The Ubiquitin Ligase Rsp5p is Required for the Modification and Sorting of Biosynthetic Membrane Proteins into Multivesicular Bodies in S. cerevisiae.

Precursor forms of vacuolar proteins with transmembrane domains, such as the carboxypeptidase S Cps1p and the polyphosphatase Phm5p, are selectively sorted in endosomal compartments to vesicles that invaginate, budding into the lumen of the late endosomes, resulting in the formation of multivesicular bodies (MVBs). These proteins are then delivered to the vacuolar lumen following fusion of the MVBs with the vacuole. The sorting of Cps1p and Phm5p to these structures is mediated by ubiquitylation, and in a doa4 mutant, which has reduced level of free ubiquitin, these proteins are missorted to the vacuolar membrane. A RING-finger ubiquitin ligase Tul1p has been shown to participate to the ubiquitylation of Cps1p and Phm5p. We show here that the HECT-ubiquitin ligase Rsp5p is also required for the ubiquitylation of these proteins, and therefore for their sorting to MVBs. Rsp5p is an essential ubiquitin ligase containing an N-terminal C2 domain followed by three WW domains, and a C-terminal catalytic HECT domain. In cells with low levels of Rsp5p (npi1 mutant cells), vacuolar hydrolases do not reach the vacuolar lumen and are instead missorted to the vacuolar membrane. The C2 domain and the second WW domain of Rsp5p are important determinants for sorting to MVBs. Removal of the Bul proteins, two components of the Rsp5p ubiquitin ligase complex, also impairs the sorting of these proteins, but to a lesser extent. Ubiquitylation of Cps1p was strongly reduced in the npi1 mutant strain and ubiquitylation was completely abolished in the npi1 tul1 double mutant. These data demonstrate that Rsp5p plays a novel and key role in intracellular trafficking, and extend the currently very short list of substrates ubiquitylated in vivo by several different ubiquitin ligases acting cooperatively

The vacuolar proton-ATPase plays a major role in several membrane-bounded organelles in Paramecium

The vacuolar proton-ATPase (V-ATPase) is a multisubunit enzyme complex that is able to transfer protons over membranes against an electrochemical potential under ATP hydrolysis. The enzyme consists of two subcomplexes: V0, which is membrane embedded; and V1, which is cytosolic. V0 was also reported to be involved in fusion of vacuoles in yeast. We identified six genes encoding c-subunits (proteolipids) of V0 and two genes encoding F-subunits of V1 and studied the role of the V-ATPase in trafficking in Paramecium. Green fluorescent protein (GFP) fusion proteins allowed a clear subcellular localization of c- and F-subunits in the contractile vacuole complex of the osmoregulatory system and in food vacuoles. Several other organelles were also detected, in particular dense core secretory granules (trichocysts). The functional significance of the V-ATPase in Paramecium was investigated by RNA interference (RNAi), using a recently developed feeding method. A novel strategy was used to block the expression of all six c- or both F-subunits simultaneously. The V-ATPase was found to be crucial for osmoregulation, the phagocytotic pathway and the biogenesis of dense core secretory granules. No evidence was found supporting participation of V0 in membrane fusion

Heterologous expression of a plant uracil transporter in yeast: improvement of plasma membrane targeting in mutants of the Rsp5p ubiquitin protein ligase.

Plasma membrane proteins involved in transport processes play a crucial role in cell physiology. On account of these properties, these molecules are ideal targets for development of new therapeutic and agronomic agents. However, these proteins are of low abundance, which limits their study. Although yeast seems ideal for expressing heterologous transporters, plasma membrane proteins are often retained in intracellular compartments. We tried to find yeast mutants potentially able to improve functional expression of a whole set of heterologous transporters. We focused on Arabidopsis thaliana ureide transporter 1 (AtUPS1), previously cloned by functional complementation in yeast. Tagged versions of AtUPS1 remain mostly trapped in the endoplasmic reticulum and were able to reach slowly the plasma membrane. In contrast, untagged AtUPS1 is rapidly delivered to plasma membrane, where it remains in stable form. Tagged and untagged versions of AtUPS1 were expressed in cells deficient in the ubiquitin ligase Rsp5p, involved in various stages of the intracellular trafficking of membrane-bound proteins. rsp5 mutants displayed further plasma membrane stabilization of untagged AtUPS1, and improved steady state amounts of tagged versions of AtUPS1. rsp5 cells are thus powerful tools to solve the many problems inherent in heterologous expression of membrane proteins in yeast, including ER retention

Trafficking of Siderophore Transporters in Saccharomyces cerevisiae and Intracellular Fate of Ferrioxamine B Conjugates

