Glycosphingolipids are controlled by the spatial organization of their metabolism and by transport specificity. Using immunoelectron microscopy, we localize to the Golgi stack the glycosyltransferases that produce glucosylceramide (GlcCer), lactosylceramide (LacCer), and GM3. GlcCer is synthesized on the cytosolic side and must translocate across to the Golgi lumen for LacCer synthesis. However, only very little natural GlcCer translocates across the Golgi in vitro. As GlcCer reaches the cell surface when Golgi vesicular trafficking is inhibited, it must translocate across a post-Golgi membrane. Concanamycin, a vacuolar proton pump inhibitor, blocks translocation independently of multidrug transporters that are known to translocate short-chain GlcCer. Concanamycin did not reduce LacCer and GM3 synthesis. Thus, GlcCer destined for glycolipid synthesis follows a different pathway and transports back into the endoplasmic reticulum (ER) via the late Golgi protein FAPP2. FAPP2 knockdown strongly reduces GM3 synthesis. Overall, we show that newly synthesized GlcCer enters two pathways: one toward the noncytosolic surface of a post-Golgi membrane and one via the ER toward the Golgi lumen LacCer synthase.

By their metabolic activities, microorganisms have a crucial role in the biogeochemical cycles of elements. The complete understanding of these processes requires, however, the deciphering of both the structure and the function, including synecologic interactions, of microbial communities. Using a metagenomic approach, we demonstrated here that an acid mine drainage highly contaminated with arsenic is dominated by seven bacterial strains whose genomes were reconstructed. Five of them represent yet uncultivated bacteria and include two strains belonging to a novel bacterial phylum present in some similar ecosystems, and which was named 'Candidatus Fodinabacter communificans.' Metaproteomic data unravelled several microbial capabilities expressed in situ, such as iron, sulfur and arsenic oxidation that are key mechanisms in biomineralization, or organic nutrient, amino acid and vitamin metabolism involved in synthrophic associations. A statistical analysis of genomic and proteomic data and reverse transcriptase-PCR experiments allowed us to build an integrated model of the metabolic interactions that may be of prime importance in the natural attenuation of such anthropized ecosystems.

Many mammalian ABC transporters move membrane lipids to acceptor lipid assemblies in the extracellular aqueous milieu. Because the desorption from the membrane costs more energy than provided by two ATPs, the transporter probably only translocates the lipid to a partially hydrophilic site on its extracellular face. From this high-energy site, the lipid may efficiently move to the acceptor, which ideally is bound to the transporter, or, in the absence of an acceptor, fall back into the membrane. If the lipid originated from the cytosolic membrane surface, this represents lipid flop and is probably a side activity of the transporters.

HighlightWe show that the ancestor of cultivated grapevine harbours genetic factors that increase the inducibility of stilbenes correlated with increased resistance to the important pathogen grapevine downy mildew.

Eukaryotic cells control their proteome by regulating protein production and protein clearance. Protein production is determined to a large extent by mRNA levels, whereas protein degradation depends mostly upon the proteasome. Dysfunction of the proteasome leads to the accumulation of non-functional proteins that can aggregate, be toxic for the cell, and, in extreme cases, lead to cell death. mRNA levels are controlled by their rates of synthesis and degradation. Recent evidence indicates that these rates have oppositely co-evolved to ensure appropriate mRNA levels. This opposite co-evolution has been correlated with the mutations in the Ccr4-Not complex. Consistently, the deadenylation enzymes responsible for the rate-limiting step in eukaryotic mRNA degradation, Caf1 and Ccr4, are subunits of the Ccr4-Not complex. Another subunit of this complex is a RING E3 ligase, Not4. It is essential for cellular protein solubility and has been proposed to be involved in co-translational quality control. An open question has been whether this role of Not4 resides strictly in the regulation of the deadenylation module of the Ccr4-Not complex. However, Not4 is important for proper assembly of the proteasome, and the Ccr4-Not complex may have multiple functional modules that participate in protein quality control in different ways. In this work we studied how the functions of the Caf1/Ccr4 and Not4 modules are connected. We concluded that Not4 plays a role in protein quality control independently of the Ccr4 deadenylase, and that it is involved in clearance of aberrant proteins at least in part via the proteasome.

