scholarly journals A novel mutant of the Sup35 protein of Saccharomyces cerevisiae defective in translation termination and in GTPase activity still supports cell viability

2008 ◽  
Vol 9 (1) ◽  
Author(s):  
Céline Fabret ◽  
Bruno Cosnier ◽  
Sergey Lekomtsev ◽  
Sylvie Gillet ◽  
Isabelle Hatin ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Viability ◽  
Translation Termination ◽  
Gtpase Activity

scholarly journals GTP Hydrolysis by eRF3 Facilitates Stop Codon Decoding during Eukaryotic Translation Termination

2004 ◽  
Vol 24 (17) ◽  
pp. 7769-7778 ◽  
Author(s):  
Joe Salas-Marco ◽  
David M. Bedwell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Polypeptide Chain ◽  
Stop Codon ◽  
Translation Termination ◽  
Gtpase Activity ◽  
Functional Importance ◽  
Gtp Hydrolysis ◽  
Nascent Polypeptide ◽  
Eukaryotic Translation ◽  
Termination Process

ABSTRACT Translation termination in eukaryotes is mediated by two release factors, eRF1 and eRF3. eRF1 recognizes each of the three stop codons (UAG, UAA, and UGA) and facilitates release of the nascent polypeptide chain. eRF3 is a GTPase that stimulates the translation termination process by a poorly characterized mechanism. In this study, we examined the functional importance of GTP hydrolysis by eRF3 in Saccharomyces cerevisiae. We found that mutations that reduced the rate of GTP hydrolysis also reduced the efficiency of translation termination at some termination signals but not others. As much as a 17-fold decrease in the termination efficiency was observed at some tetranucleotide termination signals (characterized by the stop codon and the first following nucleotide), while no effect was observed at other termination signals. To determine whether this stop signal-dependent decrease in the efficiency of translation termination was due to a defect in either eRF1 or eRF3 recycling, we reduced the level of eRF1 or eRF3 in cells by expressing them individually from the CUP1 promoter. We found that the limitation of either factor resulted in a general decrease in the efficiency of translation termination rather than a decrease at a subset of termination signals as observed with the eRF3 GTPase mutants. We also found that overproduction of eRF1 was unable to increase the efficiency of translation termination at any termination signals. Together, these results suggest that the GTPase activity of eRF3 is required to couple the recognition of translation termination signals by eRF1 to efficient polypeptide chain release.

scholarly journals Eukaryotic Release Factor 1 Phosphorylation by CK2 Protein Kinase Is Dynamic but Has Little Effect on the Efficiency of Translation Termination in Saccharomyces cerevisiae

2006 ◽  
Vol 5 (8) ◽  
pp. 1378-1387 ◽  
Author(s):  
Adam K. Kallmeyer ◽  
Kim M. Keeling ◽  
David M. Bedwell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Synthesis ◽  
Protein Kinase ◽  
Stop Codon ◽  
Translation Termination ◽  
Release Factor ◽  
Codon Recognition ◽  
Number Of Factors ◽  
Stop Codon Recognition

ABSTRACT Protein synthesis requires a large commitment of cellular resources and is highly regulated. Previous studies have shown that a number of factors that mediate the initiation and elongation steps of translation are regulated by phosphorylation. In this report, we show that a factor involved in the termination step of protein synthesis is also subject to phosphorylation. Our results indicate that eukaryotic release factor 1 (eRF1) is phosphorylated in vivo at serine 421 and serine 432 by the CK2 protein kinase (previously casein kinase II) in the budding yeast Saccharomyces cerevisiae. Phosphorylation of eRF1 has little effect on the efficiency of stop codon recognition or nonsense-mediated mRNA decay. Also, phosphorylation is not required for eRF1 binding to the other translation termination factor, eRF3. In addition, we provide evidence that the putative phosphatase Sal6p does not dephosphorylate eRF1 and that the state of eRF1 phosphorylation does not influence the allosuppressor phenotype associated with a sal6Δ mutation. Finally, we show that phosphorylation of eRF1 is a dynamic process that is dependent upon carbon source availability. Since many other proteins involved in protein synthesis have a CK2 protein kinase motif near their extreme C termini, we propose that this represents a common regulatory mechanism that is shared by factors involved in all three stages of protein synthesis.

Observation of Amyloid-Like Fibers of the Sup35 Protein from Saccharomyces Cerevisiae

2000 ◽  
Vol 6 (S2) ◽  
pp. 664-665
Author(s):  
Anthony S. Kowal ◽  
Thomas Scheibel ◽  
Susan L. Lindquist
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Conformation ◽  
Prion Proteins ◽  
Translation Termination ◽  
Polarized Light ◽  
Yeast Cells ◽  
Yeast Prion ◽  
Epigenetic Modifier ◽  
Insoluble Form

In the yeast Saccharomyces cerevisiae, [PST] acts as an epigenetic modifier of translation termination efficiency. [PSI+] can be passed through generations of yeast cells via changes in protein conformation rather than changes in DNA or RNA, and has thus been referred to as a yeast prion. The [PSI+] determinant is the Sup35 protein. Sup35 can exist in two states - soluble and insoluble. Soluble Sup35 functions in translation termination, but when insoluble, stop codons are read through, resulting in incorrect protein products.Sup35 is composed of three distinct domains, N, M, and C. The N region is rich in glutamine and asparagine and is required for the [PST] phenotype to exist. M is a highly charged domain, and no specific function has been assigned to it. C is essential in yeast, as it is responsible for translation termination. The insoluble form of Sup35 has characteristics reminiscent of other prion proteins - in vitro it binds to the dye Congo Red and it exhibits apple green birefringence in polarized light.

