Descripción: Epsins are endocytic proteins with a structured epsin N-terminal homology (ENTH) domain that binds phosphoinositides and a poorly structured C-terminal region that interacts with ubiquitin and endocytic machinery, including clathrin and endocytic scaffolding proteins. Yeast has two redundant genes encoding epsins, ENT1 and ENT2; deleting both genes is lethal. We demonstrate that the ENTH domain is both necessary and sufficient for viability of ent1Δent2Δ cells. Mutational analysis of the ENTH domain revealed a surface patch that is essential for viability and that binds guanine nucleotide triphosphatase-activating proteins for Cdc42, a critical regulator of cell polarity in all eukaryotes. Furthermore, the epsins contribute to regulation of specific Cdc42 signaling pathways in yeast cells. These data support a model in which the epsins function as spatial and temporal coordinators of endocytosis and cell polarity. © 2006 by The National Academy of Sciences of the USA.

Descripción: Asr1, a tomato gene induced by abiotic stress, belongs to a family, composed by at least three members, involved in adaptation to dry climates. To understand the mechanism by which proteins of this family seem to protect cells from water loss in plants, we expressed Asr1 in the heterologous expression system Saccharomyces cerevisiae under the control of a galactose-inducible promoter. In a mutant yeast strain deficient in one component of the stress-responsive high-osmolarity glycerol (HOG) pathway, namely the MAP kinase Hog1, the synthesis of ASR1 protein restores growth under osmotic stress conditions such as 0.5 M NaCl and 1.2 M sorbitol. In contrast, the rescuing of this phenotype was less evident using a wild-type strain or the upstream MAP kinase kinase (Pbs2)-deficient strain. In both knock-out strains impaired in glycerol synthesis because of a dysfunctional HOG pathway, but not in wild-type, ASR1 led to the accumulation of endogenous glycerol in an osmotic stress-independent and unrestrained manner. These data suggest that ASR1 complements yeast HOG-deficient phenotypes by inducing downstream components of the HOG pathway. The results are discussed in terms of the function of ASR proteins in planta at the molecular and cellular level. Copyright © Physiologia Plantarum 2006.

Descripción: Saccharomyces cerevisiae pyruvate kinase 1 (Pyk1) was demonstrated to be associated to an immunoprecipitate of yeast protein kinase A holoenzyme (HA. Tpk1-Bcy1) and to be phosphorylated in a cAMP-dependent process. Both glutathione S-transferase (GST)-Pyk1 and GST-Pyk2 were phosphorylated in vitro by the bovine heart protein kinase A (PKA) catalytic subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GST-Pyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated in vivo, in intact cells overexpressing the protein, or in vitro using crude extracts, as source of protein kinase A, when a wild type strain was used but were not phosphorylated when using a strain with only one TPK gene with an attenuated mutation (tpk1w1). The effect of phosphorylation on Pyk activity was assayed in partially purified preparations from three strains, containing different endogenous protein kinase A activity levels. Pyk1 activity was measured at different phosphoenolpyruvate concentrations in the absence or in the presence of the activator fructose 1,6-bisphosphate at 1.5 mM. Preliminary kinetic results derived from the comparison of Pyk1 obtained from extracts with the highest versus those from the lowest protein kinase A activity indicate that the enzyme is more active upon phosphorylation conditions; in the absence of the activator it shows a shift in the titration curve for phosphoenolpyruvate to the left and an increase in the Hill coefficient, whereas in the presence of fructose 1,6-bisphosphate it shows an nH value of 1.4, as compared with an nH of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity.

Descripción: An enzyme, aminolaevulinate dehydratase (5-aminolaevulinate hydro-lyase (adding 5-aminolaevulinate and cyclizing), EC 4.2.1.24), which catalyzes the following reaction: {A figure is presented} has been isolated from yeast. Its purification and some of its properties have been studied. 5-Aminolaevulinate synthetase, another enzyme involved in porphyrin biosynthesis, has been detected in the mitochondrial particles. © 1967.

