RCS1, a gene involved in controlling cell size in Saccharomyces cerevisiae

Cloning and sequencing of RCS1, Saccharomyces cerevisiae gene whose product seems to be involved in timing the budding event of the cell cycle, is described. A haploid strain in which the 3'-terminal region of the chromosomal copy of the gene has been disrupted produces cells that are, on average, twice the size of cells of the parental strain. The critical size for budding in the mutant is similarly increased, and the disruption mutation is dominant in a diploid heterozygous for the RCS1 gene. Spores from this diploid have a reduced ability to germinate, the effect being more pronounced in the spores carrying the disrupted copy of RCS1. However, disrupted cells recover from alpha-factor treatment equally as well as wild-type cells.     

The Saccharomyces cerevisiae TGL2 gene encodes a protein with lipolytic activity and can complement an Escherichia coli diacylglycerol kinase disruptant

Escherichia coli cells with a disrupted diacylglycerol kinase gene are unable to grow on media containing arbutin due to a lethal accumulation of diacylglycerol. In order to isolate genes from the yeast Saccharomyces cerevisiae involved in diacylglycerol metabolism we complemented an E. coli diacylglycerol kinase disruptant with a yeast genomic library and transformants were selected capable of growing in the presence of arbutin. Using this method, a gene (TGL2) was isolated coding for a protein resembling lipases from Pseudomonas. After expression of the TGL2 gene in E. coli, lipolytic activity towards triacylglycerols and diacylglycerols with short-chain fatty acids could be measured. Therefore, it is very likely that the TGL2 gene can complement the E. coli diacylglycerol kinase disruptant, because it encodes a protein that degrades the diacylglycerol accumulated after growth in the presence of arbutin. Disruption of the TGL2 gene in S. cerevisiae did not result in a detectable phenotype. The role of the Tgl2 protein in lipid degradation in yeast is still unclear. The nucleotide sequence published here has been submitted to the EMBL sequence data bank and is available under accession number X98000. © 1998 John Wiley & Sons, Ltd.     

Cloning and genetic analysis of the gene encoding a new protein kinase in Saccharomyces cerevisiae

We have isolated a single gene from the yeast Saccharomyces cerevisiae encoding a potential 800 amino acid polypeptide of calculated M_r 90 098 Da. This protein consists of an N-terminal region that shares significant homology with the catalytic domains of several serine- and threonine-specific protein kinases, as well as a large, unique, C-terminal domain of unknown function. Haploid disruption mutants are viable and do not exhibit any readily observable growth defects under varying conditions of temperature, nutrients or osmotic strength. Due to the apparent structural similarity between this kinase and the protein products of the KIN1 and KIN2 genes, we have chosen to name this new gene KIN3.     

Cloning of CDC33: a gene essential for growth and sporulation which does not interfere with cAMP production in Saccharomyces cerevisiae

The CDC33 gene of Saccharomyces cerevisiae belongs to the class II 'START' genes. Its product is required for the initiation of a new cell division cycle (Hartwell, 1974). Many results suggest that the cAMP signalling pathway is one of the major controlling elements of 'START'. Components of this pathway are encoded by class II 'START' genes. The aim of the present study is to determine whether or not the CDC33 gene interferes with the cAMP signalling pathway. We report here the molecular cloning of the CDC33 gene by complementation of the cdc33-1 thermosensitive mutant. The identity of the cloned gene is confirmed by site-specific reintegration and segregation analysis. This gene is transcribed into a 900-nucleotides mRNA and appears to be relatively abundant in the cell. We also show that the CDC33 gene product is essential for sporulation. cdc33-1 mutant cells are able to enter into the resting state. The cAMP intracellular pool is not modified when the cdc33-1 mutant is shifted to the restrictive temperature. The cdc33-1 mutation is not suppressed by other known elements of the cAMP cascade. All these results suggest that the CDC33 'START' gene does not interfere with the cAMP signalling pathway which controls cell division.     

The product of the KIN1 locus in Saccharomyces cerevisiae is a serine/threonine-specific protein kinase.

