DPH5, a methyltransferase gene required for diphthamide biosynthesis in Saccharomyces cerevisiae.

A mutant of Saccharomyces cerevisiae defective in the S-adenosylmethionine (AdoMet)-dependent methyltransferase step of diphthamide biosynthesis was selected by intracellular expression of the F2 fragment of diphtheria toxin (DT) and shown to belong to complementation group DPH5. The DPH5 gene was cloned, sequenced, and found to encode a 300-residue protein with sequence similarity to bacterial AdoMet:uroporphyrinogen III methyltransferases, enzymes involved in cobalamin (vitamin B12) biosynthesis. Both DPH5 and AdoMet:uroporphyrinogen III methyltransferases lack sequence motifs commonly found in other methyltransferases and may represent a new family of AdoMet:methyltransferases. The DPH5 protein was produced in Escherichia coli and shown to be active in methylation of elongation factor 2 partially purified from the dph5 mutant. A null mutation of the chromosomal DPH5 gene did not affect cell viability, in agreement with other studies indicating that diphthamide is not required for cell survival. The dph5 null mutant survived expression of three enzymically attenuated DT fragments but was killed by expression of fully active DT fragment A. Consistent with these results, elongation factor 2 from the dph5 null mutant was found to have weak ADP-ribosyl acceptor activity, which was detectable only in the presence of high concentrations of fragment A.

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Saccharomyces cerevisiae elongation factor 2. Genetic cloning, characterization of expression, and G-domain modeling.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)48413-x ◽

1992 ◽

Vol 267 (2) ◽

pp. 1190-1197 ◽

Cited By ~ 3

Author(s):

J P Perentesis ◽

L D Phan ◽

W B Gleason ◽

D C LaPorte ◽

D M Livingston ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Elongation Factor ◽

Domain Modeling ◽

Elongation Factor 2

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Cloning and characterization of FAD1, the structural gene for flavin adenine dinucleotide synthetase of Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.15.1.264 ◽

1995 ◽

Vol 15 (1) ◽

pp. 264-271 ◽

Cited By ~ 63

Author(s):

M Wu ◽

B Repetto ◽

D M Glerum ◽

A Tzagoloff

Keyword(s):

Saccharomyces Cerevisiae ◽

Flavin Adenine Dinucleotide ◽

Genomic Library ◽

Sequence Similarity ◽

Complementation Group ◽

Flavin Mononucleotide ◽

Synthetase Activity ◽

Multicopy Plasmid ◽

Multiple Copies ◽

Extragenic Suppression

The FAD1 gene of Saccharomyces cerevisiae has been selected from a genomic library on the basis of its ability to partially correct the respiratory defect of pet mutants previously assigned to complementation group G178. Mutants in this group display a reduced level of flavin adenine dinucleotide (FAD) and an increased level of flavin mononucleotide (FMN) in mitochondria. The restoration of respiratory capability by FAD1 is shown to be due to extragenic suppression. FAD1 codes for an essential yeast protein, since disruption of the gene induces a lethal phenotype. The FAD1 product has been inferred to be yeast FAD synthetase, an enzyme that adenylates FMN to FAD. This conclusion is based on the following evidence. S. cerevisiae transformed with FAD1 on a multicopy plasmid displays an increase in FAD synthetase activity. This is also true when the gene is expressed in Escherichia coli. Lastly, the FAD1 product exhibits low but significant primary sequence similarity to sulfate adenyltransferase, which catalyzes a transfer reaction analogous to that of FAD synthetase. The lower mitochondrial concentration of FAD in G178 mutants is proposed to be caused by an inefficient exchange of external FAD for internal FMN. This is supported by the absence of FAD synthetase activity in yeast mitochondria and the presence of both extramitochondrial and mitochondrial riboflavin kinase, the preceding enzyme in the biosynthetic pathway. A lesion in mitochondrial import of FAD would account for the higher concentration of mitochondrial FMN in the mutant if the transport is catalyzed by an exchange carrier. The ability of FAD1 to suppress impaired transport of FAD is explained by mislocalization of the synthetase in cells harboring multiple copies of the gene. This mechanism of suppression is supported by the presence of mitochondrial FAD synthetase activity in S. cerevisiae transformed with FAD1 on a high-copy-number plasmid but not in mitochondrial of a wild-type strain.