Descripción: The major cAMP receptors in eukaryotes are the regulatory (R) subunits of PKA, an allosteric enzyme conserved in fungi through mammals. While mammals have four R-subunit genes, Saccharomyces cerevisiae has only one, Bcy1. To achieve a molecular understanding of PKA activation in yeast and to explore the evolution of cyclic-nucleotide binding (CNB) domains, we solved the structure of cAMP-bound Bcy1(168-416). Surprisingly, the relative orientation of the two CNB domains in Bcy1 is very different from mammalian R-subunits. This quaternary structure is defined primarily by a fungi-specific sequence in the hinge between the αB/αC helices of the CNB-A domain. The unique interface between the two CNB domains in Bcy1 defines the allosteric mechanism for cooperative activation of PKA by cAMP. Some interface motifs are isoform-specific while others, although conserved, play surprisingly different roles in each R-subunit. Phylogenetic analysis shows that structural differences in Bcy1 are shared by fungi of the subphylum Saccharomycotina. © 2010 Elsevier Ltd.

Descripción: The first specific precursor of porphyrin biosynthesis is δ-aminolevulinic acid. δ-Aminolevulinic acid enters Saccharomyces cerevisiae cells through the γ-aminobutyric acid specific permease Uga4p. It was described that this permease is inducible by γ-aminobutyric acid and its regulation involves several specific and pleiotropic transcriptional factors. However, some studies showed that under certain growth conditions the synthesis of Uga4p was not dependent on the presence of γ-aminobutyric acid. To study the effect of the trans-acting factors Uga43p, Uga3p, Uga35p, Ure2p and Gln3p on the expression of UGA4, we measured γ-aminobutyric acid and δ-aminolevulinic acid uptake in yeast mutant cells, lacking one of these regulatory factors, grown under different conditions. Experiments analyzing the UGA4 promoter using a fusion construction UGA4::lacZ were also carried out. The results show that the constitutive expression of the UGA4 gene found in cells under certain growth conditions depends on the presence of Uga3p and Uga35p. In contrast, Gln3p and Ure2p do not seem to have any effect on this constitutive mechanism. Copyright (C) 2000 Federation of European Microbiological Societies.

Descripción: Trypanosoma cruzi, the aetiological agent of Chagas' disease, is exposed to extremely different environment conditions during its life cycle, and transporters are key molecules for its adaptive regulation. Amino acids, and particularly arginine, are essential components in T. cruzi metabolism. In this work, a novel T. cruzi arginine permease was identified by screening different members of the AAAP family (amino acid/auxin permeases) in yeast complementation assays using a toxic arginine analogue. One gene candidate, TcAAAP411, was characterized as a very specific, high-affinity, l-arginine permease. This work is the first identification of the molecular components involved specifically in amino acid transport in T. cruzi and provides new insights for further validation of the TcAAAP family as functional permeases. © 2010 Federation of European Microbiological Societies.

Descripción: Whereas biosensors have been usually proposed as analytical tools, used to investigate the surrounding media pursuing an analytical answer, we have used a biosensor-like device to characterize the microbial cells immobilized on it. We have studied the kinetics of transport and degradation of glucose at different concentrations and temperatures. When glucose concentrations of 15 and 1.5 mM were assayed, calculated activation energies were 25.2 and 18.4 kcal mol-1, respectively, in good agreement with previously published data. The opportunity and convenience of using Arrhenius plots to estimate the activation energy in metabolic-related processes is also discussed. © 2009 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland.

Descripción: The specificity in phosphorylation by kinases is determined by the molecular recognition of the peptide target sequence. In Saccharomyces cerevisiae, the protein kinase A (PKA) specificity determinants are less studied than in mammalian PKA. The catalytic turnover numbers of the catalytic subunits isoforms Tpk1 and Tpk2 were determined, and both enzymes are shown to have the same value of 3 s-1. We analyze the substrate behavior and sequence determinants around the phosphorylation site of three protein substrates, Pyk1, Pyk2, and Nth1. Nth1 protein is a better substrate than Pyk1 protein, and both are phosphorylated by either Tpk1 or Tpk2. Both enzymes also have the same selectivity toward the protein substrates and the peptides derived from them. The three substrates contain one or more Arg-Arg-X-Ser consensus motif, but not all of them are phosphorylated. The determinants for specificity were studied using the peptide arrays. Acidic residues in the position P+1 or in the N-terminal flank are deleterious, and positive residues present beyond P-2 and P-3 favor the catalytic reaction. A bulky hydrophobic residue in position P+1 is not critical. The best substrate has in position P+4 an acidic residue, equivalent to the one in the inhibitory sequence of Bcy1, the yeast regulatory subunit of PKA. The substrate effect in the holoenzyme activation was analyzed, and we demonstrate that peptides and protein substrates sensitized the holoenzyme to activation by cAMP in different degrees, depending on their sequences. The results also suggest that protein substrates are better co-activators than peptide substrates. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

