.

A new glucose-repressible gene identified from the analysis of chromatin structure in deletion mutants of yeast SUC2 locus.

We have previously shown that some changes occur in the chromatin structure of the 3' flank of the yeast SUC2 gene in going from a repressed to an active state. In an attempt to find out the causes of these changes, we have carried out experiments in which mutant copies of SUC2 locus lacking either 5' or 3' flanks have been analysed for their transcriptional activity and chromatin structure. These experiments allowed us to discard any relationship between SUC2 transcription and chromatin changes within its 3'flank. Sequencing of this flank and mRNA analysis, however, resulted in the location of a putative peroxisomal 3-oxoacyl-CoA thiolase gene (POT1), which is repressible by glucose. The disruption of the gene produced a yeast strain unable to use oleic acid as a carbon source. This is the first time that chromatin structure analysis has permitted the identification of a new gene.