scholarly journals Bypassing the Catalytic Activity of SIR2 for SIR Protein Spreading in Saccharomyces cerevisiae

2006 ◽  
Vol 17 (12) ◽  
pp. 5287-5297 ◽  
Author(s):  
Bo Yang ◽  
Ann L. Kirchmaier
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Catalytic Activity ◽  
Histone Deacetylase ◽  
Genetic Evidence ◽  
Enzymatic Function ◽  
Lysine Residues ◽  
Histone Hypoacetylation ◽  
In Cells

Sir protein spreading along chromosomes and silencing in Saccharomyces cerevisiae requires the NAD+-dependent histone deacetylase activity of Sir2p. We tested whether this requirement could be bypassed at the HM loci and telomeres in cells containing a stably expressed, but catalytically inactive mutant of Sir2p, sir2-345p, plus histone mutants that mimic the hypoacetylated state normally created by Sir2p. Sir protein spreading was rescued in sir2-345 mutants expressing histones in which key lysine residues in their N-termini had been mutated to arginine. Mating in these mutants was also partially restored upon overexpression of Sir3p. Together, these results indicate that histone hypoacetylation is sufficient for Sir protein spreading in the absence of production of 2′-O-acetyl-ADP ribose by sir2p and Sir2p's enzymatic function for silencing can be bypassed in a subset of cells in a given population. These results also provide genetic evidence for the existence of additional critical substrates of Sir2p for silencing in vivo.

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.

scholarly journals Nicotinamide Clearance by Pnc1 Directly Regulates Sir2-Mediated Silencing and Longevity

2004 ◽  
Vol 24 (3) ◽  
pp. 1301-1312 ◽  
Author(s):  
Christopher M. Gallo ◽  
Daniel L. Smith ◽  
Jeffrey S. Smith
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nicotinic Acid ◽  
Life Span ◽  
Histone Deacetylase ◽  
Transcriptional Repression ◽  
Transcriptional Silencing ◽  
Inhibitory Effect ◽  
Salvage Pathway

ABSTRACT The Saccharomyces cerevisiae Sir2 protein is an NAD+-dependent histone deacetylase (HDAC) that functions in transcriptional silencing and longevity. The NAD+ salvage pathway protein, Npt1, regulates Sir2-mediated processes by maintaining a sufficiently high intracellular NAD+ concentration. However, another NAD+ salvage pathway component, Pnc1, modulates silencing independently of the NAD+ concentration. Nicotinamide (NAM) is a by-product of the Sir2 deacetylase reaction and is a natural Sir2 inhibitor. Pnc1 is a nicotinamidase that converts NAM to nicotinic acid. Here we show that recombinant Pnc1 stimulates Sir2 HDAC activity in vitro by preventing the accumulation of NAM produced by Sir2. In vivo, telomeric, rDNA, and HM silencing are differentially sensitive to inhibition by NAM. Furthermore, PNC1 overexpression suppresses the inhibitory effect of exogenously added NAM on silencing, life span, and Hst1-mediated transcriptional repression. Finally, we show that stress suppresses the inhibitory effect of NAM through the induction of PNC1 expression. Pnc1, therefore, positively regulates Sir2-mediated silencing and longevity by preventing the accumulation of intracellular NAM during times of stress.

scholarly journals Glycerophosphocholine-dependent Growth Requires Gde1p (YPL110c) and Git1p in Saccharomyces cerevisiae

2005 ◽  
Vol 280 (43) ◽  
pp. 36110-36117 ◽  
Author(s):  
Edward Fisher ◽  
Claudia Almaguer ◽  
Roman Holic ◽  
Peter Griac ◽  
Jana Patton-Vogt
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Wild Type Strain ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Intracellular Fraction ◽  
Dependent Growth ◽  
Phosphate Source ◽  
Glycerophosphocholine Phosphodiesterase ◽  
In Cells