Descripción: Aims: Analysis of the physiology and metabolism of Escherichia coli arcA and creC mutants expressing a bifunctional alcohol-acetaldehyde dehydrogenase from Leuconostoc mesenteroides growing on glycerol under oxygen-restricted conditions. The effect of an ldhA mutation and different growth medium modifications was also assessed. Methods and Results: Expression of adhE in E. coli CT1061 [arcA creC(Con)] resulted in a 1·4-fold enhancement in ethanol synthesis. Significant amounts of lactate were produced during micro-oxic cultures and strain CT1061LE, in which fermentative lactate dehydrogenase was deleted, produced up to 6·5 ± 0·3 g l-1 ethanol in 48 h. Escherichia coli CT1061LE derivatives resistant to >25 g l-1 ethanol were obtained by metabolic evolution. Pyruvate and acetaldehyde addition significantly increased both biomass and ethanol concentrations, probably by overcoming acetyl-coenzyme A (CoA) shortage. Yeast extract also promoted growth and ethanol synthesis, and this positive effect was mainly attributable to its vitamin content. Two-stage bioreactor cultures were conducted in a minimal medium containing 100 μg l-1 calcium d-pantothenate to evaluate oxic acetyl-CoA synthesis followed by a switch into fermentative conditions. Ethanol reached 15·4 ± 0·9 g l-1 with a volumetric productivity of 0·34 ± 0·02 g l-1 h-1. Conclusions: Escherichia coli responded to adhE over-expression by funnelling carbon and reducing equivalents into a highly reduced metabolite, ethanol. Acetyl-CoA played a key role in micro-oxic ethanol synthesis and growth. Significance and Impact of the Study: Insight into the micro-oxic metabolism of E. coli growing on glycerol is essential for the development of efficient industrial processes for reduced biochemicals production from this substrate, with special relevance to biofuels synthesis. © 2010 The Society for Applied Microbiology.

Descripción: The epsins are a family of adaptors involved in recruiting other endocytic proteins, binding of ubiquitylated cargo and induction of membrane curvature. These molecules bear a characteristic epsin N-terminal homology (ENTH) domain and multiple peptide motifs that mediate protein-protein interactions. We have previously demonstrated that the ENTH domain of epsin is involved in Cdc42 signaling regulation. Here, we present evidence that yeast epsin 2 (Ent2) plays a signaling role during cell division. We observed that overexpression of the ENTH domain of Ent2 (ENTH2), but not Ent1, promoted the formation of chains of cells and aberrant septa. This dominant-negative effect resulted from ENTH2-mediated interference with septin assembly pathways. We mapped the ENTH2 determinants responsible for induction of the phenotype and found them to be important for efficient binding to the septin regulatory protein, Bem3. Supporting a physiological role for epsin 2 in cell division, the protein localized to sites of polarized growth and cytokinesis and rescued a defect in cell division induced by Bem3 misregulation. Collectively, our findings provide a potential molecular mechanism linking endocytosis (via epsin 2) with signaling pathways regulating cell division.

Descripción: Concentration gradients inside cells are involved in key processes such as cell division and morphogenesis. Here we show that a model of the enzymatic step catalized by phosphofructokinase (PFK), a step which is responsible for the appearance of homogeneous oscillations in the glycolytic pathway, displays Turing patterns with an intrinsic length-scale that is smaller than a typical cell size. All the parameter values are fully consistent with classic experiments on glycolytic oscillations and equal diffusion coefficients are assumed for ATP and ADP. We identify the enzyme concentration and the glycolytic flux as the possible regulators of the pattern. To the best of our knowledge, this is the first closed example of Turing pattern formation in a model of a vital step of the cell metabolism, with a built-in mechanism for changing the diffusion length of the reactants, and with parameter values that are compatible with experiments. Turing patterns inside cells could provide a check-point that combines mechanical and biochemical information to trigger events during the cell division process. © 2007 Strier, Ponce Dawson.

