scholarly journals Significance of C-terminal cysteine modifications to the biological activity of the Saccharomyces cerevisiae a-factor mating pheromone.

1991 ◽  
Vol 11 (7) ◽  
pp. 3603-3612 ◽  
Author(s):  
S Marcus ◽  
G A Caldwell ◽  
D Miller ◽  
C B Xue ◽  
F Naider ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Biological Activity ◽  
Methyl Ester ◽  
Total Synthesis ◽  
Biologically Active ◽  
Structural Genes ◽  
Wild Type ◽  
Mating Pheromone ◽  
Carboxy Terminal

We have undertaken total synthesis of the Saccharomyces cerevisiae a-factor (NH2-YIIKGVFWDPAC[S-farnesyl]-COOCH3) and several Cys-12 analogs to determine the significance of S-farnesylation and carboxy-terminal methyl esterification to the biological activity of this lipopeptide mating pheromone. Replacement of either the farnesyl group or the carboxy-terminal methyl ester by a hydrogen atom resulted in marked reduction but not total loss of bioactivity as measured by a variety of assays. Moreover, both the farnesyl and methyl ester groups could be replaced by other substituents to produce biologically active analogs. The bioactivity of a-factor decreased as the number of prenyl units on the cysteine sulfur decreased from three to one, and an a-factor analog having the S-farnesyl group replaced by an S-hexadecanyl group was more active than an S-methyl a-factor analog. Thus, with two types of modifications, a-factor activity increased as the S-alkyl group became bulkier and more hydrophobic. MATa cells having deletions of the a-factor structural genes (mfal1 mfa2 mutants) were capable of mating with either sst2 or wild-type MAT alpha cells in the presence of exogenous a-factor, indicating that it is not absolutely essential for MATa cells to actively produce a-factor in order to mate. Various a-factor analogs were found to partially restore mating to these strains as well, and their relative activities in the mating restoration assay were similar to their activities in the other assays used in this study. Mating was not restored by addition of exogenous a-factor to a cross of a wild-type MAT alpha strain and a MATaste6 mutant, indicating a role of the STE6 gene product in mating in addition to its secretion of a-factor.

Significance of C-terminal cysteine modifications to the biological activity of the Saccharomyces cerevisiae a-factor mating pheromone

1991 ◽  
Vol 11 (7) ◽  
pp. 3603-3612
Author(s):  
S Marcus ◽  
G A Caldwell ◽  
D Miller ◽  
C B Xue ◽  
F Naider ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Biological Activity ◽  
Methyl Ester ◽  
Total Synthesis ◽  
Biologically Active ◽  
Structural Genes ◽  
Wild Type ◽  
Mating Pheromone ◽  
Carboxy Terminal

We have undertaken total synthesis of the Saccharomyces cerevisiae a-factor (NH2-YIIKGVFWDPAC[S-farnesyl]-COOCH3) and several Cys-12 analogs to determine the significance of S-farnesylation and carboxy-terminal methyl esterification to the biological activity of this lipopeptide mating pheromone. Replacement of either the farnesyl group or the carboxy-terminal methyl ester by a hydrogen atom resulted in marked reduction but not total loss of bioactivity as measured by a variety of assays. Moreover, both the farnesyl and methyl ester groups could be replaced by other substituents to produce biologically active analogs. The bioactivity of a-factor decreased as the number of prenyl units on the cysteine sulfur decreased from three to one, and an a-factor analog having the S-farnesyl group replaced by an S-hexadecanyl group was more active than an S-methyl a-factor analog. Thus, with two types of modifications, a-factor activity increased as the S-alkyl group became bulkier and more hydrophobic. MATa cells having deletions of the a-factor structural genes (mfal1 mfa2 mutants) were capable of mating with either sst2 or wild-type MAT alpha cells in the presence of exogenous a-factor, indicating that it is not absolutely essential for MATa cells to actively produce a-factor in order to mate. Various a-factor analogs were found to partially restore mating to these strains as well, and their relative activities in the mating restoration assay were similar to their activities in the other assays used in this study. Mating was not restored by addition of exogenous a-factor to a cross of a wild-type MAT alpha strain and a MATaste6 mutant, indicating a role of the STE6 gene product in mating in addition to its secretion of a-factor.

scholarly journals CHS5, a gene involved in chitin synthesis and mating in Saccharomyces cerevisiae.