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Diphtheria toxin-resistant mutants of Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.5.12.3357 ◽

1985 ◽

Vol 5 (12) ◽

pp. 3357-3360 ◽

Cited By ~ 60

Author(s):

J Y Chen ◽

J W Bodley ◽

D M Livingston

Keyword(s):

Saccharomyces Cerevisiae ◽

Diphtheria Toxin ◽

Elongation Factor ◽

Selection Procedure ◽

Prolonged Storage ◽

Elongation Factor 2 ◽

Resistant Phenotype ◽

Complementation Groups ◽

Mendelian Character

We developed a selection procedure based on the observation that diphtheria toxin kills spheroplasts of Saccharomyces cerevisiae (Murakami et al., Mol. Cell. Biol. 2:588-592, 1982); this procedure yielded mutants resistant to the in vitro action of the toxin. Spheroplasts of mutagenized S. cerevisiae were transformed in the presence of diphtheria toxin, and the transformed survivors were screened in vitro for toxin-resistant elongation factor 2. Thirty-one haploid ADP ribosylation-negative mutants comprising five complementation groups were obtained by this procedure. The mutants grew normally and were stable to prolonged storage. Heterozygous diploids produced by mating wild-type sensitive cells with the mutants revealed that in each case the resistant phenotype was recessive to the sensitive phenotype. Sporulation of these diploids yielded tetrads in which the resistant phenotype segregated as a single Mendelian character. From these observations, we concluded that these mutants are defective in the enzymatic steps responsible for the posttranslational modification of elongation factor 2 which is necessary for recognition by diphtheria toxin.

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Diphtheria toxin-resistant mutants of Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.5.12.3357-3360.1985 ◽

1985 ◽

Vol 5 (12) ◽

pp. 3357-3360

Author(s):

J Y Chen ◽

J W Bodley ◽

D M Livingston

Keyword(s):

Saccharomyces Cerevisiae ◽

Diphtheria Toxin ◽

Elongation Factor ◽

Selection Procedure ◽

Prolonged Storage ◽

Elongation Factor 2 ◽

Resistant Phenotype ◽

Complementation Groups ◽

Mendelian Character

We developed a selection procedure based on the observation that diphtheria toxin kills spheroplasts of Saccharomyces cerevisiae (Murakami et al., Mol. Cell. Biol. 2:588-592, 1982); this procedure yielded mutants resistant to the in vitro action of the toxin. Spheroplasts of mutagenized S. cerevisiae were transformed in the presence of diphtheria toxin, and the transformed survivors were screened in vitro for toxin-resistant elongation factor 2. Thirty-one haploid ADP ribosylation-negative mutants comprising five complementation groups were obtained by this procedure. The mutants grew normally and were stable to prolonged storage. Heterozygous diploids produced by mating wild-type sensitive cells with the mutants revealed that in each case the resistant phenotype was recessive to the sensitive phenotype. Sporulation of these diploids yielded tetrads in which the resistant phenotype segregated as a single Mendelian character. From these observations, we concluded that these mutants are defective in the enzymatic steps responsible for the posttranslational modification of elongation factor 2 which is necessary for recognition by diphtheria toxin.

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Brettanomyces bruxellensis SSU1 Haplotypes Confer Different Levels of Sulfite Tolerance When Expressed in a Saccharomyces cerevisiae SSU1 Null Mutant

Applied and Environmental Microbiology ◽

10.1128/aem.02429-18 ◽

2018 ◽

Vol 85 (4) ◽

Cited By ~ 2

Author(s):

C. Varela ◽

C. Bartel ◽

M. Roach ◽

A. Borneman ◽

C. Curtin

Keyword(s):

