scholarly journals The Saccharomyces cerevisiae CDC25 gene product binds specifically to catalytically inactive ras proteins in vivo.

1992 ◽  
Vol 12 (5) ◽  
pp. 2091-2099 ◽  
Author(s):  
T Munder ◽  
P Fürst
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Product ◽  
Cell Cycle Control ◽  
Bound State ◽  
Negative Regulator ◽  
Yeast Cells ◽  
Nucleotide Exchange ◽  
Protein Protein Interaction ◽  
Upstream Regulator

Genetic data suggest that the yeast cell cycle control gene CDC25 is an upstream regulator of RAS2. We have been able to show for the first time that the guanine nucleotide exchange proteins Cdc25 and Sdc25 from Saccharomyces cerevisiae bind directly to their targets Ras1 and Ras2 in vivo. Using the characteristics of the yeast Ace1 transcriptional activator to probe for protein-protein interaction, we found that the CDC25 gene product binds specifically to wild-type Ras2 but not to the mutated Ras2Val-19 and Ras2 delta Val-19 proteins. The binding properties of Cdc25 to Ras2 were strongly diminished in yeast cells expressing an inactive Ira1 protein, which normally acts as a negative regulator of Ras activity. On the basis of these data, we propose that the ability of Cdc25 to interact with Ras2 proteins is strongly dependent on the activation state of Ras2. Cdc25 binds predominantly to the catalytically inactive GDP-bound form of Ras2, whereas a conformational change of Ras2 to its activated GTP-bound state results in its loss of binding affinity to Cdc25.

The Saccharomyces cerevisiae CDC25 gene product binds specifically to catalytically inactive ras proteins in vivo

1992 ◽  
Vol 12 (5) ◽  
pp. 2091-2099
Author(s):  
T Munder ◽  
P Fürst
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Product ◽  
Cell Cycle Control ◽  
Bound State ◽  
Negative Regulator ◽  
Yeast Cells ◽  
Nucleotide Exchange ◽  
Protein Protein Interaction ◽  
Upstream Regulator

Genetic data suggest that the yeast cell cycle control gene CDC25 is an upstream regulator of RAS2. We have been able to show for the first time that the guanine nucleotide exchange proteins Cdc25 and Sdc25 from Saccharomyces cerevisiae bind directly to their targets Ras1 and Ras2 in vivo. Using the characteristics of the yeast Ace1 transcriptional activator to probe for protein-protein interaction, we found that the CDC25 gene product binds specifically to wild-type Ras2 but not to the mutated Ras2Val-19 and Ras2 delta Val-19 proteins. The binding properties of Cdc25 to Ras2 were strongly diminished in yeast cells expressing an inactive Ira1 protein, which normally acts as a negative regulator of Ras activity. On the basis of these data, we propose that the ability of Cdc25 to interact with Ras2 proteins is strongly dependent on the activation state of Ras2. Cdc25 binds predominantly to the catalytically inactive GDP-bound form of Ras2, whereas a conformational change of Ras2 to its activated GTP-bound state results in its loss of binding affinity to Cdc25.

scholarly journals Prenylation of Saccharomyces cerevisiae Chs4p Affects Chitin Synthase III Activity and Chitin Chain Length

2006 ◽  
Vol 6 (2) ◽  
pp. 328-336 ◽  
Author(s):  
Kariona A. Grabińska ◽  
Paula Magnelli ◽  
Phillips W. Robbins
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chain Length ◽  
Attachment Site ◽  
Chitin Synthase ◽  
Yeast Cells ◽  
Average Chain Length ◽  
Calcofluor White ◽  
Protein Factor

ABSTRACT Chs4p (Cal2/Csd4/Skt5) was identified as a protein factor physically interacting with Chs3p, the catalytic subunit of chitin synthase III (CSIII), and is indispensable for its enzymatic activity in vivo. Chs4p contains a putative farnesyl attachment site at the C-terminal end (CVIM motif) conserved in Chs4p of Saccharomyces cerevisiae and other fungi. Several previous reports questioned the role of Chs4p prenylation in chitin biosynthesis. In this study we reinvestigated the function of Chs4p prenylation. We provide evidence that Chs4p is farnesylated by showing that purified Chs4p is recognized by anti-farnesyl antibody and is a substrate for farnesyl transferase (FTase) in vitro and that inactivation of FTase increases the amount of unmodified Chs4p in yeast cells. We demonstrate that abolition of Chs4p prenylation causes a ∼60% decrease in CSIII activity, which is correlated with a ∼30% decrease in chitin content and with increased resistance to the chitin binding compound calcofluor white. Furthermore, we show that lack of Chs4p prenylation decreases the average chain length of the chitin polymer. Prenylation of Chs4p, however, is not a factor that mediates plasma membrane association of the protein. Our results provide evidence that the prenyl moiety attached to Chs4p is a factor modulating the activity of CSIII both in vivo and in vitro.

