Cell size modulation by CDC25 and RAS2 genes in Saccharomyces cerevisiae.

A detailed kinetic analysis of the cell cycle of cdc25-1, RAS2Val-19, or cdc25-1/RAS2Val-19 mutants during exponential growth is presented. At the permissive temperature (24 degrees C), cdc25-1 cells show a longer G1/unbudded phase of the cell cycle and have a smaller critical cell size required for budding without changing the growth rate in comparison to an isogenic wild type. The RAS2Val-19 mutation efficiently suppresses the ts growth defect of the cdc25-1 mutant at 36 degrees C and the increase of G1 phase at 24 degrees C. Moreover, it causes a marked increase of the critical cell mass required to enter into a new cell division cycle compared with that of the wild type. Since the critical cell mass is physiologically modulated by nutritional conditions, we have also studied the behavior of these mutants in different media. The increase in cell size caused by the RAS2Val-19 mutation is evident in all tested growth conditions, while the effect of cdc25-1 is apparently more pronounced in rich culture media. CDC25 and RAS2 gene products have been showed to control cell growth by regulating the cyclic AMP metabolic pathway. Experimental evidence reported herein suggests that the modulation of the critical cell size by CDC25 and RAS2 may involve adenylate cyclase.     

Multiple mechanisms regulate expression of low temperature responsive (LOT) genes in Saccharomyces cerevisiae

Using cDNA subtraction screening, we identified five Saccharomyces cerevisiae genes whose expressions is up-regulated when culture temperature was down-shifted from 30 to 10 degrees C. Among these LOT (low temperature-responsive) genes, three (LOT1, LOT2, and LOT3) were identical to FBA1, RPL2B, and NOP1, encoding a fructose biphosphate aldolase, a ribosomal protein L2B, and a nucleolar protein for rRNA processing, respectively. No functions were assigned for LOT5 and LOT6, which are identical to YKL183w and YLR011w, respectively. Northern hybridization analysis revealed that these genes are not uniformly regulated in response to the change of growth temperature. In addition, all the LOT genes, except for LOT1/FBA1, were induced by a low concentration of cycloheximide. The data indicate that multiple mechanisms, including translational functionality may be involved in the regulation of LOT gene expression in yeast.     

Regulatory function of the Saccharomyces cerevisiae RAS C-terminus.

Activating mutations (valine 19 or leucine 68) were introduced into the Saccharomyces cerevisiae RAS1 and RAS2 genes. In addition, a deletion was introduced into the wild-type gene and into an activated RAS2 gene, removing the segment of the coding region for the unique C-terminal domain that lies between the N-terminal 174 residues and the penultimate 8-residue membrane attachment site. At low levels of expression, a dominant activated phenotype, characterized by low glycogen levels and poor sporulation efficiency, was observed for both full-length RAS1 and RAS2 variants having impaired GTP hydrolytic activity. Lethal CDC25 mutations were bypassed by the expression of mutant RAS1 or RAS2 proteins with activating amino acid substitutions, by expression of RAS2 proteins lacking the C-terminal domain, or by normal and oncogenic mammalian Harvey ras proteins. Biochemical measurements of adenylate cyclase in membrane preparations showed that the expression of RAS2 proteins lacking the C-terminal domain can restore adenylate cyclase activity to cdc25 membranes.     

Positive control of sporulation-specific genes by the IME1 and IME2 products in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, meiosis and spore formation require the induction of sporulation-specific genes. Two genes are thought to activate the sporulation program: IME1 and IME2 (inducer of meiosis). Both genes are induced upon entry into meiosis, and IME1 is required for IME2 expression. We report here that IME1 is essential for expression of four sporulation-specific genes. In contrast, IME2 is not absolutely essential for expression of the sporulation-specific genes, but contributes to their rapid induction. Expression of IME2 from a heterologous promoter permits the expression of these sporulation-specific genes, meiotic recombination, and spore formation in the absence of IME1. We propose that the IME1 and IME2 products can each activate sporulation-specific genes independently. In addition, the IME1 product stimulates sporulation-specific gene expression indirectly through activation of IME2 expression.     

