scholarly journals Farnesyl cysteine C-terminal methyltransferase activity is dependent upon the STE14 gene product in Saccharomyces cerevisiae.

1990 ◽  
Vol 10 (10) ◽  
pp. 5071-5076 ◽  
Author(s):  
C A Hrycyna ◽  
S Clarke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cysteine Residue ◽  
Peptide Sequence ◽  
Direct Result ◽  
Wild Type ◽  
Methyltransferase Activity ◽  
Ras Protein ◽  
Carboxyl Methyltransferase ◽  
C Terminus ◽  
Mutant Cells

Membrane extracts of sterile Saccharomyces cerevisiae strains containing the a-specific ste14 mutation lack a farnesyl cysteine C-terminal carboxyl methyltransferase activity that is present in wild-type a and alpha cells. Other a-specific sterile strains with ste6 and ste16 mutations also have wild-type levels of the farnesyl cysteine carboxyl methyltransferase activity. This enzyme activity, detected by using a synthetic peptide sequence based on the C-terminus of a ras protein, may be responsible not only for the essential methylation of the farnesyl cysteine residue of a mating factor, but also for the methylation of yeast RAS1 and RAS2 proteins and possibly other polypeptides with similar C-terminal structures. We demonstrate that the farnesylation of the cysteine residue in the peptide is required for the methyltransferase activity, suggesting that methyl esterification follows the lipidation reaction in the cell. To show that the loss of methyltransferase activity is a direct result of the ste14 mutation, we transformed ste14 mutant cells with a plasmid complementing the mating defect of this strain and found that active enzyme was produced. Finally, we demonstrated that a similar transformation of cells possessing the wild-type STE14 gene resulted in sixfold overproduction of the enzyme. Although more complicated possibilities cannot be ruled out, these results suggest that STE14 is a candidate for the structural gene for a methyltransferase involved in the formation of isoprenylated cysteine alpha-methyl ester C-terminal structures.

Farnesyl cysteine C-terminal methyltransferase activity is dependent upon the STE14 gene product in Saccharomyces cerevisiae

1990 ◽  
Vol 10 (10) ◽  
pp. 5071-5076
Author(s):  
C A Hrycyna ◽  
S Clarke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cysteine Residue ◽  
Peptide Sequence ◽  
Direct Result ◽  
Wild Type ◽  
Methyltransferase Activity ◽  
Ras Protein ◽  
Carboxyl Methyltransferase ◽  
C Terminus ◽  
Mutant Cells

Membrane extracts of sterile Saccharomyces cerevisiae strains containing the a-specific ste14 mutation lack a farnesyl cysteine C-terminal carboxyl methyltransferase activity that is present in wild-type a and alpha cells. Other a-specific sterile strains with ste6 and ste16 mutations also have wild-type levels of the farnesyl cysteine carboxyl methyltransferase activity. This enzyme activity, detected by using a synthetic peptide sequence based on the C-terminus of a ras protein, may be responsible not only for the essential methylation of the farnesyl cysteine residue of a mating factor, but also for the methylation of yeast RAS1 and RAS2 proteins and possibly other polypeptides with similar C-terminal structures. We demonstrate that the farnesylation of the cysteine residue in the peptide is required for the methyltransferase activity, suggesting that methyl esterification follows the lipidation reaction in the cell. To show that the loss of methyltransferase activity is a direct result of the ste14 mutation, we transformed ste14 mutant cells with a plasmid complementing the mating defect of this strain and found that active enzyme was produced. Finally, we demonstrated that a similar transformation of cells possessing the wild-type STE14 gene resulted in sixfold overproduction of the enzyme. Although more complicated possibilities cannot be ruled out, these results suggest that STE14 is a candidate for the structural gene for a methyltransferase involved in the formation of isoprenylated cysteine alpha-methyl ester C-terminal structures.