The activation of adenylate cyclase by guanyl nucleotides in Saccharomyces cerevisiae is controlled by the CDC25 start gene product

1987 ◽  
Vol 7 (10) ◽  
pp. 3857-3861
Author(s):  
J Daniel ◽  
J M Becker ◽  
E Enari ◽  
A Levitzki
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Adenylate Cyclase ◽  
Gene Product ◽  
Ternary Complex ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Terminal Portion ◽  
Altered Regulation ◽  
Cdc25 Protein

In the thermosensitive cdc25 start mutant of Saccharomyces cerevisiae, the regulation of adenylate cyclase by guanyl nucleotides was rapidly nullified when the enzyme was prepared from nonsynchronized cells shifted to the restrictive temperature. In agreement with previous in vivo complementation studies, this biochemical defect was fully suppressed by the expression of either the whole cloned CDC25 gene or its C-terminal portion. Moreover, membranes prepared from cdc25(Ts) cells grown at the permissive temperature evinced an altered regulation of adenylate cyclase by guanyl nucleotides. These results indicate that the CDC25 protein, together with RAS, is involved in the regulation of adenylate cyclase by guanyl nucleotides and raise the possibility that adenylate cyclase might form a ternary complex with RAS and CDC25.

Functional interaction between p21rap1A and components of the budding pathway in Saccharomyces cerevisiae

1992 ◽  
Vol 12 (9) ◽  
pp. 4084-4092
Author(s):  
P C McCabe ◽  
H Haubruck ◽  
P Polakis ◽  
F McCormick ◽  
M A Innis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Bound States ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Amino Terminal ◽  
Loop Region

The rap1A gene encodes a 21-kDa, ras-related GTP-binding protein (p21rap1A) of unknown function. A close structural homolog of p21rap1A (65% identity in the amino-terminal two-thirds) is the RSR1 gene product (Rsr1p) of Saccharomyces cerevisiae. Although Rsr1p is not essential for growth, its presence is required for nonrandom selection of bud sites. To assess the similarity of these proteins at the functional level, wild-type and mutant forms of p21rap1A were tested for complementation of activities known to be fulfilled by Rsr1p. Expression of p21rap1A, like multicopy expression of RSR1, suppressed the conditional lethality of a temperature-sensitive cdc24 mutation. Point mutations predicted to affect the localization of p21rap1A or its ability to cycle between GDP and GTP-bound states disrupted suppression of cdc24ts, while other mutations in the 61-65 loop region improved suppression. Expression of p21rap1A could not, however, suppress the random budding phenotype of rsr1 cells. p21rap1A also apparently interfered with the normal activity of Rsrlp, causing random budding in diploid wild-type cells, suggesting an inability of p21rap1A to interact appropriately with Rsr1p regulatory proteins. Consistent with this hypothesis, we found an Rsr1p-specific GTPase-activating protein (GAP) activity in yeast membranes which was not active toward p21rap1A, indicating that p21rap1A may be predominantly GTP bound in yeast cells. Coexpression of human Rap1-specific GAP suppressed the random budding due to expression of p21rap1A or its derivatives, including Rap1AVal-12. Although Rap1-specific GAP stimulated the GTPase of Rsr1p in vitro, it did not dominantly interfere with Rsr1p function in vivo. A chimera consisting of Rap1A1-165::Rsr1p166-272 did not exhibit normal Rsr1p function in the budding pathway. These results indicated that p21rap1A and Rsr1p share at least partial functional homology, which may have implications for p21rap1A function in mammalian cells.

scholarly journals Identification of a mouse protein whose homolog in Saccharomyces cerevisiae is a component of the CCR4 transcriptional regulatory complex.

1995 ◽  
Vol 15 (7) ◽  
pp. 3487-3495 ◽  
Author(s):  
M P Draper ◽  
C Salvadore ◽  
C L Denis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Yeast Cells ◽  
Significant Similarity ◽  
Eukaryotic Transcription ◽  
C Elegans ◽  
Mouse Protein ◽  
Transcriptional Regulatory ◽  
Evolutionarily Conserved ◽  
Regulatory Complex

The CCR4 protein from Saccharomyces cerevisiae is a component of a multisubunit complex that is required for the regulation of a number of genes in yeast cells. We report here the identification of a mouse protein (mCAF1 [mouse CCR4-associated factor 1]) which is capable of interacting with and binding to the yeast CCR4 protein. The mCAF1 protein was shown to have significant similarity to proteins from humans, Caenorhabditis elegans, Arabidopsis thaliana, and S. cerevisiae. The yeast gene (yCAF1) had been previously cloned as the POP2 gene, which is required for expression of several genes. Both yCAF1 (POP2) and the C. elegans homolog of CAF1 were shown to genetically interact with CCR4 in vivo, and yCAF1 (POP2) physically associated with CCR4. Disruption of the CAF1 (POP2) gene in yeast cells gave phenotypes and defects in transcription similar to those observed with disruptions of CCR4, including the ability to suppress spt10-enhanced ADH2 expression. In addition, yCAF1 (POP2) when fused to LexA was capable of activating transcription. mCAF1 could also activate transcription when fused to LexA and could functionally substitute for yCAF1 in allowing ADH2 expression in an spt10 mutant background. These data imply that CAF1 is a component of the CCR4 protein complex and that this complex has retained evolutionarily conserved functions important to eukaryotic transcription.