Multiple genes encode the translation elongation factor EF-1 gamma in Saccharomyces cerevisiae.

A gene encoding a yeast homologue of translation elongation factor 1 gamma (EF-1 gamma), TEF3, was isolated as a gene dosage extragenic suppressor of the cold-sensitive phenotype of the Saccharomyces cerevisiae drs2 mutant. The drs2 mutant is deficient in the assembly of 40S ribosomal subunits. We have identified a second gene, TEF4, that encodes a protein highly related to both the Tef3p protein (Tef3p), and EF-1 gamma isolated from other organisms. In contrast to TEF3, the TEF4 gene contains an intron. Gene disruptions showed that neither gene is required for mitotic growth. Haploid spores containing disruptions of both genes are viable and have no defects in ribosomal subunit composition or polyribosomes. Unlike TEF3, extra copies of TEF4 do not suppress the cold-sensitive 40S ribosomal subunit deficiency of a drs2 strain. Low-stringency genomic Southern hybridization analysis indicates there may be additional yeast genes related to TEF3 and TEF4.     

Allelism of IMP1 and GAL2 genes of Saccharomyces cerevisiae.

Cloning and characterization of the previously described Saccharomyces cerevisiae IMP1 gene, which was assumed to be a nuclear determinant involved in the nucleomitochondrial control of the utilization of galactose, demonstrate allelism to the GAL2 gene. Galactose metabolism does not necessarily involve the induction of the specific transport system coded by GAL2/IMP1, because a null mutant takes up galactose and grows on it. Data on galactose uptake are presented, and the dependence on ATP for constitutive and inducible galactose transport is discussed. These results can account for the inability of imp1/gal2 mutants to grow on galactose in a respiration-deficient background. Under these conditions, uptake was affected at the functional level but not at the biosynthetic level.     

A positive regulatory site and a negative regulatory site control the expression of the Saccharomyces cerevisiae CYC7 gene.

A series of BAL31 deletions were constructed in vitro in the upstream region of the Saccharomyces cerevisiae CYC7 gene, encoding the iso-2-cytochrome c protein. These deletions identified two sites which play a role in governing the expression of this gene. A positive site, the deletion of which led to decreased CYC7 expression, lay ca. 240 base pairs 5' to the translational initiation codon (-240). A negative site, the deletion of which led to greatly increased levels of CYC7 expression, lay at ca. -300 bp. Deletion of both these sites resulted in low wild-type-like expression of the gene. Therefore, these two sites appear to act antagonistically to give the low wild-type levels of CYC7 expression. Within the region defined as containing the positive site, there is a sequence which bears some homology to the upstream activation sites in the regulated gene, CYC1, encoding the iso-1-cytochrome c protein.     

The INO2 and INO4 loci of Saccharomyces cerevisiae are pleiotropic regulatory genes.

We isolated a mutant of Saccharomyces cerevisiae defective in the formation of phosphatidylcholine via methylation of phosphatidylethanolamine. The mutant synthesized phosphatidylcholine at a reduced rate and accumulated increased amounts of methylated phospholipid intermediates. It was also found to be auxotrophic for inositol and allelic to an existing series of ino4 mutants. The ino2 and ino4 mutants, originally isolated on the basis of an inositol requirement, are unable to derepress the cytoplasmic enzyme inositol-1-phosphate synthase (myo-inositol-1-phosphate synthase; EC 5.5.1.4). The INO4 and INO2 genes were, thus, previously identified as regulatory genes whose wild-type product is required for expression of the INO1 gene product inositol-1-phosphate synthase (T. Donahue and S. Henry, J. Biol. Chem. 256:7077-7085, 1981). In addition to the identification of a new ino4-allele, further characterization of the existing series of ino4 and ino2 mutants, reported here, demonstrated that they all have a reduced capacity to convert phosphatidylethanolamine to phosphatidylcholine. The pleiotropic phenotype of the ino2 and ino4 mutants described in this paper suggests that the INO2 and INO4 loci are involved in the regulation of phospholipid methylation in the membrane as well as inositol biosynthesis in the cytoplasm.     