Yeast dom34 Mutants Are Defective in Multiple Developmental Pathways and Exhibit Decreased Levels of Polyribosomes

Genetics ◽  
1998 ◽  
Vol 149 (1) ◽  
pp. 45-56
Author(s):  
Luther Davis ◽  
JoAnne Engebrecht
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Heterologous Expression ◽  
Protein Translation ◽  
Developmental Pathways ◽  
G1 Phase ◽  
Growth Defect ◽  
Wild Type ◽  
Drosophila Homolog ◽  
Mutant Cells

Abstract The DOM34 gene of Saccharomyces cerevisiae is similar togenes found in diverse eukaryotes and archaebacteria. Analysis of dom34 strains shows that progression through the G1 phase of the cell cycle is delayed, mutant cells enter meiosis aberrantly, and their ability to form pseudohyphae is significantly diminished. RPS30A, which encodes ribosomal protein S30, was identified in a screen for high-copy suppressors of the dom34Δ growth defect. dom34Δ mutants display an altered polyribosome profile that is rescued by expression of RPS30A. Taken together, these data indicate that Dom34p functions in protein translation to promote G1 progression and differentiation. A Drosophila homolog of Dom34p, pelota, is required for the proper coordination of meiosis and spermatogenesis. Heterologous expression of pelota in dom34Δ mutants restores wild-type growth and differentiation, suggesting conservation of function between the eukaryotic members of the gene family.

scholarly journals CSE4 Genetically Interacts With the Saccharomyces cerevisiae Centromere DNA Elements CDE I and CDE II but Not CDE III: Implications for the Path of the Centromere DNA Around a Cse4p Variant Nucleosome

Genetics ◽  
2000 ◽  
Vol 156 (3) ◽  
pp. 973-981
Author(s):  
Kevin C Keith ◽  
Molly Fitzgerald-Hayes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Loss ◽  
Structural Studies ◽  
Wild Type ◽  
Histone Fold ◽  
Histone Fold Domain ◽  
Histone H3 Variant ◽  
Dna Elements ◽  
Mutant Cells ◽  
Loss Rates

Abstract Each Saccharomyces cerevisiae chromosome contains a single centromere composed of three conserved DNA elements, CDE I, II, and III. The histone H3 variant, Cse4p, is an essential component of the S. cerevisiae centromere and is thought to replace H3 in specialized nucleosomes at the yeast centromere. To investigate the genetic interactions between Cse4p and centromere DNA, we measured the chromosome loss rates exhibited by cse4 cen3 double-mutant cells that express mutant Cse4 proteins and carry chromosomes containing mutant centromere DNA (cen3). When compared to loss rates for cells carrying the same cen3 DNA mutants but expressing wild-type Cse4p, we found that mutations throughout the Cse4p histone-fold domain caused surprisingly large increases in the loss of chromosomes carrying CDE I or CDE II mutant centromeres, but had no effect on chromosomes with CDE III mutant centromeres. Our genetic evidence is consistent with direct interactions between Cse4p and the CDE I-CDE II region of the centromere DNA. On the basis of these and other results from genetic, biochemical, and structural studies, we propose a model that best describes the path of the centromere DNA around a specialized Cse4p-nucleosome.

Mild temperature shock alters the transcription of a discrete class of Saccharomyces cerevisiae genes

1983 ◽  
Vol 3 (3) ◽  
pp. 457-465
Author(s):  
C H Kim ◽  
J R Warner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Temperature Shift ◽  
Restrictive Temperature ◽  
Ribosomal Protein Genes ◽  
Wild Type ◽  
Temperature Shock ◽  
Mild Temperature ◽  
Mutant Cells

In Saccharomyces cerevisiae the synthesis of ribosomal proteins declines temporarily after a culture has been subjected to a mild temperature shock, i.e., a shift from 23 to 36 degrees C, each of which support growth. Using cloned genes for several S. cerevisiae ribosomal proteins, we found that the changes in the synthesis of ribosomal proteins parallel the changes in the concentration of mRNA of each. The disappearance and reappearance of the mRNA is due to a brief but severe inhibition of the transcription of each of the ribosomal protein genes, although the total transcription of mRNA in the cells is relatively unaffected by the temperature shock. The precisely coordinated response of these genes, which are scattered throughout the genome, suggests that either they or the enzyme which transcribes them has unique properties. In certain S. cerevisiae mutants, the synthesis of ribosomal proteins never recovers from a temperature shift. Yet both the decline and the resumption of transcription of these genes during the 30 min after the temperature shift are indistinguishable from those in wild-type cells. The failure of the mutant cells to grow at the restrictive temperature appears to be due to their inability to process the RNA transcribed from genes which have introns (Rosbash et al., Cell 24:679-686, 1981), a large proportion of which appear to be ribosomal protein genes.