scholarly journals Yeast repressor alpha 2 binds to its operator cooperatively with yeast protein Mcm1.

1989 ◽  
Vol 9 (11) ◽  
pp. 5228-5230 ◽  
Author(s):  
C A Keleher ◽  
S Passmore ◽  
A D Johnson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Product ◽  
Strong Evidence ◽  
Regulatory Proteins ◽  
Yeast Cells ◽  
Specific Gene ◽  
Yeast Gene ◽  
Yeast Protein ◽  
Transcriptional Regulatory

To bring about repression of a family fo genes in Saccharomyces cerevisiae called the a-specific genes, two transcriptional regulatory proteins, alpha 2 and GRM (general regulator of matin type), bind cooperatively to an operator found upstream of each a-specific gene. To date, GRM has been defined only biochemically. In this communication we show that the product of a single yeast gene (MCM1) is sufficient to bind cooperatively with alpha 2 to the operator. We also show that antiserum raised against the MCM1 gene product recognizes GRM from yeast cells. These results, in combination with previous observations, provide strong evidence that MCM1 encodes the GRM activity.

The N-terminal TPR region is the functional domain of SSN6, a nuclear phosphoprotein of Saccharomyces cerevisiae

1990 ◽  
Vol 10 (9) ◽  
pp. 4744-4756
Author(s):  
J Schultz ◽  
L Marshall-Carlson ◽  
M Carlson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Function Analysis ◽  
Normal Growth ◽  
Negative Regulator ◽  
Tetratricopeptide Repeat ◽  
Functional Domain ◽  
Snf1 Kinase ◽  
C Terminus ◽  
Protein Functions

The SSN6 protein functions as a negative regulator of a variety of genes in Saccharomyces cerevisiae and is required for normal growth, mating, and sporulation. It is a member of a family defined by a repeated amino acid sequence, the TPR (tetratricopeptide repeat) motif. Here, we have used specific antibody to identify and characterize the SSN6 protein. Both SSN6 and a bifunctional SSN6-beta-galactosidase fusion protein were localized in the nucleus by immunofluorescence staining. The N-terminal one-third of the protein containing the TPR units was identified as the region that is important for SSN6 function. Analysis of four nonsense alleles, isolated as intragenic suppressors of an ssn6::URA3 insertion, revealed that polypeptides truncated after TPR unit 7 provide SSN6 function. Deletion analysis suggested that TPR units are required but that 4 of the 10 TPR units are sufficient. In addition, deletion studies indicated that three very long, homogeneous tracts of polyglutamine and poly(glutamine-alanine) are dispensable. Previous genetic evidence suggested the SSN6 protein as a possible target of the SNF1 protein kinase. Here, we show that the C terminus of SSN6 is phosphorylated in vivo and that the SNF1 kinase is not responsible for most of the phosphorylation. Finally, SSN6 has a modest effect on the maintenance of minichromosomes.

Molecular genetic analysis of Saccharomyces cerevisiae C1-tetrahydrofolate synthase mutants reveals a noncatalytic function of the ADE3 gene product and an additional folate-dependent enzyme

1990 ◽  
Vol 10 (11) ◽  
pp. 5679-5687
Author(s):  
C K Barlowe ◽  
D R Appling
Keyword(s):  
Saccharomyces Cerevisiae ◽  
De Novo ◽  
Point Mutations ◽  
Gene Replacement ◽  
Molecular Genetic Analysis ◽  
Yeast Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Purine Synthesis

