Characterization of Gtf1p, the Connector Subunit of Yeast Mitochondrial tRNA-dependent Amidotransferase

The bacterial GatCAB operon for tRNA-dependent amidotransferase (AdT) catalyzes the transamidation of mischarged glutamyl-tRNA(Gln) to glutaminyl-tRNA(Gln). Here we describe the phenotype of temperature-sensitive (ts) mutants of GTF1, a gene proposed to code for subunit F of mitochondrial AdT in Saccharomyces cerevisiae. The ts gtf1 mutants accumulate an electrophoretic variant of the mitochondrially encoded Cox2p subunit of cytochrome oxidase and an unstable form of the Atp8p subunit of the F(1)-F(0) ATP synthase that is degraded, thereby preventing assembly of the F(0) sector. Allotopic expression of recoded ATP8 and COX2 did not significantly improve growth of gtf1 mutants on respiratory substrates. However, ts gft1 mutants are partially rescued by overexpression of PET112 and HER2 that code for the yeast homologues of the catalytic subunits of bacterial AdT. Additionally, B66, a her2 point mutant has a phenotype similar to that of gtf1 mutants. These results provide genetic support for the essentiality, in vivo, of the GatF subunit of the heterotrimeric AdT that catalyzes formation of glutaminyl-tRNA(Gln) (Frechin, M., Senger, B., Braye, M., Kern, D., Martin, R. P., and Becker, H. D. (2009) Genes Dev. 23, 1119-1130).

The Trypanosoma cruzi proteins TcCox10 and TcCox15 catalyze the formation of heme A in the yeast Saccharomyces cerevisiae

Trypanosoma cruzi, the etiologic agent for Chagas` disease, has requirements for several cofactors, one of which is heme. Because this organism is unable to synthesize heme, which serves as a prosthetic group for several heme proteins (including the respiratory chain complexes), it therefore must be acquired from the environment. Considering this deficiency, it is an open question as to how heme A, the essential cofactor for eukaryotic CcO enzymes, is acquired by this parasite. In the present work, we provide evidence for the presence and functionality of genes coding for heme O and heme A synthases, which catalyze the synthesis of heme O and its conversion into heme A, respectively. The functions of these T. cruzi proteins were evaluated using yeast complementation assays, and the mRNA levels of their respective genes were analyzed at the different T. cruzi life stages. It was observed that the amount of mRNA coding for these proteins changes during the parasite life cycle, suggesting that this variation could reflect different respiratory requirements in the different parasite life stages.

Structural modeling and mutational analysis of yeast eukaryotic translation initiation factor 5A reveal new critical residues and reinforce its involvement in protein synthesis

Eukaryotic translation initiation factor 5A (eIF5A) is a protein that is highly conserved and essential for cell viability. This factor is the only protein known to contain the unique and essential amino acid residue hypusine. This work focused on the structural and functional characterization of Saccharomyces cerevisiae eIF5A. The tertiary structure of yeast eIF5A was modeled based on the structure of its Leishmania mexicana homologue and this model was used to predict the structural localization of new site-directed and randomly generated mutations. Most of the 40 new mutants exhibited phenotypes that resulted from eIF-5A protein-folding defects. Our data provided evidence that the C-terminal alpha-helix present in yeast eIF5A is an essential structural element, whereas the eIF5A N-terminal 10 amino acid extension not present in archaeal eIF5A homologs, is not. Moreover, the mutants containing substitutions at or in the vicinity of the hypusine modification site displayed nonviable or temperature-sensitive phenotypes and were defective in hypusine modification. Interestingly, two of the temperature-sensitive strains produced stable mutant eIF5A proteins - eIF5A(K56A) and eIF5A(Q22H,L93F)- and showed defects in protein synthesis at the restrictive temperature. Our data revealed important structural features of eIF5A that are required for its vital role in cell viability and underscored an essential function of eIF5A in the translation step of gene expression.