Saccharomyces Cerevisiae ◽

Efflux Pump ◽

Wine Industry ◽

Null Mutant ◽

Brettanomyces Bruxellensis ◽

Content Type ◽

Relative Contribution ◽

Wine Spoilage ◽

Allelic Differences ◽

High Concentrations

ABSTRACT The addition of SO2 is practiced in the wine industry to mitigate the risk of microbial spoilage and to extend wine shelf-life. Generally, this strategy does not interfere with primary alcoholic fermentation, as wine strains of Saccharomyces cerevisiae exhibit significant SO2 tolerance, largely driven by the efflux pump Ssu1p. One of the key yeast species responsible for wine spoilage is Brettanomyces bruxellensis, which also exhibits strain-dependent SO2 tolerance, although this occurs via unknown mechanisms. To evaluate the factors responsible for the differential sulfite tolerance observed in B. bruxellensis strains, we employed a multifaceted approach to examine both expression and allelic differences in the BbSSU1 gene. Transcriptomic analysis following exposure to SO2 highlighted different inducible responses in two B. bruxellensis strains. It also revealed disproportionate transcription of one putative BbSSU1 haplotype in both genetic backgrounds. Here, we confirm the functionality of BbSSU1 by complementation of a null mutant in a S. cerevisiae wine strain. The expression of four distinct BbSSU1 haplotypes in the S. cerevisiae ΔSSU1 mutant revealed up to a 3-fold difference in conferred SO2 tolerance. Substitution of key amino acids distinguishing the encoded proteins was performed to evaluate their relative contribution to SO2 tolerance. Protein modeling of two haplotypes which differed in two amino acid residues suggested that these substitutions affect the binding of Ssu1p ligands near the channel opening. Taken together, preferential transcription of a BbSSU1 allele that encodes a more efficient Ssu1p transporter may represent one mechanism that contributes to differences in sulfite tolerances between B. bruxellensis strains. IMPORTANCE Brettanomyces bruxellensis is one of the most important wine spoilage microorganisms, with the use of sulfite being the major method to control spoilage. However, this species displays a wide intraspecies distribution in sulfite tolerance, with some strains capable of tolerating high concentrations of SO2, with relatively high concentrations of this antimicrobial needed for their control. Although SO2 tolerance has been studied in several organisms and particularly in S. cerevisiae, little is known about the mechanisms that confer SO2 tolerance in B. bruxellensis. Here, we confirmed the functionality of the sulfite efflux pump encoded by BbSSU1 and determined the efficiencies of four different BbSSU1 haplotypes. Gene expression analysis showed greater expression of the haplotype conferring greater SO2 tolerance. Our results suggest that a combination of BbSSU1 haplotype efficiency, copy number, and haplotype expression levels likely contributes to the diverse SO2 tolerances observed for different B. bruxellensis strains.

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A Chemical Genomic Screen in Saccharomyces cerevisiae Reveals a Role for Diphthamidation of Translation Elongation Factor 2 in Inhibition of Protein Synthesis by Sordarin

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01603-07 ◽

2008 ◽

Vol 52 (5) ◽

pp. 1623-1629 ◽

Cited By ~ 19

Author(s):

Javier Botet ◽

María Rodríguez-Mateos ◽

Juan P. G. Ballesta ◽

José Luis Revuelta ◽

Miguel Remacha

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Synthesis ◽

Elongation Factor ◽

Translation Elongation Factor ◽

Cellular Mechanisms ◽

Translation Elongation ◽

Chemical Genomic ◽

Elongation Factor 2 ◽

Fungal Organisms ◽

Inhibitor Of Protein Synthesis

ABSTRACT Sordarin and its derivatives are antifungal compounds of potential clinical interest. Despite the highly conserved nature of the fungal and mammalian protein synthesis machineries, sordarin is a selective inhibitor of protein synthesis in fungal organisms. In cells sensitive to sordarin, its mode of action is through preventing the release of translation elongation factor 2 (eEF2) during the translocation step, thus blocking protein synthesis. To further investigate the cellular components required for the effects of sordarin in fungal cells, we have used the haploid deletion collection of Saccharomyces cerevisiae to systematically identify genes whose deletion confers sensitivity or resistance to the compound. Our results indicate that genes in a number of cellular pathways previously unknown to play a role in sordarin response are involved in its growth effects on fungal cells and reveal a specific requirement for the diphthamidation pathway of cells in causing eEF2 to be sensitive to the effects of sordarin on protein synthesis. Our results underscore the importance of the powerful genomic tools developed in yeast (Saccharomyces cerevisiae) to more comprehensively understanding the cellular mechanisms involved in the response to therapeutic agents.