1997 ◽  
Vol 17 (5) ◽  
pp. 2485-2496 ◽  
Author(s):  
B Santos ◽  
A Duran ◽  
M H Valdivieso
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Fusion ◽  
Chitin Synthase ◽  
Activity Levels ◽  
Wild Type ◽  
Neurofilament Proteins ◽  
Chitin Synthesis ◽  
Mating Efficiency ◽  
Carboxy Terminal

The CHS5 locus of Saccharomyces cerevisiae is important for wild-type levels of chitin synthase III activity. chs5 cells have reduced levels of this activity. To further understand the role of CHS5 in yeast, the CHS5 gene was cloned by complementation of the Calcofluor resistance phenotype of a chs5 mutant. Transformation of the mutant with a plasmid carrying CHS5 restored Calcofluor sensitivity, wild-type cell wall chitin levels, and chitin synthase III activity levels. DNA sequence analysis reveals that CHS5 encodes a unique polypeptide of 671 amino acids with a molecular mass of 73,642 Da. The predicted sequence shows a heptapeptide repeated 10 times, a carboxy-terminal lysine-rich tail, and some similarity to neurofilament proteins. The effects of deletion of CHS5 indicate that it is not essential for yeast cell growth; however, it is important for mating. Deletion of CHS3, the presumptive structural gene for chitin synthase III activity, results in a modest decrease in mating efficiency, whereas chs5delta cells exhibit a much stronger mating defect. However, chs5 cells produce more chitin than chs3 mutants, indicating that CHS5 plays a role in other processes besides chitin synthesis. Analysis of mating mixtures of chs5 cells reveals that cells agglutinate and make contact but fail to undergo cell fusion. The chs5 mating defect can be partially rescued by FUS1 and/or FUS2, two genes which have been implicated previously in cell fusion, but not by FUS3. In addition, mating efficiency is much lower in fus1 fus2 x chs5 than in fus1 fus2 x wild type crosses. Our results indicate that Chs5p plays an important role in the cell fusion step of mating.

scholarly journals Loss of Ubp3 increases silencing, decreases unequal recombination in rDNA, and shortens the replicative life span in Saccharomyces cerevisiae

2014 ◽  
Vol 25 (12) ◽  
pp. 1916-1924 ◽  
Author(s):  
David Öling ◽  
Rehan Masoom ◽  
Kristian Kvint
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Life Span ◽  
Rna Polymerase Ii ◽  
Ribosomal Dna ◽  
Genetic Evidence ◽  
Wild Type ◽  
Replicative Life Span ◽  
Unequal Recombination

Ubp3 is a conserved ubiquitin protease that acts as an antisilencing factor in MAT and telomeric regions. Here we show that ubp3∆ mutants also display increased silencing in ribosomal DNA (rDNA). Consistent with this, RNA polymerase II occupancy is lower in cells lacking Ubp3 than in wild-type cells in all heterochromatic regions. Moreover, in a ubp3∆ mutant, unequal recombination in rDNA is highly suppressed. We present genetic evidence that this effect on rDNA recombination, but not silencing, is entirely dependent on the silencing factor Sir2. Further, ubp3∆ sir2∆ mutants age prematurely at the same rate as sir2∆ mutants. Thus our data suggest that recombination negatively influences replicative life span more so than silencing. However, in ubp3∆ mutants, recombination is not a prerequisite for aging, since cells lacking Ubp3 have a shorter life span than isogenic wild-type cells. We discuss the data in view of different models on how silencing and unequal recombination affect replicative life span and the role of Ubp3 in these processes.