In eucaryotes, 10-formyltetrahydrofolate (formyl-THF) synthetase, 5,10-methenyl-THF cyclohydrolase, and NADP(+)-dependent 5,10-methylene-THF dehydrogenase activities are present on a single polypeptide termed C1-THF synthase. This trifunctional enzyme, encoded by the ADE3 gene in the yeast Saccharomyces cerevisiae, is thought to be responsible for the synthesis of the one-carbon donor 10-formyl-THF for de novo purine synthesis. Deletion of the ADE3 gene causes adenine auxotrophy, presumably as a result of the lack of cytoplasmic 10-formyl-THF. In this report, defined point mutations that affected one or more of the catalytic activities of yeast C1-THF synthase were generated in vitro and transferred to the chromosomal ADE3 locus by gene replacement. In contrast to ADE3 deletions, point mutations that inactivated all three activities of C1-THF synthase did not result in an adenine requirement. Heterologous expression of the Clostridium acidiurici gene encoding a monofunctional 10-formyl-THF synthetase in an ade3 deletion strain did not restore growth in the absence of adenine, even though the monofunctional synthetase was catalytically competent in vivo. These results indicate that adequate cytoplasmic 10-formyl-THF can be produced by an enzyme(s) other than C1-THF synthase, but efficient utilization of that 10-formyl-THF for purine synthesis requires a nonenzymatic function of C1-THF synthase. A monofunctional 5,10-methylene-THF dehydrogenase, dependent on NAD+ for catalysis, has been identified and purified from yeast cells (C. K. Barlowe and D. R. Appling, Biochemistry 29:7089-7094, 1990). We propose that the characteristics of strains expressing full-length but catalytically inactive C1-THF synthase could result from the formation of a purine-synthesizing multienzyme complex involving the structurally unchanged C1-THF synthase and that production of the necessary one-carbon units in these strains is accomplished by an NAD+ -dependent 5,10-methylene-THF dehydrogenase.

Evidence that POB1, a Saccharomyces cerevisiae protein that binds to DNA polymerase alpha, acts in DNA metabolism in vivo

1992 ◽  
Vol 12 (12) ◽  
pp. 5724-5735
Author(s):  
J Miles ◽  
T Formosa
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Yeast Cells ◽  
Chromosome Loss ◽  
Dna Metabolism ◽  
Checkpoint Gene ◽  
Dna Polymerase Alpha ◽  
In Cells

Potential DNA replication accessory factors from the yeast Saccharomyces cerevisiae have previously been identified by their ability to bind to DNA polymerase alpha protein affinity matrices (J. Miles and T. Formosa, Proc. Natl. Acad. Sci. USA 89:1276-1280, 1992). We have now used genetic methods to characterize the gene encoding one of these DNA polymerase alpha-binding proteins (POB1) to determine whether it plays a role in DNA replication in vivo. We find that yeast cells lacking POB1 are viable but display a constellation of phenotypes indicating defective DNA metabolism. Populations of cells lacking POB1 accumulate abnormally high numbers of enlarged large-budded cells with a single nucleus at the neck of the bud. The average DNA content in a population of cells lacking POB1 is shifted toward the G2 value. These two phenotypes indicate that while the bulk of DNA replication is completed without POB1, mitosis is delayed. Deleting POB1 also causes elevated levels of both chromosome loss and genetic recombination, enhances the temperature sensitivity of cells with mutant DNA polymerase alpha genes, causes increased sensitivity to UV radiation in cells lacking a functional RAD9 checkpoint gene, and causes an increased probability of death in cells carrying a mutation in the MEC1 checkpoint gene. The sequence of the POB1 gene indicates that it is identical to the CTF4 (CHL15) gene identified previously in screens for mutations that diminish the fidelity of chromosome transmission. These phenotypes are consistent with defective DNA metabolism in cells lacking POB1 and strongly suggest that this DNA polymerase alpha-binding protein plays a role in accurately duplicating the genome in vivo.

Identification of pre-mRNA polyadenylation sites in Saccharomyces cerevisiae

1992 ◽  
Vol 12 (9) ◽  
pp. 4215-4229
Author(s):  
S Heidmann ◽  
B Obermaier ◽  
K Vogel ◽  
H Domdey
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Signal Sequence ◽  
Site Directed Mutagenesis ◽  
Yeast Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Adh1 Gene ◽  
Polyadenylation Sites ◽  
Yeast Genes

In contrast to higher eukaryotes, little is known about the nature of the sequences which direct 3'-end formation of pre-mRNAs in the yeast Saccharomyces cerevisiae. The hexanucleotide AAUAAA, which is highly conserved and crucial in mammals, does not seem to have any functional importance for 3'-end formation in yeast cells. Instead, other elements have been proposed to serve as signal sequences. We performed a detailed investigation of the yeast ACT1, ADH1, CYC1, and YPT1 cDNAs, which showed that the polyadenylation sites used in vivo can be scattered over a region spanning up to 200 nucleotides. It therefore seems very unlikely that a single signal sequence is responsible for the selection of all these polyadenylation sites. Our study also showed that in the large majority of mRNAs, polyadenylation starts directly before or after an adenosine residue and that 3'-end formation of ADH1 transcripts occurs preferentially at the sequence PyAAA. Site-directed mutagenesis of these sites in the ADH1 gene suggested that this PyAAA sequence is essential for polyadenylation site selection both in vitro and in vivo. Furthermore, the 3'-terminal regions of the yeast genes investigated here are characterized by their capacity to act as signals for 3'-end formation in vivo in either orientation.

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