Glycerophosphocholine is formed via the deacylation of the phospholipid phosphatidylcholine. The protein encoded by Saccharomyces cerevisiae open reading frame YPL110c effects glycerophosphocholine metabolism in vivo, most likely by acting as a glycerophosphocholine phosphodiesterase. Deletion of YPL110c causes an accumulation of glycerophosphocholine in cells prelabeled with [14C]choline. Correspondingly, overexpression of YPL110c results in reduced intracellular glycerophosphocholine in cells prelabeled with [14C]choline. Glycerophospho[3H]choline supplied in the growth medium accumulates to a much greater extent in the intracellular fraction of a YPL110Δ strain than in a wild type strain. Furthermore, glycerophospho[3H]choline accumulation requires the transporter encoded by GIT1, a known glycerophosphoinositol transporter. Growth on glycerophosphocholine as the sole phosphate source requires YPL110c and the Git1p permease. In contrast to glycerophosphocholine, glycerophosphoinositol metabolism is unaffected by deletion of YPL110c. The open reading frame YPL110c has been termed GDE1.

scholarly journals Calcineurin/Crz1 destabilizes Msn2 and Msn4 in the nucleus in response to Ca2+ in Saccharomyces cerevisiae

2010 ◽  
Vol 427 (2) ◽  
pp. 275-287 ◽  
Author(s):  
Yoshifumi Takatsume ◽  
Takumi Ohdate ◽  
Kazuhiro Maeta ◽  
Wataru Nomura ◽  
Shingo Izawa ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitogen Activated Protein Kinase ◽  
Gene Encoding ◽  
Induced Expression ◽  
Glyoxalase 1 ◽  
Cis Element ◽  
Mitogen Activated Protein ◽  
In The Wild ◽  
In Cells

Although methylglyoxal is derived from glycolysis, it has adverse effects on cellular function. Hence, the intrinsic role of methylglyoxal in vivo remains to be determined. Glyoxalase 1 is a pivotal enzyme in the metabolism of methylglyoxal in all types of organisms. To learn about the physiological roles of methylglyoxal, we have screened conditions that alter the expression of the gene encoding glyoxalase 1, GLO1, in Saccharomyces cerevisiae. We show that the expression of GLO1 is induced following treatment with Ca2+ and is dependent on the MAPK (mitogen-activated protein kinase) Hog1 protein and the Msn2/Msn4 transcription factors. Intriguingly, the Ca2+-induced expression of GLO1 was enhanced in the presence of FK506, a potent inhibitor of calcineurin. Consequently, the Ca2+-induced expression of GLO1 in a mutant that is defective in calcineurin or Crz1, the sole transcription factor downstream of calcineurin, was much greater than that in the wild-type strain even without FK506. This phenomenon was dependent upon a cis-element, the STRE (stress-response element), in the promoter that is able to mediate the response to Ca2+ signalling together with Hog1 and Msn2/Msn4. The level of Ca2+-induced expression of GLO1 reached a maximum in cells overexpressing MSN2 even when FK506 was not present, whereas in cells overexpressing CRZ1 the level was greatly reduced and increased markedly when FK506 was present. We also found that the levels of Msn2 and Msn4 proteins in Ca2+-treated cells decreased gradually and that FK506 blocked the degradation of Msn2/Msn4. We propose that Crz1 destabilizes Msn2/Msn4 in the nuclei of cells in response to Ca2+ signalling.

scholarly journals Uptake of radiolabeled GlcNAc into Saccharomyces cerevisiae via native hexose transporters and its in vivo incorporation into GPI precursors in cells expressing heterologous GlcNAc kinase

2012 ◽  
Vol 12 (3) ◽  
pp. 305-316 ◽  
Author(s):  
John J. Scarcelli ◽  
Paul A. Colussi ◽  
Anne-Lise Fabre ◽  
Eckhard Boles ◽  
Peter Orlean ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Hexose Transporters ◽  
In Cells

scholarly journals Novel Gal3 proteins showing altered Gal80p binding cause constitutive transcription of Gal4p-activated genes in Saccharomyces cerevisiae.