Stoichiometry of G protein subunits affects the Saccharomyces cerevisiae mating pheromone signal transduction pathway

1990 ◽  
Vol 10 (2) ◽  
pp. 510-517
Author(s):  
G M Cole ◽  
D E Stone ◽  
S I Reed
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Signal Transduction ◽  
G Protein ◽  
Signal Transduction Pathway ◽  
Mating Pheromone ◽  
Gal1 Promoter ◽  
Gamma Subunits ◽  
Adaptation Response ◽  
High Level

The Saccharomyces cerevisiae GPA1, STE4, and STE18 genes encode products homologous to mammalian G-protein alpha, beta, and gamma subunits, respectively. All three genes function in the transduction of the signal generated by mating pheromone in haploid cells. To characterize more completely the role of these genes in mating, we have conditionally overexpressed GPA1, STE4, and STE18, using the galactose-inducible GAL1 promoter. Overexpression of STE4 alone, or STE4 together with STE18, generated a response in haploid cells suggestive of pheromone signal transduction: arrest in G1 of the cell cycle, formation of cellular projections, and induction of the pheromone-inducible transcript FUS1 25- to 70-fold. High-level STE18 expression alone had none of these effects, nor did overexpression of STE4 in a MATa/alpha diploid. However, STE18 was essential for the response, since overexpression of STE4 was unable to activate a response in a ste18 null strain. GPA1 hyperexpression suppressed the phenotype of STE4 overexpression. In addition, cells that overexpressed GPA1 were more resistant to pheromone and recovered more quickly from pheromone than did wild-type cells, which suggests that GPA1 may function in an adaptation response to pheromone.

Isolation of the thymidylate synthetase gene (TMP1) by complementation in Saccharomyces cerevisiae

1982 ◽  
Vol 2 (4) ◽  
pp. 437-442
Author(s):  
G R Taylor ◽  
B J Barclay ◽  
R K Storms ◽  
J D Friesen ◽  
R H Haynes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
High Frequency ◽  
Wild Type Strain ◽  
Biologically Active ◽  
Thymidylate Synthetase ◽  
Temperature Sensitive ◽  
Synthetase Activity ◽  
Enzymatic Conversion ◽  
Wild Type ◽  
Sensitive Mutant

The structural gene (TMP1) for yeast thymidylate synthetase (thymidylate synthase; EC 2.1.1.45) was isolated from a chimeric plasmid bank by genetic complementation in Saccharomyces cerevisiae. Retransformation of the dTMP auxotroph GY712 and a temperature-sensitive mutant (cdc21) with purified plasmid (pTL1) yielded Tmp+ transformants at high frequency. In addition, the plasmid was tested for the ability to complement a bacterial thyA mutant that lacks functional thymidylate synthetase. Although it was not possible to select Thy+ transformants directly, it was found that all pTL1 transformants were phenotypically Thy+ after several generations of growth in nonselective conditions. Thus, yeast thymidylate synthetase is biologically active in Escherichia coli. Thymidylate synthetase was assayed in yeast cell lysates by high-pressure liquid chromatography to monitor the conversion of [6-3H]dUMP to [6-3H]dTMP. In protein extracts from the thymidylate auxotroph (tmp1-6) enzymatic conversion of dUMP to dTMP was barely detectable. Lysates of pTL1 transformants of this strain, however, had thymidylate synthetase activity that was comparable to that of the wild-type strain.

scholarly journals Expression of the Schwanniomyces occidentalis SWA2 amylase in Saccharomyces cerevisiae: role of N-glycosylation on activity, stability and secretion

1998 ◽  
Vol 329 (1) ◽  
pp. 65-71 ◽  
Author(s):  
Esther YÁÑEZ ◽  
A. Teresa CARMONA ◽  
Mercedes TIEMBLO ◽  
Antonio JIMÉNEZ ◽  
María FERNÁNDEZ-LOBATO
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Catalytic Efficiency ◽  
Extracellular Enzyme ◽  
Site Directed Mutagenesis ◽  
Activity Levels ◽  
Wild Type ◽  
Schwanniomyces Occidentalis ◽  
Type Protein ◽  
Wild Type Protein