scholarly journals Barrier Activity in Candida albicans Mediates Pheromone Degradation and Promotes Mating

2007 ◽  
Vol 6 (6) ◽  
pp. 907-918 ◽  
Author(s):  
Dana Schaefer ◽  
Pierre Côte ◽  
Malcolm Whiteway ◽  
Richard J. Bennett
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
Cell Growth ◽  
Cell Fusion ◽  
Aspartyl Protease ◽  
Wild Type ◽  
Pheromone Activity ◽  
Important Regulator ◽  
Mutant Cells ◽  
Type Strains

ABSTRACT Mating in Candida albicans and Saccharomyces cerevisiae is regulated by the secretion of peptide pheromones that initiate the mating process. An important regulator of pheromone activity in S. cerevisiae is barrier activity, involving an extracellular aspartyl protease encoded by the BAR1 gene that degrades the alpha pheromone. We have characterized an equivalent barrier activity in C. albicans and demonstrate that the loss of C. albicans BAR1 activity results in opaque a cells exhibiting hypersensitivity to alpha pheromone. Hypersensitivity to pheromone is clearly seen in halo assays; in response to alpha pheromone, a lawn of C. albicans Δbar1 mutant cells produces a marked zone in which cell growth is inhibited, whereas wild-type strains fail to show halo formation. C. albicans mutants lacking BAR1 also exhibit a striking mating defect in a cells, but not in α cells, due to overstimulation of the response to alpha pheromone. The block to mating occurs prior to cell fusion, as very few mating zygotes were observed in mixes of Δbar1 a and α cells. Finally, in a barrier assay using a highly pheromone-sensitive strain, we were able to demonstrate that barrier activity in C. albicans is dependent on Bar1p. These studies reveal that a barrier activity to alpha pheromone exists in C. albicans and that the activity is analogous to that caused by Bar1p in S. cerevisiae.

scholarly journals A Saccharomyces cerevisiae Mutant Lacking a K+/H+ Exchanger

1998 ◽  
Vol 180 (22) ◽  
pp. 5860-5865 ◽  
Author(s):  
Jorge Ramírez ◽  
Oscar Ramírez ◽  
Carlos Saldaña ◽  
Roberto Coria ◽  
Antonio Peña
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Core Protein ◽  
Kinetic Studies ◽  
Flow Cytometry Analysis ◽  
Wild Type ◽  
Reading Frame ◽  
Duplication Rate ◽  
Significant Difference ◽  
Internal Ph ◽  
Mutant Cells

ABSTRACT The KHA1 gene corresponding to the open reading frame YJL094c (2.62 kb) encoding a putative K+/H+antiporter (873 amino acids) in Saccharomyces cerevisiaewas disrupted by homologous recombination. The core protein is similar to the putative Na+/H+ antiporters fromEnterococcus hirae (NAPA gene) andLactococcus lactis (LLUPP gene) and the putative K+/H+ exchanger from Escherichia coli (KEFC gene). Disruption of the KHA1gene resulted in an increased K+ accumulation and net influx without a significant difference in efflux, as well as an increased growth rate, smaller cells, and twice the cell yield per glucose used. Flow cytometry analysis showed an increase of the DNA duplication rate in the mutant. Kinetic studies of86Rb+ uptake showed the same saturable system for wild-type and disruptant strains. Mutant cells also produced a greater acidification of the medium coincident with an internal pH alkalinization and showed a higher oxygen consumption velocity. We speculate that higher K+ accumulation and increased osmotic pressure accelerate the cell cycle and metabolic activity.

Size matters ? yeast petite colonie mutants

2007 ◽  
Vol 28 (2) ◽  
pp. 44
Author(s):  
Des Clark-Walker
Keyword(s):  
Saccharomyces Cerevisiae ◽  
High Frequency ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Brewers Yeast ◽  
Mutant Cells

It is well known that brewers yeast can grow by fermentation but it can also respire using mitochondria. However, damage to mitochondria can permanently block respiration. Such damaged or mutant cells can still grow, although more slowly than the wild-type, producing ?petite colonie? forms on agar plates. Remarkably, these small colonies appear spontaneously at the high frequency of 1% per generation. Indeed, petite colonie forms had frequently been observed in plated cultures of brewers or bakers yeast Saccharomyces cerevisiae by a number of groups but it was not until 1949 that Boris Ephrussi and colleagues in Paris described how these mutants differed in many properties from wild-type cells. Most saliently, they were found to lack respiration, the mutation was cytoplasmic (i.e. not associated with nuclear chromosomes), and as well as occurring spontaneously mutants could be produced in high frequency by treatment with acriflavine.