The catalytic domain (30 kDa) of all protein kinases can be aligned for maximum homology, thereby revealing both invariant and highly conserved residues. The KIN1 locus from Saccharomyces cerevisiae was isolated by hybridization to a degenerate oligonucleotide encoding the conserved protein kinase domain, DVWSFG. The predicted amino acid sequence revealed significant homology to the catalytic domain of protein kinases. Using antibodies raised against a bacterial LacZ/KIN1 fusion protein, we have identified by immunoprecipitation the yeast KIN1 gene product as a 145,000 dalton protein (p145KIN1). In exponentially growing yeast cells, the KIN1 protein is phosphorylated primarily on serine residues. The gene product of KIN1 was shown to be a serine/threonine-specific protein kinase in immune complexes, as determined by the transfer of label from [gamma-32P]ATP to either pp145KIN1 or to an exogenously added substrate, alpha-casein. The optimal metal ion concentration in this assay was 20 mM-MnCl2. Subsequent phosphoamino acid analysis of the radiolabelled product, pp145KIN1, demonstrated that this autophosphorylation was specific for serine/threonine residues. There is no apparent difference between wild-type cells and cells containing a disrupted KIN1 gene. The biochemical characterization of protein kinases in simple eukaryotes such as yeast will aid us in determining the role of phosphorylation in cellular growth and physiology.     

The SKS1 protein kinase is a multicopy suppressor of the snf3 mutation of Saccharomyces cerevisiae

Saccharomyces cerevisiae strains carrying snf3 are defective in high affinity glucose transport, and thus are unable to grow fermentatively on media with low concentrations of glucose. A multicopy suppressor of the snf3 growth defect, SKS1 (suppressor kinase of snf3), was found to encode a putative ser/thr protein kinase homologous to Ran1p, a kinase that regulates the switch between meiosis and vegetative growth in Schizosaccharomyces pombe. Overexpression of the SKS1 open reading frame is sufficient for suppression of the growth defects of snf3 mutants. Disruption of the open reading frame eliminates this suppression; as does the mutation of the consensus ATP binding site of Sks1p. A DDSE (DNA dependent snf3 suppressor element) was found to be present in the SKS1 promoter region. The suppression by this DDSE occurs in the absence of SKS1 coding region, that is, the DDSE can suppress a snf3 sks1 double null mutant which fails to grow fermentatively on low glucose as a snf3 mutant does. Both SKS1 and its DDSE can additionally suppress the growth defects of grr1 mutants, which are also impaired in high affinity glucose transport. The snf3 genomic suppressors, rgt1, RGT2 and ssn6, are also capable of suppressing snf3 associated growth defects in a strain lacking sks1.     

Importance of cell wall mannoproteins for septum formation in Saccharomyces cerevisiae

The mannosyltransferase mutants mnn9 and mnn10 were isolated in a genetic screen for septation defects in Saccharomyces cerevisiae. Ultrastructural examination of mutant cell walls revealed markedly thin septal structures and occasional failure to construct trilaminar septa, which then led to the formation of bulky default septa at the bud neck. In the absence of a functional septation apparatus, mnn10 mutants are unable to complete cytokinesis and die as cell chains with incompletely separated cytoplasms, indicating that mannosylation defects impair the ability to form remedial septa. We could not detect N-linked glycosylation of the beta(1,3)glucan synthase Fks1p and mnn10 defects do not change the molecular weight or abundance of the protein. We discuss a model explaining the pleiotropic effects of impaired N-linked protein glycosylation on septation in S. cerevisiae.     

Genetic evidence for a functional interaction between Saccharomyces cerevisiae CDC24 and CDC42

Cdc24p and Cdc42p are involved in the control of cell polarity during the Saccharomyces cerevisiae cell cycle. Cdc42p is a member of the Ras superfamily of GTPases and Cdc24p displays limited amino-acid sequence similarity with the Dbl proto-oncoprotein, which acts to stimulate guanine-nucleotide exchange on human Cdc42p. We have performed several genetic experiments to test whether Cdc24p and Cdc42p interact within the cell. First, overexpression of Cdc24p suppressed the dominant-negative cdc42^D118A allele. Second, overexpression of wild-type CDC24 and CDC42 genes together was a lethal event resulting in a morphological phenotype of large, round, unbudded cells, indicating a loss of cell polarity. Third, a cdc24^ts cdc42^ts double mutant exhibited a synthetic-lethal phenotype at the semi-permissive temperature of 30°C. These data suggest that Cdc24p and Cdc42p interact within the cell and that Cdc24p may be involved in the regulation of Cdc42p activity.     