We have studied the intracellular trafficking of Sit1 [ferrioxamine B (FOB) transporter] and Enb1 (enterobactin transporter) in Saccharomyces cerevisiae using green fluorescent protein (GFP) fusion proteins. Enb1 was constitutively targeted to the plasma membrane. Sit1 was essentially targeted to the vacuolar degradation pathway when synthesized in the absence of substrate. Massive plasma membrane sorting of Sit1 was induced by various siderophore substrates of Sit1, and by coprogen, which is not a substrate of Sit1. Thus, different siderophore transporters use different regulated trafficking processes. We also studied the fate of Sit1-mediated internalized siderophores. Ferrioxamine B was recovered in isolated vacuolar fractions, where it could be detected spectrophotometrically. Ferrioxamine B coupled to an inhibitor of mitochondrial protoporphyrinogen oxidase (acifluorfen) could not reach its target unless the cells were disrupted, confirming the tight compartmentalization of siderophores within cells. Ferrioxamine B coupled to a fluorescent moiety, FOB-nitrobenz-2-oxa-1,3-diazole, used as a Sit1-dependent iron source, accumulated in the vacuolar lumen even in mutants displaying a steady-state accumulation of Sit1 at the plasma membrane or in endosomal compartments. Thus, the fates of siderophore transporters and siderophores diverge early in the trafficking process

Extracellular-matrix-like proteins associated with the secretory compartment are essential to organize functional exocytotic microdomains in Paramecium

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Yeasts: Mini-factories Producing Tailored Lipids For Green Chemistry. When Infrared Light Reveals Cell Metabolism

Yeasts have been used for centuries in biotechnological applications, for food processing industry (wine, beer, cider, bread, cheese, etc.). For the past few decades, they have also been studied with the aim of producing oil to replace fossil resources. Through a genetic modification of baking yeast (S. cerevisiae), i.e. expression of proteins involved in oil storage in seeds, we obtained strains able to produce more lipids. To better understand the mechanisms involved in oil accumulation in these yeasts, we analysed these strains and control strains (with low oil content) by infrared microscopy (Fourier Transformed Infrared, FT-IR). This technique provides an overview of the cellular metabolism thanks to a spectral fingerprint of biological macromolecules (lipids, nucleic acids, sugars, etc.). FT-IR can replace traditional biochemical tests, which use large quantities of biomass and solvents. Large populations can be tested (high throughput approaches). Combined with the high spectral and spatial resolution offered by synchrotron light, FT-IR can be used for an analysis at the single cell scale, for statistical and population heterogeneity analyses. Thanks to the presence of the biology lab in the SOLEIL facility, we were able to develop a protocol allowing the FT-IR analysis of freshly-sampled cells at different growth times. A drop of the cell suspension was dried on a ZnSe ATR hemisphere 4 mm in diameter. The spectra were recorded using the Continuum XL microscope available on the SMIS beamline, with a spatial resolution of 4x4 µm, which is the mean size of a cell. The spectra were then analysed (spectrum correction and statistical analyses) to evaluate spectrum variations between strains. We have shown that an increase in oil content leads to significant metabolic changes, in particular on carbon pools. An inverse correlation between oil content and carbohydrates reserves (glycogen) was revealed. This inverse correlation between storage lipids and storage carbohydrates has been confirmed by biochemical analysis

Chromato'graphique ! Séparons et visualisons les pigments végétaux !

La chromatographie est une technique analytique utilisée dans les laboratoires ou présente dans les séries télévisées scientifiques. L’atelier propose une présentation de végétaux contenant des pigments colorés. Un atelier pratique est prévu pour l’extraction de ces pigments et leur observation en milieu acide ou basique. Une chromatographie des pigments extraits (dépôts et/ou dessins sur papier chromatographie) sera effectuée. Chaque participant pourra laisser libre cours à son imagination et ainsi créer son œuvre chromatographique

DISCOmega 2: Application of DISCO beamline technical developments for membrane fluidity measurements on yeasts with Omega3 contrasted content

We will use DUV DISCO beamline to perform measurement of fluorescence anisotropy using TMA-DPH membrane staining. With fluorescence anisotropy data on yeast strains with contrasted omega 3 content, we will explore the link between omega 3 content and membrane fluidity. This work will provide fundamental data on the physiology of Debaryomyces species which are key yeasts for food and non-food biotechnologies. Membrane fluidity data obtained on DISCO beamline, combine with a set of phylogeny, genomic and biochemical existing results will allow major advances on omega 3 metabolism in Debaryomyces species and will contribute to our largest project on the study of the Saccharomycotina subphylum

SRCD-FTIR coupled study of hydrophobic integral protein fold in lipid bodies

Highly hydrophobic proteins, including integral membrane proteins are of major interest for the scientific community due to their economical and biotechnological importance. Among them, a particular class is associated with the phospholipids monolayer of lipid bodies (LBs). These proteins share structural properties and are also connected to agronomic stakes (oleosins) or health issues (apolipoproteins and PAT proteins). Thanks to an heterologous expression system we developed in yeast, we are able to produce purified lipid bodies harboring these proteins and suitable for structural analysis. We will use this powerful tool to study the fold of hydrophobic integral protein in lipid bodies using SRCD-FTIR coupled approaches. This study will confirm our first results obtained in 2010 on AtS3 oleosin