Sec59 encodes a membrane protein required for core glycosylation in Saccharomyces cerevisiae

1989 ◽  
Vol 9 (3) ◽  
pp. 1191-1199
Author(s):  
M Bernstein ◽  
F Kepes ◽  
R Schekman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Product ◽  
Carboxypeptidase Y ◽  
Secretory Proteins ◽  
Restrictive Temperature ◽  
Wild Type ◽  
Partial Restoration ◽  
Alpha Factor ◽  
Mutant Cells

When incubated at a restrictive temperature, Saccharomyces cerevisiae sec59 mutant cells accumulate inactive and incompletely glycosylated forms of secretory proteins. Three different secretory polypeptides (invertase, pro-alpha-factor, and pro-carboxypeptidase Y) accumulated within a membrane-bounded organelle, presumably the endoplasmic reticulum, and resisted proteolytic degradation unless the membrane was permeabilized with detergent. Molecular cloning and DNA sequence analysis of the SEC59 gene predicted an extremely hydrophobic protein product of 59 kilodaltons. This prediction was confirmed by reconstitution of the sec59 defect in vitro. The alpha-factor precursor, which was translated in a soluble fraction from wild-type cells, was translocated into, but inefficiently glycosylated within, membranes from sec59 mutant cells. Residual glycosylation activity of membranes of sec59 cells was thermolabile compared with the activity of wild-type membranes. Partial restoration of glycosylation was obtained in reactions that were supplemented with mannose or GDP-mannose, but not those supplemented with other sugar nucleotides. These results were consistent with a role for the Sec59 protein in the transfer of mannose to dolichol-linked oligosaccharide.

scholarly journals Plk is a functional homolog of Saccharomyces cerevisiae Cdc5, and elevated Plk activity induces multiple septation structures.

1997 ◽  
Vol 17 (6) ◽  
pp. 3408-3417 ◽  
Author(s):  
K S Lee ◽  
R L Erikson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cell Division ◽  
Specific Activity ◽  
Ectopic Expression ◽  
Sf9 Cells ◽  
Wild Type ◽  
C Terminus ◽  
M Phase

Plk is a mammalian serine/threonine protein kinase whose activity peaks at the onset of M phase. It is closely related to other mammalian kinases, Snk, Fnk, and Prk, as well as to Xenopus laevis Plx1, Drosophila melanogaster polo, Schizosaccharomyces pombe Plo1, and Saccharomyces cerevisiae Cdc5. The M phase of the cell cycle is a highly coordinated process which insures the equipartition of genetic and cellular materials during cell division. To enable understanding of the function of Plk during M phase progression, various Plk mutants were generated and expressed in Sf9 cells and budding yeast. In vitro kinase assays with Plk immunoprecipitates prepared from Sf9 cells indicate that Glu206 and Thr210 play equally important roles for Plk activity and that replacement of Thr210 with a negatively charged residue elevates Plk specific activity. Ectopic expression of wild-type Plk (Plk WT) complements the cell division defect associated with the cdc5-1 mutation in S. cerevisiae. The degree of complementation correlates closely with the Plk activity measured in vitro, as it is enhanced by a mutationally activated Plk, T210D, but is not observed with the inactive forms K82M, D194N, and D194R. In a CDC5 wild-type background, expression of Plk WT or T210D, but not of inactive forms, induced a sharp accumulation of cells in G1. Consistent with elevated Plk activity, this phenomenon was enhanced by the C-terminally deleted forms WT deltaC and T210D deltaC. Expression of T210D also induced a class of cells with unusually elongated buds which developed multiple septal structures. This was not observed with the C-terminally deleted form T210D deltaC, however. It appears that the C terminus of Plk is not required for the observed cell cycle influence but may be important for polarized cell growth and septal structure formation.

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