The role of N-linked glycosylation on the biological activity of Schwanniomyces occidentalis SWA2 α-amylase, as expressed in Saccharomyces cerevisiae, was analysed by site-directed mutagenesis of the two potential N-glycosylation sites, Asn-134 and Asn-229. These residues were replaced by Ala or Gly individually or in various combinations and the effects on the activity, secretion and thermal stability of the enzyme were studied. Any Asn-229 substitution caused a drastic decrease in activity levels of the extracellular enzyme. In contrast, substitutions of Asn-134 had little or no effect. The use of antibodies showed that α-amylase was secreted in all the mutants tested, although those containing substitutions at Asn-229 seemed to have a lower rate of synthesis and/or higher degradation than the wild-type strain. α-Amylases with substitution at Asn-229 had a 2 kDa lower molecular mass than the wild-type protein, as did the wild-type protein itself after treatment with endoglycosidase F. These findings indicate that Asn-229 is the single glycosylated residue in SWA2. Thermostability analysis of both purified wild-type (T50 = 50 °C, where T50 is the temperature resulting in 50% loss of activity) and mutant enzymes indicated that removal of carbohydrate from the 229 position results in a decrease of approx. 3 °C in the T50 of the enzyme. The Gly-229 mutation does not change the apparent affinity of the enzyme for starch (Km) but decreases to 1/22 its apparent catalytic efficiency (kcat/Km). These results therefore indicate that glycosylation at the 229 position has an important role in the extracellular activity levels, kinetics and stability of the Sw. occidentalis SWA2 α-amylase in both its wild-type and mutant forms.

scholarly journals Optimal Replication of Human Cytomegalovirus Correlates with Endocytosis of Glycoprotein gpUL132

2010 ◽  
Vol 84 (14) ◽  
pp. 7039-7052 ◽  
Author(s):  
Barbara Kropff ◽  
Yvonne Koedel ◽  
William Britt ◽  
Michael Mach
Keyword(s):  
Amino Acid ◽  
Human Cytomegalovirus ◽  
Intracellular Localization ◽  
Surface Expression ◽  
Wild Type Virus ◽  
Wild Type ◽  
Recombinant Viruses ◽  
Carboxy Terminal ◽  
Golgi Network

ABSTRACT Envelopment of a herpesvirus particle is a complex process of which much is still to be learned. We previously identified the glycoprotein gpUL132 of human cytomegalovirus (HCMV) as an envelope component of the virion. In its carboxy-terminal portion, gpUL132 contains at least four motifs for sorting of transmembrane proteins to endosomes; among them are one dileucine-based signal and three tyrosine-based signals of the YXXØ and NPXY (where X stands for any amino acid, and Ø stands for any bulky hydrophobic amino acid) types. To investigate the role of each of these trafficking signals in intracellular localization and viral replication, we constructed a panel of expression plasmids and recombinant viruses in which the signals were rendered nonfunctional by mutagenesis. In transfected cells wild-type gpUL132 was mainly associated with the trans-Golgi network. Consecutive mutation of the trafficking signals resulted in increasing fractions of the protein localized at the cell surface, with gpUL132 mutated in all four trafficking motifs predominantly associated with the plasma membrane. Concomitant with increased surface expression, endocytosis of mutant gpUL132 was reduced, with a gpUL132 expressing all four motifs in mutated form being almost completely impaired in endocytosis. The replication of recombinant viruses harboring mutations in single trafficking motifs was comparable to replication of wild-type virus. In contrast, viruses containing mutations in three or four of the trafficking signals showed pronounced deficits in replication with a reduction of approximately 100-fold. Moreover, recombinant viruses expressing gpUL132 with three or four trafficking motifs mutated failed to incorporate the mutant protein into the virus particle. These results demonstrate a role of endocytosis of an HCMV envelope glycoprotein for incorporation into the virion and optimal virus replication.

scholarly journals Identification of nonessential disulfide bonds and altered conformations in the v-sis protein, a homolog of the B chain of platelet-derived growth factor.

1988 ◽  
Vol 8 (3) ◽  
pp. 1011-1018 ◽  
Author(s):  
M K Sauer ◽  
D J Donoghue
Keyword(s):  
Biological Activity ◽  
Growth Factor ◽  
Disulfide Bonds ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Biologically Active ◽  
Platelet Derived Growth Factor ◽  
Wild Type ◽  
Type V