In this study, new strains were isolated from an environment with elevated arsenic levels, Sainte-Marieaux-Mines (France), and the diversity of aoxB genes encoding the arsenite oxidase large subunit was investigated. The distribution of bacterial aoxB genes is wider than what was previously thought. AoxB subfamilies characterized by specific signatures were identified. An exhaustive analysis of AoxB sequences from this study and from public databases shows that horizontal gene transfer has likely played a role in the spreading of aoxB in prokaryotic communities.Arsenic, which is one of the most toxic metalloids, is distributed ubiquitously but not uniformly around the world. Levels of arsenic differ considerably from one geographical region to another, depending on the geochemical characteristics of the soil (natural contamination) and the industrial activities carried out in the vicinity (anthropogenic contamination) (22). In aquatic environments, arsenic occurs mainly in the form of the inorganic species arsenate [As(V)] and arsenite [As(III)]; the latter species, which is more bioavailable, is usually thought to have more-toxic effects on prokaryotes than As(V) (34). As(III) oxidation leads to the formation of the less available form As(V), which can either precipitate with iron [Fe(III)] or be adsorbed by ferrihydrite. The oxidation process may be mediated by microbial activities, which contribute to the natural remediation processes observed in contaminated environments (21,26,27,34). Consequently, bioprocesses for the treatment of arsenic-contaminated waters have been developed based on the precipitation or adsorption of the As(V) produced by bacteria (4, 9, 21). Some well-known prokaryotes oxidize As(III) into As(V) under aerobic (e.g., Herminiimonas arsenicoxydans, Thiomonas spp., or Rhizobium sp. strain NT26) or anaerobic (e.g., Alkalilimnicola ehrlichii) conditions as part of a detoxification process (12,17,31,32,39). Some chemolithotrophs also use arsenite as an electron donor (e.g., Rhizobium sp. strain NT26 or Thiomonas arsenivorans) (5, 32). The aerobic arsenite oxidases involved in such processes are heterodimers consisting of a large subunit with a molybdenum center and a [3Fe-4S] cluster (AroA, AsoA, and AoxB) and a small subunit containing a Rieske-type [2Fe-2S] cluster (AroB, AsoB, and AoxA) (1, 13). The large subunit in these enzymes is similar to that found in other members of the dimethyl sulfoxide (DMSO) reductase family of molybdenum enzymes but is clearly phylogenetically divergent from the respiratory arsenate reductases (ArrA) or other proteins of the DMSO reductase family of molybdenum oxidoreductases, such as the new arsenite reductase described recently for Alkalilimnicola ehrlichii (25,31,40).aox genes have been identified in 25 bacterial and archaeal genera isolated from various arsenic-rich environments, most of which belong to the Alpha-, Beta-, or Gammaproteobacteria phylum (7,10,12,14,23,25,29,32,37). Recent studies based on environmental DNA extracted from soils, sediments, a...

SummaryMonoterpenes are important constituents of the aromas of food and beverages, including wine. Among monoterpenes in wines, wine lactone has the most potent odor. It was proposed to form via acid-catalyzed cyclization of (E)-8-carboxylinalool during wine maturation. It only reaches very low concentrations in wine but its extremely low odor detection threshold makes it an important aroma compound.Using LC-MS/MS, we show here that the (E)-8-carboxylinalool content in wines correlates with their wine lactone content and estimate the kinetic constant for the very slow formation of wine lactone from (E)-8-carboxylinalool. We show that (E)-8-carboxylinalool is accumulated as a glycoside in grape (Vitis vinifera) berries and that one of the cytochrome P450 enzymes most highly expressed in maturing berries, CYP76F14, efficiently oxidizes linalool to (E)-8-carboxylinalool.Our analysis of (E)-8-carboxylinalool in Riesling 9 Gewurztraminer grapevine progeny established that the CYP76F14 gene co-locates with a quantitative trait locus for (E)-8-carboxylinalool content in grape berries.Our data support the role of CYP76F14 as the major (E)-8-carboxylinalool synthase in grape berries and the role of (E)-8-carboxylinalool as a precursor to wine lactone in wine, providing new insights into wine and grape aroma metabolism, and new methods for food and aroma research and production.

Euglena mutabilis is a photosynthetic protist found in acidic aquatic environments such as peat bogs, volcanic lakes and acid mine drainages (AMDs). Through its photosynthetic metabolism, this protist is supposed to have an important role in primary production in such oligotrophic ecosystems. Nevertheless, the exact contribution of E. mutabilis in organic matter synthesis remains unclear and no evidence of metabolite secretion by this protist has been established so far. Here we combined in situ proteo-metabolomic approaches to determine the nature of the metabolites accumulated by this protist or potentially secreted into an AMD. Our results revealed that the secreted metabolites are represented by a large number of amino acids, polyamine compounds, urea and some sugars but no fatty acids, suggesting a selective organic matter contribution in this ecosystem. Such a production may have a crucial impact on the bacterial community present on the study site, as it has been suggested previously that prokaryotes transport and recycle in situ most of the metabolites secreted by E. mutabilis. Consequently, this protist may have an indirect but important role in AMD ecosystems but also in other ecological niches often described as nitrogen-limited.