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Translational Roles of Elongation Factor 2 Protein Lysine Methylation

Journal of Biological Chemistry ◽

10.1074/jbc.m114.605527 ◽

2014 ◽

Vol 289 (44) ◽

pp. 30511-30524 ◽

Cited By ~ 15

Author(s):

Maria C. Dzialo ◽

Kyle J. Travaglini ◽

Sean Shen ◽

Kevin Roy ◽

Guillaume F. Chanfreau ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Synthesis ◽

Elongation Factor ◽

Amino Acid Sequences ◽

Fine Tuning ◽

Lysine Methylation ◽

Translational Machinery ◽

Elongation Factor 2 ◽

Translational Apparatus

Methylation of various components of the translational machinery has been shown to globally affect protein synthesis. Little is currently known about the role of lysine methylation on elongation factors. Here we show that in Saccharomyces cerevisiae, the product of the EFM3/YJR129C gene is responsible for the trimethylation of lysine 509 on elongation factor 2. Deletion of EFM3 or of the previously described EFM2 increases sensitivity to antibiotics that target translation and decreases translational fidelity. Furthermore, the amino acid sequences of Efm3 and Efm2, as well as their respective methylation sites on EF2, are conserved in other eukaryotes. These results suggest the importance of lysine methylation modification of EF2 in fine tuning the translational apparatus.

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Identification and Characterization of a Novel Evolutionarily Conserved Lysine-specific Methyltransferase Targeting Eukaryotic Translation Elongation Factor 2 (eEF2)

Journal of Biological Chemistry ◽

10.1074/jbc.m114.601658 ◽

2014 ◽

Vol 289 (44) ◽

pp. 30499-30510 ◽

Cited By ~ 26

Author(s):

Erna Davydova ◽

Angela Y. Y. Ho ◽

Jedrzej Malecki ◽

Anders Moen ◽

Jorrit M. Enserink ◽

...

Keyword(s):

Ribosomal Proteins ◽

Sequence Similarity ◽

Elongation Factor ◽

Protein Translation ◽

Cellular Protein ◽

Yeast Cells ◽

Lysine Methylation ◽

Elongation Factor 2

The components of the cellular protein translation machinery, such as ribosomal proteins and translation factors, are subject to numerous post-translational modifications. In particular, this group of proteins is frequently methylated. However, for the majority of these methylations, the responsible methyltransferases (MTases) remain unknown. The human FAM86A (family with sequence similarity 86) protein belongs to a recently identified family of protein MTases, and we here show that FAM86A catalyzes the trimethylation of eukaryotic elongation factor 2 (eEF2) on Lys-525. Moreover, we demonstrate that the Saccharomyces cerevisiae MTase Yjr129c, which displays sequence homology to FAM86A, is a functional FAM86A orthologue, modifying the corresponding residue (Lys-509) in yeast eEF2, both in vitro and in vivo. Finally, Yjr129c-deficient yeast cells displayed phenotypes related to eEF2 function (i.e. increased frameshifting during protein translation and hypersensitivity toward the eEF2-specific drug sordarin). In summary, the present study establishes the function of the previously uncharacterized MTases FAM86A and Yjr129c, demonstrating that these enzymes introduce a functionally important lysine methylation in eEF2. Based on the previous naming of similar enzymes, we have redubbed FAM86A and Yjr129c as eEF2-KMT and Efm3, respectively.

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Translation Elongation Factor 2 Is Part of the Target for a New Family of Antifungals

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.10.2694 ◽

1998 ◽

Vol 42 (10) ◽

pp. 2694-2699 ◽

Cited By ~ 56

Author(s):

Laura Capa ◽

Alfonso Mendoza ◽

José L. Lavandera ◽

Federico Gómez de las Heras ◽

José F. García-Bustos

Keyword(s):

Mutant Cell ◽

Complex Structure ◽

Elongation Factor ◽

Drug Binding ◽

Translation Elongation Factor ◽

Three Dimensional Model ◽

Translation Elongation ◽

New Family ◽

Cell Extracts ◽

Elongation Factor 2

ABSTRACT Translation elongation factor 2 (EF2), which in Saccharomyces cerevisiae is expressed from the EFT1 andEFT2 genes, has been found to be targeted by a new family of highly specific antifungal compounds derived from the natural product sordarin. Two complementation groups of mutants resistant to the semisynthetic sordarin derivative GM193663 were found. The major one (21 members) consisted of isolates with mutations onEFT2. The minor one (four isolates) is currently being characterized but it is already known that resistance in this group is not due to mutations on EFT1, pointing to the complex structure of the functional target for these compounds. Mutations on EF2 clustered, forming a possible drug binding pocket on a three-dimensional model of EF2, and mutant cell extracts lost the capacity to bind to the inhibitors. This new family of antifungals holds the promise to be a much needed and potent addition to current antimicrobial treatments, as well as a useful tool for dissection of the elongation process in ribosomal protein synthesis.

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