1997 ◽  
Vol 17 (5) ◽  
pp. 2566-2575 ◽  
Author(s):  
T E Blank ◽  
M P Woods ◽  
C M Lebo ◽  
P Xin ◽  
J E Hopper
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Strong Support ◽  
Constitutive Expression ◽  
Mutant Proteins ◽  
Binding Characteristics ◽  
Isolation And Characterization ◽  
In Cells

Gal4p-mediated activation of galactose gene expression in Saccharomyces cerevisiae normally requires both galactose and the activity of Gal3p. Recent evidence suggests that in cells exposed to galactose, Gal3p binds to and inhibits Ga180p, an inhibitor of the transcriptional activator Gal4p. Here, we report on the isolation and characterization of novel mutant forms of Gal3p that can induce Gal4p activity independently of galactose. Five mutant GAL3(c) alleles were isolated by using a selection demanding constitutive expression of a GAL1 promoter-driven HIS3 gene. This constitutive effect is not due to overproduction of Gal3p. The level of constitutive GAL gene expression in cells bearing different GAL3(c) alleles varies over more than a fourfold range and increases in response to galactose. Utilizing glutathione S-transferase-Gal3p fusions, we determined that the mutant Gal3p proteins show altered Gal80p-binding characteristics. The Gal3p mutant proteins differ in their requirements for galactose and ATP for their Gal80p-binding ability. The behavior of the novel Gal3p proteins provides strong support for a model wherein galactose causes an alteration in Gal3p that increases either its ability to bind to Gal80p or its access to Gal80p. With the Gal3p-Gal80p interaction being a critical step in the induction process, the Gal3p proteins constitute an important new reagent for studying the induction mechanism through both in vivo and in vitro methods.

scholarly journals Saccharomyces cerevisiae Yak1p protein kinase autophosphorylates on tyrosine residues and phosphorylates myelin basic protein on a C-terminal serine residue

2000 ◽  
Vol 348 (2) ◽  
pp. 263-272 ◽  
Author(s):  
Shouki KASSIS ◽  
Tiffany MELHUISH ◽  
Roland S. ANNAN ◽  
Susan L. CHEN ◽  
John C. LEE ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Catalytic Activity ◽  
Protein Kinase ◽  
Myelin Basic Protein ◽  
Basic Protein ◽  
Full Length ◽  
Tandem Ms ◽  
Tyrosine Residues

The serine/threonine protein kinase, Yak1p, functions as a negative regulator of the cell cycle in Saccharomyces cerevisiae, acting downstream of the cAMP-dependent protein kinase. In the present work we report that overexpression of haemagglutinin-tagged full-lengthYak1p and an N-terminally truncated form (residues 148-807) lead to growth arrest in PKA compromised yak1 null yeast cells. Both forms of recombinant Yak1p kinase were catalytically active and preferred myelin basic protein (MBP) as a substrate over several other proteins. Phosphopeptide analysis of bovine MBP by tandem MS revealed two major Yak1p phosphorylation sites, Thr-97 and Ser-164. Peptides containing each site were obtained and tested as Yak1p substrates. Both forms of Yak1p phosphorylated a peptide containing the Ser-164 residue with far more efficient kinetics than MBP. The maximal velocity (Vmax) values of the full-length Yak1p reaction were 110±21 (Ser-164) and 8.7±1.7 (MBP), and those of N-terminally truncated Yak1p were 560.7±74.8 (Ser-164) and 34.4±2.2 (MBP) pmol/min per mg of protein. Although neither form of Yak1p was able to phosphorylate two generic protein tyrosine kinase substrates, both were phosphorylated on tyrosine residues in vivo and underwent tyrosine autophosphorylation when reacted with ATP in vitro. Tandem MS showed that Tyr-530 was phosphorylated both in vivo and in vitro after reaction with ATP. Pre-treatment with protein tyrosine phosphatase 1B removed all of Yak1p phosphotyrosine content and drastically reduced Yak1p activity against exogenous substrates, suggesting that the phosphotyrosine content of the enzyme is essential for its catalytic activity. Although the N-terminally truncated Yak1p was expressed at a lower level than the full-length protein, its catalytic activity and phosphotyrosine content were significantly higher than those of the full-length enzyme. Taken together, our results suggest that Yak1p is a dual specificity protein kinase which autophosphorylates on Tyr-530 and phosphorylates exogenous substrates on Ser/Thr residues.

scholarly journals Interactions among the subunits of the G protein involved in Saccharomyces cerevisiae mating.