Descripción: The yeast Yarrowia lipolytica accumulates oils and is able to produce extracellular lipases when growing in different carbon sources including glycerol, the principal by-product of the biodiesel industry. In this study, biomass production of a novel mutant strain of Y. lipolytica was statistically optimized by Response Surface Methodology in media containing biodiesel-derived glycerol as main carbon source. This strain exhibited distinctive morphological and fatty acid profile characteristics, and showed an increased extracellular lipase activity. An organic source of nitrogen and the addition of 1.0 g/l olive oil were necessary for significant lipase production. Plackett-Burman and Central Composite Statistical Designs were employed for screening and optimization of fermentation in shaken flasks cultures, and the maximum values obtained were 16.1 g/l for biomass and 12.2 Units/ml for lipase, respectively. Optimized batch bioprocess was thereafter scaled in aerated bioreactors and the values reached for lipase specific activity after 95 % of the glycerol had been consumed, were three-fold higher than those obtained in shaken flasks cultures. A sustainable bioprocess to obtain biomass and extracellular lipase activity was attained by maximizing the use of the by-products of biodiesel industry.

Descripción: Removal of the poly(A) tail is the first step in the degradation of many eukaryotic mRNAs. In metazoans and yeast, the Ccr4/Caf1/Not complex has the predominant deadenylase activity, while the Pan2/Pan3 complex may trim poly(A) tails to the correct size, or initiate deadenylation. In trypanosomes, turnover of several constitutively-expressed or long-lived mRNAs is not affected by depletion of the 5'-3' exoribonuclease XRNA, but is almost completely inhibited by depletion of the deadenylase CAF1. In contrast, two highly unstable mRNAs, encoding EP procyclin and a phosphoglycerate kinase, PGKB, accumulate when XRNA levels are reduced. We here show that degradation of EP mRNA was partially inhibited after CAF1 depletion. RNAi-targeting trypanosome PAN2 had a mild effect on global deadenylation, and on degradation of a few mRNAs including EP. By amplifying and sequencing degradation intermediates, we demonstrated that a reduction in XRNA had no effect on degradation of a stable mRNA encoding a ribosomal protein, but caused accumulation of EP mRNA fragments that had lost substantial portions of the 5' and 3' ends. The results support a model in which trypanosome mRNAs can be degraded by at least two different, partially independent, cytoplasmic degradation pathways attacking both ends of the mRNA. © 2009 The Author(s).

Descripción: As in most insect groups, host plant shifts in cactophilic Drosophila represent environmental challenges as flies must adjust their developmental programme to the presence of different chemical compounds and/or to a microflora that may differ in the diversity and abundance of yeasts and bacteria. In this context, wing morphology provides an excellent opportunity to investigate the factors that may induce changes during development. In this work, we investigated phenotypic plasticity and developmental instability of wing morphology in flies on the cactophilic Drosophila buzzatii and Drosophila koepferae raised on alternative breeding substrates. We detected significant differences in wing size between and within species, and between flies reared on different cactus hosts. However, differences in wing shape between flies emerged from different cactus hosts were not significant either in D. buzzatii or in D. koepferae. Our results also showed that morphological responses involved the entire organ, as variation in size and shape correlated between different portions of the wing. Finally, we studied the effect of the rearing cactus host on developmental instability as measured by the degree of fluctuating asymmetry (FA). Levels of FA in wing size were significantly greater in flies of both species reared in non-preferred when compared with those reared in preferred host cacti. Our results are discussed in the framework of an integrative view aimed at investigating the relevance of host plant shifts in the evolution of the guild of cactophilic Drosophila species that diversified in South America. © 2007 The Authors.