Fibroblast growth factor 2 restrains ras-driven proliferation of malignant cells by triggering RhoA-mediated senescence

Fibroblast growth factor 2 (FGF2) is considered to be a bona fide oncogenic factor, although results from our group and others call this into question. Here, we report that exogenous recombinant FGF2 irreversibly inhibits proliferation by inducing senescence in Ras-dependent malignant mouse cells, but not in immortalized nontumorigenic cell lines. We report the following findings in K-Ras-dependent malignant YI adrenocortical cells and H-Ras V12-transformed BALB-3T3 fibroblasts: (a) FGF2 inhibits clonal growth and tumor onset in nude and immunocompetent BALB/c mice, (b) FGF2 irreversibly blocks the cell cycle, and (c) FGF2 induces the senescence-associated -galactosidase with no accompanying signs of apoptosis or necrosis. The tyrosine kinase inhibitor PD173074 completely protected malignant cells from FGF2. In Yl adrenal cells, reducing the constitutively high levels of K-Ras-GTP using the dominant-negative RasN17 mutant made cells resistant to FGF2 cytotoxicity. In addition, transfection of the dominant-negative RhoA-N19 into either YI or 3T3-B61 malignant cell lines yielded stable clonal transfectants that were unable to activate RhoA and were resistant to the FGF2 stress response. We conclude that in Rasdependent malignant cells, FGF2 interacts with its cognate receptors to trigger a senescence-like process involving RboAGTP. Surprisingly, attempts to select FGF2-resistant cells from the Yl and 3T3-B61 cell lines yielded only rare clones that (a) had lost the overexpressed ras oncogene, (b) were dependent on FGF2 for proliferation, and (c) were poorly tumorigenic. Thus, FGF2 exerted a strong negative selection that Rasdependent malignant cells could rarely overcome.

Arginine vasopressin controls p27(KiP1) protein expression by PKC activation and irreversibly inhibits the proliferation of K-Ras-dependent mouse Y1 adrenocortical malignant cells

The neurohypophyseal hormone arginine vasopressin (AVP) is a classic mitogen in many cells. In K-Ras-dependent mouse Y1 adrenocortical malignant cells, AVP elicits antagonistic responses such as the activation of the PKC and the ERK1/2 mitogenic pathways to down-regulate cyclin D1 gene expression, which induces senescence-associated beta-galactosidase (SA-beta Gal) and leads to cell cycle arrest. Here, we report that in the metabolic background of Y1 cells, PKC activation either by AVP or by PMA inhibits the PI3K/Akt pathway and stabilises the p27(Kip1) protein even in the presence of the mitogen fibroblast growth factor 2 (FGF2). These results suggest that p27(Kip1) is a critical signalling node in the mechanisms underlying the survival of the Y1 cells. In Y1 cells that transiently express wild-type p27(Kip1), AVP caused a severe reduction in cell survival, as shown by clonogenic assays. However, AVP promoted the survival of Y1 cells transiently expressing mutant p27-S10A or mutant p27-T187A, which cannot be phosphorylated at Ser10 and Thr187, respectively. In addition, PKC activation by PMA mimics the toxic effect caused by AVP in Y1 cells, and inhibition of PKC completely abolishes the effects caused by both PMA and AVP in clonogenic assays. The vulnerability of Y1 cells during PKC activation is a phenotype conditioned upon K-ras oncogene amplification because K-Ras down-regulation with an inducible form of the dominant-negative mutant H-RasN17 has resulted in Y1 cells that are resistant to AVP`s deleterious effects. These data show that the survival destabilisation of K-Ras-dependent Y1 malignant cells by AVP requires large quantities of the p27(Kip1) protein as well as phosphorylation of the p27(Kip1) protein at both Ser10 and Thr187. (C) 2011 Elsevier B.V. All rights reserved.

Sdo1p, the yeast orthologue of Shwachman-Bodian-Diamond syndrome protein, binds RNA and interacts with nuclear rRNA-processing factors

The Shwachman-Bodian-Diamond syndrome protein (SBDS) is a member of a highly conserved protein family of not well understood function, with putative orthologues found in different organisms ranging from Archaea, yeast and plants to vertebrate animals. The yeast orthologue of SBDS, Sdo1p, has been previously identified in association with the 60S ribosomal subunit and is proposed to participate in ribosomal recycling. Here we show that Sdo1p interacts with nucleolar rRNA processing factors and ribosomal proteins, indicating that it might bind the pre-60S complex and remain associated with it during processing and transport to the cytoplasm. Corroborating the protein interaction data, Sdo1p localizes to the nucleus and cytoplasm and co-immunoprecipitates precursors of 60S and 40S subunits, as well as the mature rRNAs. Sdo1p binds RNA directly, suggesting that it may associate with the ribosomal subunits also through RNA interaction. Copyright (C) 2009 John Wiley & Sons, Ltd.

Oroidin Inhibits the Activity of the Multidrug Resistance Target Pdr5p from Yeast Plasma Membranes

Oroidin was isolated from the marine sponge Agelas sventres and inhibited the activity and function of Pdr5p, an enzyme responsible for the multidrug resistance phenotype in Saccharomyces cerevisiae. This compound may help in the development of new drugs that reverse this dangerous phenotype of pathogenic yeast and fungi.