Multiple protein tyrosine phosphatase-encoding genes in the yeast Saccharomyces cerevisiae.

In higher eukaryotic organisms, the regulation of tyrosine phosphorylation is known to play a major role in the control of cell division. Recently, a wide variety of protein tyrosine phosphatase (PTPase)-encoding genes (PTPs) have been identified to accompany the many tyrosine kinases previously studied. However, in the yeasts, where the cell cycle has been most extensively studied, identification of the genes involved in the direct regulation of tyrosine phosphorylation has been difficult. We have identified a pair of genes in the yeast Saccharomyces cerevisiae, which we call PTP1 and PTP2, whose products are highly homologous to PTPases identified in other systems. Both genes are poorly expressed, and contain sequence elements consistent with low-abundance proteins. We have carried out an extensive genetic analysis of PTP1 and PTP2, and found that they are not essential either singly or in combination. Neither deletion nor overexpression results in any strong phenotypes in a number of assays. Deletions also do not affect the mitotic blockage caused by deletion of the MIH1 gene (encoding a positive regulator of mitosis) and induction of the heterologous Schizosaccharomyces pombe wee1+ gene (encoding a negative regulator of mitosis). Molecular analysis has shown that PTP1 and PTP2 are quite different structurally and are not especially well conserved at the amino acid sequence level. Low-stringency Southern blots indicate that yeast may contain a family of PTPase-encoding genes. These results suggest that yeast may contain other PTPase-encoding genes that overlap functionally with PTP1 and PTP2.     

RAS genes in Saccharomyces cerevisiae: signal transduction in search of a pathway.

Ras proteins in budding yeasts initially appeared to regulate initiation of the cell cycle in response to nutrient availability. More recent work, while clarifying the mechanism of Ras-mediated signal transduction, has undermined our notion of the signal Ras transmits. We now suspect that Ras helps to coordinate cellular metabolism and mass accumulation, but what Ras responds to is not clear.     

A dominant interfering mutation in RAS1 of Saccharomyces cerevisiae

A mutant allele of RAS1 that dominantly interferes with the wild-type Ras function in the yeast Saccharomyces cerevisiae was discovered during screening of mutants that suppress an ira2 disruption mutation. A single amino acid substitution, serine for glycine at position 22, was found to cause the mutant phenotype. The inhibitory effect of the RAS1 ^Ser22 gene could be overcome either by overexpression of CDC25 or by the ira2 disruption mutation. These results suggest that the RAS1^Ser22 gene product interferes with the normal interaction of Ras with Cdc25 by forming a dead-end complex between Ras1^Ser22 and Cdc25 proteins.     

In vitro interaction between Saccharomyces cerevisiae CDC25 and RAS2 proteins.

In Saccharomyces cerevisiae the CDC25 protein is a positive regulator of RAS/cAMP pathway [1-4], enhancing the GDP-releasing rate of RAS2 protein [5]. In this work we have tried to detect a direct interaction between CDC25 and RAS2 gene products. The results indicate that both the whole RAS2 protein and a truncated version that lacks approximately 25 C-terminal residues interact specifically with the CDC25 protein. On the contrary, a derivative of RAS2 that lacks the 112 C-terminal residues as well as the p21TI-ras is not able to bind the CDC25 protein in our assay conditions. The 310 C-terminal aminoacids of CDC25 bind RAS2 while a C-terminus deletion within this aminoacid stretch abolishes the binding. The possible physiological significance of these findings is discussed.