Effects of a novel DNA-damaging agent on the budding yeast Saccharomyces cerevisiae cell cycle

We have investigated the effects on Saccharomyces cerevisiae of a novel antitumour agent (FCE24517 or Tallimustine) which causes selective alkylations to adenines in the minor groove of DNA. Tallimustine, added to wild-type cells for short periods, reduced the growth rate and increased the percentage of budded cells and delayed the cell cycle in the late S+G₂+M phases. In the rad9Δ null mutant cells, Tallimustine treatment did not affect growth rate and the percentage of budded cells but greatly reduced cell viability compared to isogenic cells. Consistent with a role of RAD9 in inducing a transient delay in G₂ phase which preserves cell viability, the potent cytotoxic effect of the drug on rad9Δ cells was alleviated by treatment with nocodazole. Tallimustine was also found to delay the resumption from G₁ arrest of wild-type but not of rad9Δ cells. These data indicate that the effects of Tallimustine on cell cycle progression in yeast are mediated by the RAD9 gene product. From our data it appears that yeast could be a valuable model system to study the mode of action of this alkylating drug and of minor groove alkylators in general.     

Para-aminobenzoate synthase gene of Saccharomyces cerevisiae encodes a bifunctional enzyme

The Saccharomyces cerevisiae gene for para-aminobenzoate (PABA) synthase has been identified based upon its ability to confer sulfonamide resistance when present on a multicopy episomal vector. The 3840 bp DNA sequence fragment reported here contains a 2199 bp open reading frame encoding a 733 amino acid protein with similarity to the two components of PABA synthase described for prokaryotes (Escherichia coli PabA and PabB), suggesting that PABA synthase is bifunctional in yeast. The cloned sequence was confirmed to be PABA synthase by gene disruption. Chromosome gel analysis places the gene for PABA synthase on chromosome XIV.     

Identification of multicopy suppressors of cell cycle arrest at the G1-S transition in Saccharomyces cerevisiae

Inactivation of HAL3 in the absence of SIT4 function leads to cell cycle arrest at the G(1)-S transition. To identify genes potentially involved in the control of this phase of the cell cycle, a screening for multicopy suppressors of a conditional sit4 hal3 mutant (strain JC002) has been developed. The screening yielded several genes known to perform key roles in cell cycle events, such as CLN3, BCK2 or SWI4, thus proving its usefulness as a tool for this type of studies. In addition, this approach allowed the identification of additional genes, most of them not previously related to the regulation of G(1)-S transition or even without known function (named here as VHS1-3, for viable in a hal3 sit4 background). Several of these gene products are involved in phospho-dephosphorylation processes, including members of the protein phosphatase 2A and protein phosphatases 2C families, as well as components of the Hal5 protein kinase family. The ability of different genes to suppress sit4 phenotypes (such as temperature sensitivity and growth on non-fermentable carbon sources) or to mimic the functions of Hal3 was evaluated. The possible relationship between the known functions of these suppressor genes and the progress through the G(1)-S transition is discussed.     

Identification of ACT4, a novel essential actin-related gene in the yeast Saccharomyces cerevisiae

Actin molecules are major cytoskeleton components of all eukaryotic cells. All conventional actins that have been identified so far are 374–376 amino acids in size and exhibit at least 70% amino acid sequence identity when compared with one another. In the yeast Saccharomyces cerevisiae, one conventional actin gene ACT1 and three so-called actin-related genes, ACT2, ACT3 and ACT5, have been identified. We report here the discovery of a new actin-related gene in this organism, which we have named ACT4. The deduced protein, Act4, of 449 amino acids, exhibits only 33·4%, 26·7%, 23·4% and 29·2% identity to Act1, Act2, Act3 and Act5, respectively. In contrast, it is 68·4% identical to the product of the Schizosaccharomyces pombe Act2 gene and has a similar level of identity to other Sch. pombe Act2 homologues. This places Act4 in the Arp3 family of actin-related proteins. ACT4 gene disruption and tetrad analysis demonstrate that this gene is essential for the vegetative growth of yeast cells. The act4 mutants exhibit heterogenous morphological phenotypes. We hypothesize that Act4 may have multiple roles in the cell cycle. The sequence has been deposited in the Genome Sequence Data Base under Accession Number L37111.     