Descripción: Bacterial DNA activates neutrophils through a CpG- and TLR9-independent mechanism. Neutrophil activation does not require DNA internalization, suggesting that it results from the interaction of bacterial DNA with a neutrophil surface receptor. The aim of this study was to characterize the interaction of bacterial DNA with the neutrophil surface. Bacterial DNA binding showed saturation and was inhibited by unlabeled DNA but not by other polyanions like yeast tRNA and poly-A. Resembling the conditions under which bacterial DNA triggers neutrophil activation, binding was not modified in the presence or absence of calcium, magnesium or serum. Treatment of neutrophils with proteases not only dramatically reduced bacterial DNA binding but also inhibited neutrophil activation induced by bacterial DNA. Experiments performed with DNA samples of different lengths obtained after digestion of bacterial DNA with DNase showed that only DNA fragments greater than ≈170-180 nucleotides competed bacterial DNA binding and retained the ability to trigger cell activation. Treatment of neutrophils with chemoattractants or conventional agonists significantly increased bacterial DNA binding. Moreover, neutrophils that underwent transmigration through human endothelial cell monolayers even in the absence of chemoattractants, exhibited higher binding levels of bacterial DNA. Together, our findings provide evidence that binding of bacterial DNA to neutrophils is a receptor-mediated process that conditions the ability of DNA to trigger cell activation. We speculate that neutrophil recognition of bacterial DNA might be modulated by the balance of agonists present at inflammatory foci. This effect might be relevant in bacterial infections with a biofilm etiology, in which extracellular DNA could function as a potent neutrophil agonist. © 2008 USCAP, Inc All rights reserved.

Descripción: The Mediator complex is a greater than 1-megadalton complex, composed of about 30 subunits and found in most eukaryotes, whose main role is to transmit signals from DNA-bound transcription factors to RNA Polymerase II. The proteasome is emerging as an important regulator of transcription during both initiation and elongation. It is increasing the number of cases where the proteolysis of transcriptional activators by the proteasome activates their function. This counterintuitive phenomenon was called "activation by destruction." Here, we show that, in Arabidopsis (Arabidopsis thaliana), PHYTOCHROME AND FLOWERING TIME1 (PFT1), the MEDIATOR25 (MED25) subunit of the plant Mediator complex, is degraded by the proteasome and that proteasome-mediated PFT1 turnover is coupled to its role in stimulating the transcription of FLOWERING LOCUS T, the plant florigen, which is involved in the process of flowering induction. We further identify two novel RING-H2 proteins that target PFT1 for degradation. We show that MED25-BINDING RING-H2 PROTEIN1 (MBR1) and MBR2 bind to PFT1 in yeast (Saccharomyces cerevisiae) and in vitro, and they promote PFT1 degradation in vivo, in a RING-H2- dependent way, typical of E3 ubiquitin ligases. We further show that both MBR1 and MBR2 also promote flowering by PFT1- dependent and -independent mechanisms. Our findings extend the phenomenon of activation by destruction to a Mediator subunit, adding a new mechanism by which Mediator subunits may regulate downstream genes in specific pathways. Furthermore, we show that two novel RING-H2 proteins are involved in the destruction of PFT1, adding new players to this process in plants. © 2012 American Society of Plant Biologists.

Descripción: Cytoplasmic events depending on RNA-binding proteins contribute to the fine-tuning of gene expression. Sterile α motif-containing RNA-binding proteins constitute a novel family of post-transcriptional regulators that recognize a specific RNA sequence motif known as Smaug recognition element (SRE). The Drosophila member of this family, dSmaug, triggers the translational repression and deadenylation of maternal mRNAs by independent mechanisms, and the yeast homologue Vts1 stimulates degradation of SRE-containing messengers. Two homologous genes are present in the mammalian genome. Here we showed that hSmaug 1, encoded in human chromosome 14, represses the translation of reporter transcripts carrying SRE motifs. When expressed in fibroblasts, hSmaug 1 forms cytoplasmic granules that contain polyadenylated mRNA and the RNA-binding proteins Staufen, TIAR, TIA-1, and HuR. Smaug 1 foci are distinct from degradation foci. The murine protein mSmaug 1 is expressed in the central nervous system and is abundant in post-synaptic densities, a subcellular region where translation is tightly regulated by synaptic stimulation. Biochemical analysis indicated that mSmaug 1 is present in synaptoneurosomal 20 S particles. These results suggest a role for mammalian Smaug 1 in RNA granule formation and translation regulation in neurons. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.