Ubiquitin Reference Technique and Its Use in Ubiquitin-Lacking Prokaryotes

In a pulse-chase assay, the in vivo degradation of a protein is measured through a brief labeling of cells with, for example, a radioactive amino acid, followed by cessation of labeling and analysis of cell extracts prepared at different times afterward (“chase”), using immunoprecipitation, electrophoresis and autoradiography of a labeled protein of interest. A conventional pulse-chase assay is fraught with sources of data scatter, as the efficacy of labeling and immunoprecipitation can vary, and sample volumes can vary as well. The ubiquitin reference technique (URT), introduced in 1996, addresses these problems. In eukaryotes, a DNA-encoded linear fusion of ubiquitin to another protein is cleaved by deubiquitylases at the ubiquitin-protein junction. A URT assay uses a fusion in which the ubiquitin moiety is located between a downstream polypeptide (test protein) and an upstream polypeptide (a long-lived reference protein). The cotranslational cleavage of a URT fusion by deubiquitylases after the last residue of ubiquitin produces, at the initially equimolar ratio, a test protein with a desired N-terminal residue and a reference protein containing C-terminal ubiquitin moiety. In addition to being more accurate than pulse-chases without a reference, URT makes it possible to detect and measure the degradation of a test protein during the pulse (before the chase). Because prokaryotes, including Gram-negative bacteria such as, for example, Escherichia coli and Vibrio vulnificus, lack the ubiquitin system, the use of URT in such cells requires ectopic expression of a deubiquitylase. We describe designs and applications of plasmid vectors that coexpress, in bacteria, both a URT-type fusion and Ubp1, a deubiquitylase of the yeast Saccharomyces cerevisiae. This single-plasmid approach extends the accuracy-enhancing URT assay to studies of protein degradation in prokaryotes

Ubiquitylation in plants: signaling hub for the integration of environmental signals

A fundamental question in biology is how organisms integrate the plethora of environmental cues that they perceive to trigger a co-ordinated response. The regulation of protein stability, which is largely mediated by the ubiquitin–proteasome system in eukaryotes, plays a pivotal role in these processes. Due to their sessile lifestyle and the need to respond rapidly to a multitude of environmental factors, plants are thought to be especially dependent on proteolysis to regulate cellular processes. In this review, we present the complexity of the ubiquitin system in plants, and discuss the relevance of the proteolytic and non-proteolytic roles of this system in the regulation and co-ordination of plant responses to environmental signals. We also discuss the role of the ubiquitin system as a key regulator of plant signaling pathways. We focus more specifically on the functions of E3 ligases as regulators of the jasmonic acid (JA), salicylic acid (SA), and ethylene hormone signaling pathways that play important roles to mount a co-ordinated response to multiple environmental stresses. We also provide examples of new players in this field that appear to integrate different cues and signaling pathway

Evidence for Two Distinct Effector-Binding Sites in Threonine Deaminase by Site-Directed Mutagenesis, Kinetic, and Binding Experiments

A three-dimensional structure comparison between the dimeric regulatory serine-binding domain of Escherichia coli D-3-phosphoglycerate dehydrogenase [Schuller, D. J., Grant, G. A., and Banaszak, L. J. (1995) Nat. Struct. Biol. 2, 69-76] and the regulatory domain of E. coli threonine deaminase [Gallagher, D. T., Gilliland, G. L., Xiao, G., Zondlo, J., Fisher, K. E., Chinchilla, D. , and Eisenstein, E. (1998) Structure 6, 465-475] led us to make the hypothesis that threonine deaminase could have two binding sites per monomer. To test this hypothesis about the corresponding plant enzyme, site-directed mutagenesis was carried out on the recombinant Arabidopsis thaliana threonine deaminase. Kinetic and binding experiments demonstrated for the first time that each regulatory domain of the monomers of A. thaliana threonine deaminase possesses two different effector-binding sites constituted in part by Y449 and Y543. Our results demonstrate that Y449 belongs to a high-affinity binding site whose interaction with a first isoleucine induces conformational modifications yielding a conformer displaying a higher activity and with enhanced ability to bind a second isoleucine on a lower-affinity binding site containing Y543. Isoleucine interaction with this latter binding site is responsible for conformational modifications leading to final inhibition of the enzyme. Y449 interacts with both regulators, isoleucine and valine. However, interaction of valine with the high-affinity binding site induces different conformational modifications leading to reversal of isoleucine binding and reversal of inhibition

La reconnaissance rurale, l’exemple du département de la Dordogne

Depuis une vingtaine d’années, les campagnes françaises connaissent de profondes mutations et se voient attribuer de nouvelles fonctions (touristique, économique, de nature, etc.). Des espaces ruraux que l’on croyait définitivement délaissés connaissent progressivement une requalification. Celle-ci se fonde sur un glissement du sens attribué à leurs éléments socio-spatiaux considérés comme constitutifs. Ainsi, leurs faiblesses (enclavement, désertification, etc.) deviennent des atouts et des potentialités (authenticité, douceur de vie, etc.). C’est à partir de l’exemple de la Dordogne, à la fois représentatif des départements ruraux français et marqué par le phénomène original du développement d’une forte implantation britannique, que ce phénomène que nous qualifions de « reconnaissance rurale » est analysé.Rural Recognition: the Example of the Dordogne Since about twenty years, French rural areas experience very deep evolutions, and are given new and important economic, cultural, environmental and symbolic functions. Rural areas that were said to be promised to quick abandon are now strongly retrained. This process is based on a deep change of the social and cultural representations of rural landscape and heritages. Thus, what seemed to be signs of weakness – isolation, depopulation, etc – are turned into advantages – authenticity, quality of life, etc. The example of the Dordogne, representative of French rural departments, but characterized by the importance of British settlement, should help us to set light and explain this process of "rural recognition"

La frontière, le territoire et le lieu. Norme et transgression dans les Pyrénées Occidentales

Depuis qu'elles existent, les frontières nationales ont intéressé les géographes. D'ailleurs, la géographie est la seule discipline scientifique à avoir consacré un de ses axes de recherches à la seule étude des frontières : la géographie des frontières. Cette géographie s'est donc construite et a connu une histoire particulière. Au fil des années de cette construction, les géographes ont constitué un corpus théorique et méthodologique. La géographie des frontières connaît aujourd'hui un rega..