Isolation of a CDC25 family gene,MSI2/LTE1, as a multicopy suppressor of ira1

We have identified MS12 as a gene of Saccharomyces cerevisiae which, when on a multicopy vector, suppresses the heat shock sensitivity caused by the loss of the IRA1 product, a negative regulator of the RAS protein. The multicopy MSI2 also suppresses the heat shock sensitivity of cells with the RAS2^val19 mutation but not those with the bcy1 mutation, suggesting that the MSI2 protein may interfere with the activity of the RAS protein. The sequence analysis of MSI2 reveals that it is identical to LTE1 belonging to the CDC25 family: CDC25, SCD25 and BUD5, each of which encodes a guanine nucleotide exchange factor for the ras superfamily gene products. Deletion of the entire MSI2 coding region reveals that MSI2 is not essential but the disruptant shows a cold-sensitive phenotype. Under the non-permissive conditions, more than 70% of the msi2 disruptants arrested at telophase as large budded cells with two nuclei divided completely and elongated spindles, indicating that the msi2 deletion is a cell division cycle mutation. These results suggest that MSI2 is involved in the termination of M phase and that this process is regulated by a ras superfamily gene product.     

Functional,comparative and cell biological analysis of Saccharomyces cerevisiae Kre5p

Saccharomyces cerevisiae kre5delta mutants lack beta-1,6-glucan, a polymer required for proper cell wall assembly and architecture. A functional and cell biological analysis of Kre5p was conducted to further elucidate the role of this diverged protein glucosyltransferase-like protein in beta-1,6-glucan synthesis. Kre5p was found to be a primarily soluble N-glycoprotein of approximately 200 kDa, that localizes to the endoplasmic reticulum. The terminal phenotype of Kre5p-deficient cells was observed, and revealed a severe cell wall morphological defect. KRE6, encoding a glucanase-like protein, was identified as a multicopy suppressor of a temperature-sensitive kre5 allele, suggesting that these proteins may participate in a common beta-1,6-biosynthetic pathway. An analysis of truncated versions of Kre5p indicated that all major regions of the protein are required for viability. Finally, Candida albicans KRE5 was shown to partially restore growth to S. cerevisiae kre5delta cells, suggesting that these proteins are functionally related.     

Saccharomyces cerevisiae Big1p,a putative endoplasmic reticulum membrane protein required for normal levels of cell wall beta-1,6-glucan

Deletion of Saccharomyces cerevisiae BIG1 causes an approximately 95% reduction in cell wall beta-1,6-glucan, an essential polymer involved in the cell wall attachment of many surface mannoproteins. The big1 deletion mutant grows very slowly, but growth can be enhanced if cells are given osmotic support. We have begun a cell biological and genetic analysis of its product. We demonstrate, using a Big1p-GFP fusion construct, that Big1p is an N-glycosylated integral membrane protein with a Type I topology that is located in the endoplasmic reticulum (ER). Some phenotypes of a big1Delta mutant resemble those of strains disrupted for KRE5, which encodes another ER protein affecting beta-l,6-glucan levels to a similar extent. In a big1Deltakre5Delta double mutant, both the growth and alkali-soluble beta-l,6-glucan levels were reduced as compared to either single mutant. Thus, while Big1p and Kre5p may have similar effects on beta-l,6-glucan synthesis, these effects are at least partially distinct. Residual beta-l,6-glucan levels in the big1Deltakre5Delta double mutant indicate that these gene products are unlikely to be beta-l,6-glucan synthase subunits, but rather may play some ancillary roles in beta-l,6-glucan synthase assembly or function, or in modifying proteins for attachment of beta-l,6-glucan.     

Localization of a protein a-tagged kex2 protein to the vacuole of Saccharomyces cerevisiae allows rapid purification of vacuolar membranes

We have previously reported an immunoisolation procedure which allows purification of Kex2p-containing Golgi membranes from lysed yeast cells. In order to evaluate the use of tagging procedures in organelle isolation we set out to isolate the same Golgi membrane fraction using a version of the Kex2 protease that had been affinity-tagged at its C-terminus. This protein is found to be localized in the vacuole, providing the basis of a method for the affinity-purification of vacuolar membranes.