1993 ◽  
Vol 13 (1) ◽  
pp. 1-8 ◽  
Author(s):  
K L Clark ◽  
D Dignard ◽  
D Y Thomas ◽  
M Whiteway
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
G Proteins ◽  
Amino Acid Sequences ◽  
Genetic Evidence ◽  
Gene Products ◽  
Gamma Subunits ◽  
Physical Interactions ◽  
Alpha Beta

The SCG1 (GPA1), STE4, and STE18 genes of Saccharomyces cerevisiae encode mating-pathway components whose amino acid sequences are similar to those of the alpha, beta, and gamma subunits, respectively, of mammalian G proteins. Genetic evidence suggests that the STE4 and STE18 gene products interact. The mating defects of a set of ste4 mutants were partially suppressed by the overexpression of STE18, and, moreover, a combination of partially defective ste4 and ste18 alleles created a totally sterile phenotype, whereas such synthetic sterility was not observed when the ste18 allele was combined with a weakly sterile ste11 allele. Others have provided genetic evidence consistent with an interaction between the SCG1 (GPA1) and STE4 gene products. We have examined the physical interactions of these subunits by using an in vivo protein association assay. The STE4 and STE18 gene products associated with each other, and this association was disrupted by a mutation in the STE4 gene product whose phenotype was partially suppressed by overexpression of STE18. The STE4 and SCG1 (GPA1) gene products also interacted in the assay, whereas we detected no association of the SCG1 (GPA1) and STE18 gene products.

scholarly journals The dynamin-like GTPase Sey1p mediates homotypic ER fusion in S. cerevisiae

2012 ◽  
Vol 197 (2) ◽  
pp. 209-217 ◽  
Author(s):  
Kamran Anwar ◽  
Robin W. Klemm ◽  
Amanda Condon ◽  
Katharina N. Severin ◽  
Miao Zhang ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Membrane Fusion ◽  
Dependent Manner ◽  
In Cells

The endoplasmic reticulum (ER) forms a network of tubules and sheets that requires homotypic membrane fusion to be maintained. In metazoans, this process is mediated by dynamin-like guanosine triphosphatases (GTPases) called atlastins (ATLs), which are also required to maintain ER morphology. Previous work suggested that the dynamin-like GTPase Sey1p was needed to maintain ER morphology in Saccharomyces cerevisiae. In this paper, we demonstrate that Sey1p, like ATLs, mediates homotypic ER fusion. The absence of Sey1p resulted in the ER undergoing delayed fusion in vivo and proteoliposomes containing purified Sey1p fused in a GTP-dependent manner in vitro. Sey1p could be partially replaced by ATL1 in vivo. Like ATL1, Sey1p underwent GTP-dependent dimerization. We found that the residual ER–ER fusion that occurred in cells lacking Sey1p required the ER SNARE Ufe1p. Collectively, our results show that Sey1p and its homologues function analogously to ATLs in mediating ER fusion. They also indicate that S. cerevisiae has an alternative fusion mechanism that requires ER SNAREs.

Interactions among the subunits of the G protein involved in Saccharomyces cerevisiae mating

1993 ◽  
Vol 13 (1) ◽  
pp. 1-8
Author(s):  
K L Clark ◽  
D Dignard ◽  
D Y Thomas ◽  
M Whiteway
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
G Proteins ◽  
Amino Acid Sequences ◽  
Genetic Evidence ◽  
Gene Products ◽  
Gamma Subunits ◽  
Physical Interactions ◽  
Alpha Beta

The SCG1 (GPA1), STE4, and STE18 genes of Saccharomyces cerevisiae encode mating-pathway components whose amino acid sequences are similar to those of the alpha, beta, and gamma subunits, respectively, of mammalian G proteins. Genetic evidence suggests that the STE4 and STE18 gene products interact. The mating defects of a set of ste4 mutants were partially suppressed by the overexpression of STE18, and, moreover, a combination of partially defective ste4 and ste18 alleles created a totally sterile phenotype, whereas such synthetic sterility was not observed when the ste18 allele was combined with a weakly sterile ste11 allele. Others have provided genetic evidence consistent with an interaction between the SCG1 (GPA1) and STE4 gene products. We have examined the physical interactions of these subunits by using an in vivo protein association assay. The STE4 and STE18 gene products associated with each other, and this association was disrupted by a mutation in the STE4 gene product whose phenotype was partially suppressed by overexpression of STE18. The STE4 and SCG1 (GPA1) gene products also interacted in the assay, whereas we detected no association of the SCG1 (GPA1) and STE18 gene products.

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