Insights into the Specificity of Thioredoxin Reductase-Thioredoxin Interactions. A Structural and Functional Investigation of the Yeast Thioredoxin System

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cryptococcus haglerorum, sp nov., an anamorphic basidiomycetous yeast isolated from nests of the leaf-cutting ant Atta sexdens

A yeast strain (CBS 8902) was isolated from the nest of a leaf-cutting ant and was shown to be related to Cryptococcus humicola. Sequencing of the D1/D2 region of the 26S ribosomal DNA and physiological characterization revealed a separate taxonomic position. A novel species named Cryptococcus haglerorum is proposed to accommodate strain CBS 8902 that assimilates n-hexadecane and several benzene compounds. Physiological characteristics distinguishing the novel species from some other members of the C. humicola complex are presented. The phylogenetic relationship of these strains to species of the genus Trichosporon Behrend is discussed.

Sympodiomyces attinorum sp nov., a yeast species associated with nests of the leaf-cutting ant Atta sexdens

Four strains of a novel yeast species were isolated from laboratory nests of the leaf-cutting ant Atta sexdens in Brazil. Three strains were found in older sponges and one was in a waste deposit in the ant nests. Sequencing of the D1/D2 region of the large-subunit rRNA gene showed that the novel species, named Sympodiomyces attinorum sp. nov., is phylogenetically related to Sympodiomyces parvus. Unlike Sympodiomyces parvus, Sympodiomyces attinorum can ferment glucose, assimilate methyl alpha-D-glucoside, salicin and citrate, and grow at 37 degreesC, thus enabling these two species to be distinguished. Differentiation from other related species is possible on the basis of other growth characteristics. The type strain of Sympodiomyces attinorum is UNESP-S156(T) (=CBS 9734(T)=NRRL Y-27639(T)).

Synonymy of the yeast genera Moniliella and Trichosporonoides and proposal of Moniliella fonsecae sp nov and five new species combinations

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Yeast extract and prebiotic in pre-initial phase diet for broiler chickens raised under different temperatures

Objetivou-se nesta pesquisa avaliar o desempenho, o rendimento de carcaça e a morfometria intestinal de frangos de corte criados em diferentes temperaturas e que receberam na fase pré-inicial ração contendo ou não extrato de leveduras e prebiótico. Foram utilizados 1.440 pintos machos de 1 dia de idade, criados em diferentes câmaras climáticas. As rações, acrescidas ou não de extrato de leveduras e prebiótico, foram oferecidas somente na fase pré-inicial (1 a 7 dias). A partir do oitavo dia, todas as aves receberam a mesma ração, reajustada de acordo com as recomendações usuais. Adotou-se o delineamento experimental inteiramente casualizado em arranjo fatorial 3 × 2 × 2, composto de três temperaturas de criação (calor, conforto e frio) e dois níveis de extrato de leveduras (com ou sem) e prebiótico (com ou sem). O desempenho das aves foi avaliado considerando o ganho de peso, o consumo de ração, a conversão alimentar e a viabilidade aos 42 dias de idade. Também foram avaliados o rendimento de carcaça e a morfometria intestinal. O calor ambiente prejudicou o desempenho e o rendimento de carcaça. A inclusão de prebiótico na ração pré-inicial aumentou o ganho de peso e melhorou a conversão alimentar das aves criadas no calor. A inclusão dos produtos na ração de frangos de corte criados em ambiente de calor e no frio tem efeito benéfico sobre as vilosidades das aves.

Evaluation of yeast cell wall on the performance of broiles fed diets with or without mycotoxins

This experiment aimed at evaluating the effects of the interactions between aflatoxin (500 or 250 ppb) and ochratoxin (500 or 250 ppb), and the possible benefits of adding yeast cell wall to prevent the effects of these mycotoxins in broiler chickens. Relative organ weight gain and live performance were evaluated at 21 and 42 days of age. Results indicated that at the levels of mycotoxins included in the experimental diets, ochratoxin reduced feed intake and body weight gain, and aflatoxin only affect feed intake of 21-day-old birds. No interaction was observed between aflatoxin and ochratoxin at the levels used in experimental study. Yeast cell wall did not significantly reduced the deleterious effects of ochratoxins. No significant differences were observed in relative organ weight gain. Yeast cell wall improved feed conversion ratio when birds were fed either contaminated or non-contaminated feeds.