Persulfide formation on mitochondrial cysteine desulfurase: enzyme activation by a eukaryote-specific interacting protein and Fe–S cluster synthesis

2012 ◽  
Vol 448 (2) ◽  
pp. 171-187 ◽  
Author(s):  
Alok Pandey ◽  
Ramesh Golla ◽  
Heeyong Yoon ◽  
Andrew Dancis ◽  
Debkumar Pain
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Viability ◽  
Conformational Change ◽  
Active Site ◽  
Enzyme Activation ◽  
Accessory Proteins ◽  
Cysteine Desulfurase ◽  
Interacting Protein ◽  
Active Site Cysteine ◽  
Isolated Mitochondria

Cysteine desulfurases abstract sulfur from the substrate cysteine, generate a covalent persulfide on the active site cysteine of the enzyme, and then donate the persulfide sulfur to various recipients such as Fe–S clusters. In Saccharomyces cerevisiae, the Nfs1p protein is the only known cysteine desulfurase, and it forms a complex with Isd11p (Nfs1p·Isd11p). Both of these proteins are found primarily in mitochondria and both are essential for cell viability. In the present study we show, using the results of experiments with isolated mitochondria and purified proteins, that Isd11p is required for the cysteine desulfurase activity of Nfs1p. Whereas Nfs1p by itself was inactive, the Nfs1p·Isd11p complex formed persulfide and was active as a cysteine desulfurase. In the absence of Isd11p, Nfs1p was able to bind the substrate cysteine but failed to form a persulfide. Addition of Isd11p allowed Nfs1p with bound substrate to generate a covalent persulfide. We suggest that Isd11p induces an activating conformational change in Nfs1p to bring the bound substrate and the active site cysteine in proximity for persulfide formation. Thus mitochondrial Nfs1p is different from bacterial cysteine desulfurases that are active in the absence of accessory proteins. Isd11p may serve to regulate cysteine desulfurase activity in mitochondria.

Strengths and weaknesses in the determination of Saccharomyces cerevisiae cell viability by ATP-based bioluminescence assay

2013 ◽  
Vol 52 (3) ◽  
pp. 157-162 ◽  
Author(s):  
Lucia Paciello ◽  
Francesco Cristino Falco ◽  
Carmine Landi ◽  
Palma Parascandola
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Viability ◽  
Bioluminescence Assay

scholarly journals Tpa1p Is Part of an mRNP Complex That Influences Translation Termination, mRNA Deadenylation, and mRNA Turnover in Saccharomyces cerevisiae

2006 ◽  
Vol 26 (14) ◽  
pp. 5237-5248 ◽  
Author(s):  
Kim M. Keeling ◽  
Joe Salas-Marco ◽  
Lev Z. Osherovich ◽  
David M. Bedwell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mrna Decay ◽  
Potential Role ◽  
Translation Termination ◽  
Tail Length ◽  
Fold Increase ◽  
Reading Frame ◽  
Yeast Strains ◽  
Messenger Ribonucleoprotein ◽  
Nonsense Mediated Mrna Decay

ABSTRACT In this report, we show that the Saccharomyces cerevisiae protein Tpa1p (for termination and polyadenylation) influences translation termination efficiency, mRNA poly(A) tail length, and mRNA stability. Tpa1p is encoded by the previously uncharacterized open reading frame YER049W. Yeast strains carrying a deletion of the TPA1 gene (tpa1Δ) exhibited increased readthrough of stop codons, and coimmunoprecipitation assays revealed that Tpa1p interacts with the translation termination factors eRF1 and eRF3. In addition, the tpa1Δ mutation led to a 1.5- to 2-fold increase in the half-lives of mRNAs degraded by the general 5′→3′ pathway or the 3′→5′ nonstop decay pathway. In contrast, this mutation did not have any affect on the nonsense-mediated mRNA decay pathway. Examination of mRNA poly(A) tail length revealed that poly(A) tails are longer than normal in a tpa1Δ strain. Consistent with a potential role in regulating poly(A) tail length, Tpa1p was also found to coimmunoprecipitate with the yeast poly(A) binding protein Pab1p. These results suggest that Tpa1p is a component of a messenger ribonucleoprotein complex bound to the 3′ untranslated region of mRNAs that affects translation termination, deadenylation, and mRNA decay.

Sign in / Sign up

Export Citation Format

Share Document