Le vignoble au Québec, géographie d’un rêve sous contrainte

La province du Québec a vu, en moins de dix années, le nombre de ses exploitations viticoles commerciales doubler. Le vignoble québécois peut être caractérisé à la fois de «petit vignoble» par sa superficie et de «vignoble de l’extrême» par les conditions climatiques mais aussi législatives qui le touchent. Malgré ces contraintes difficiles et pesantes que d’aucuns penseraient insurmontables, les vignerons québécois ne manquent ni de ténacité ni de rêve.The Province of Québec witnessed a doubling of its commercial vineyards in less than ten years. The Québec wine industry can be described both as centered on “small vineyards” because of the average size of its operations, and as a “vineyard of the extreme” because of the climatic as well as legal conditions that affect it. Despite these difficult conditions that one would deem insuperable, Quebec wine makers do not lack tenacity or fantasy

Lessons from Comparison of Hypoxia Signaling in Plants and Mammals

Hypoxia is an important stress for organisms, including plants and mammals. In plants, hypoxia can be the consequence of flooding and causes important crop losses worldwide. In mammals, hypoxia stress may be the result of pathological conditions. Understanding the regulation of responses to hypoxia offers insights into novel approaches for crop improvement, particularly for the development of flooding-tolerant crops and for producing better therapeutics for hypoxia-related diseases such as inflammation and cancer. Despite their evolutionary distance, plants and mammals deploy strikingly similar mechanisms to sense and respond to the different aspects of hypoxia-related stress, including low oxygen levels and the resulting energy crisis, nutrient depletion, and oxidative stress. Over the last two decades, the ubiquitin/proteasome system and the ubiquitin-like protein SUMO have been identified as key regulators that act in concert to regulate core aspects of responses to hypoxia in plants and mammals. Here, we review ubiquitin and SUMO-dependent mechanisms underlying the regulation of hypoxia response in plants and mammals. By comparing and contrasting these mechanisms in plants and mammals, this review seeks to pinpoint conceptually similar mechanisms but also highlight future avenues of research at the junction between different fields of research

Le vignoble au Québec, géographie d’un rêve sous contrainte

La province du Québec a vu, en moins de dix années, le nombre de ses exploitations viticoles commerciales doubler. Le vignoble québécois peut être caractérisé à la fois de « petit vignoble » par sa superficie et de « vignoble de l’extrême » par les conditions climatiques mais aussi législatives qui le touchent. Malgré ces contraintes difficiles et pesantes que d’aucuns penseraient insurmontables, les vignerons québécois ne manquent ni de ténacité ni de rêve.The Québec wine industry, geography of a dream against all odds The Province of Québec witnessed a doubling of its commercial vineyards in less than ten years. The Québec wine industry can be described both as centered on “small vineyards” because of the average size of its operations, and as a “vineyard of the extreme” because of the climatic as well as legal conditions that affect it. Despite these difficult conditions that one would deem insuperable, Quebec wine makers do not lack tenacity or fantasy

Le vignoble au Québec, géographie d’un rêve sous contrainte

La province du Québec a vu, en moins de dix années, le nombre de ses exploitations viticoles commerciales doubler. Le vignoble québécois peut être caractérisé à la fois de «petit vignoble» par sa superficie et de «vignoble de l’extrême» par les conditions climatiques mais aussi législatives qui le touchent. Malgré ces contraintes difficiles et pesantes que d’aucuns penseraient insurmontables, les vignerons québécois ne manquent ni de ténacité ni de rêve.The Province of Québec witnessed a doubling of its commercial vineyards in less than ten years. The Québec wine industry can be described both as centered on “small vineyards” because of the average size of its operations, and as a “vineyard of the extreme” because of the climatic as well as legal conditions that affect it. Despite these difficult conditions that one would deem insuperable, Quebec wine makers do not lack tenacity or fantasy

Inter-species variation in the oligomeric states of the higher plant Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase

In darkened leaves the Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) form a regulatory multi-enzyme complex with the small chloroplast protein CP12. GAPDH also forms a high molecular weight regulatory mono-enzyme complex. Given that there are different reports as to the number and subunit composition of these complexes and that enzyme regulatory mechanisms are known to vary between species, it was reasoned that protein-protein interactions may also vary between species. Here, this variation is investigated. This study shows that two different tetramers of GAPDH (an A2B2 heterotetramer and an A4 homotetramer) have the capacity to form part of the PRK/GAPDH/CP12 complex. The role of the PRK/GAPDH/CP12 complex is not simply to regulate the 'non-regulatory' A4 GAPDH tetramer. This study also demonstrates that the abundance and nature of PRK/GAPDH/CP12 interactions are not equal in all species and that whilst NAD enhances complex formation in some species, this is not sufficient for complex formation in others. Furthermore, it is shown that the GAPDH mono-enzyme complex is more abundant as a 2(A2B2) complex, rather than the larger 4(A2B2) complex. This smaller complex is sensitive to cellular metabolites indicating that it is an important regulatory isoform of GAPDH. This comparative study has highlighted considerable heterogeneity in PRK and GAPDH protein interactions between closely related species and the possible underlying physiological basis for this is discussed. © 2011 The Author(s)