The protein encoded by v-sis, the oncogene of simian sarcoma virus, is homologous to the B chain of platelet-derived growth factor (PDGF). There are eight conserved Cys residues between PDGF-B and the v-sis protein. Both native PDGF and the v-sis protein occur as disulfide-bonded dimers, probably containing both intramolecular and intermolecular disulfide bonds. Oligonucleotide-directed mutagenesis was used to change the Cys codons to Ser codons in the v-sis gene. Four single mutants lacked detectable biological activity, indicating that Cys-127, Cys-160, Cys-171, and Cys-208 are required for formation of a biologically active v-sis protein. The other four single mutants retained biological activity as determined in transformation assays, indicating that Cys-154, Cys-163, Cys-164, and Cys-210 are dispensable for biological activity. Double and triple mutants containing three of these altered sites were constructed, some of which were transforming as well. The v-sis proteins encoded by biologically active mutants displayed significantly reduced levels of dimeric protein compared with the wild-type v-sis protein, which dimerized very efficiently. Furthermore, a mutant with a termination codon at residue 209 exhibited partial transforming activity. This study thus suggests that the minimal region required for transformation consists of residues 127 to 208. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that the v-sis proteins encoded by some of the biologically active mutants exhibited an altered conformation when compared with the wild-type v-sis protein, and suggested that Cys-154 and Cys-163 participate in a nonessential disulfide bond.

scholarly journals The Biological Activity of Natural and Mutant Ptα Alleles

2001 ◽  
Vol 194 (5) ◽  
pp. 695-704 ◽  
Author(s):  
Deena Gibbons ◽  
Nataki C. Douglas ◽  
Domingo F. Barber ◽  
Qiang Liu ◽  
Renee Sullo ◽  
...  
Keyword(s):  
Biological Activity ◽  
T Cell Receptor ◽  
Cytoplasmic Tail ◽  
Cell Receptor ◽  
Biologically Active ◽  
Primary Role ◽  
Truncated Form ◽  
Thymocyte Differentiation ◽  
Successful Expression

β selection is a major checkpoint in early thymocyte differentiation, mediated by successful expression of the pre-T cell receptor (TCR) comprising the TCRβ chain, CD3 proteins, and a surrogate TCRα chain, pTα. The mechanism of action of the pre-TCR is unresolved. In humans and mice, the pTα gene encodes two RNAs, pTαa, and a substantially truncated form, pTαb. This study shows that both are biologically active in their capacity to rescue multiple thymocyte defects in pTα−/− mice. Further active alleles of pTα include one that lacks both the major ectodomain and much of the long cytoplasmic tail (which is unique among antigen receptor chains), and another in which the cytoplasmic tail is substituted with the short tail of TCR Cα. Thus, very little of the pTα chain is required for function. These data support a hypothesis that the primary role of pTα is to stabilize the pre-TCR, and that much of the conserved structure of pTα probably plays a critical regulatory role.

scholarly journals Role of Saccharomyces cerevisiae Oxidoreductases Bdh1p and Ara1p in the Metabolism of Acetoin and 2,3-Butanediol

2009 ◽  
Vol 76 (3) ◽  
pp. 670-679 ◽  
Author(s):  
Eva González ◽  
M. Rosario Fernández ◽  
Didac Marco ◽  
Eduard Calam ◽  
Lauro Sumoy ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Type Strain ◽  
Wild Type Strain ◽  
Growth Conditions ◽  
Wild Type ◽  
Genome Expression ◽  
Whole Genome Expression ◽  
Histidine Tag ◽  
Butanediol Dehydrogenase

ABSTRACT NAD-dependent butanediol dehydrogenase (Bdh1p) from Saccharomyces cerevisiae reversibly transforms acetoin to 2,3-butanediol in a stereospecific manner. Deletion of BDH1 resulted in an accumulation of acetoin and a diminution of 2,3-butanediol in two S. cerevisiae strains under two different growth conditions. The concentrations of (2R,3R)-2,3-butanediol are mostly dependent on Bdh1p activity, while those of (meso)-2,3-butanediol are also influenced by the activity of NADP(H)-dependent oxidoreductases. One of them has been purified and shown to be d-arabinose dehydrogenase (Ara1p), which converts (R/S)-acetoin to meso-2,3-butanediol and (2S,3S)-2,3-butanediol. Deletion of BDH2, a gene adjacent to BDH1, whose encoded protein is 51% identical to Bdh1p, does not significantly alter the levels of acetoin or 2,3-butanediol in comparison to the wild-type strain. Furthermore, we have expressed Bdh2p with a histidine tag and have shown it to be inactive toward 2,3-butanediol. A whole-genome expression analysis with microarrays demonstrates that BDH1 and BDH2 are reciprocally regulated.

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