Browsing by Author "Ripoche, P."
Now showing 1 - 5 of 5
Results Per Page
Sort Options
- Existence of a tightly regulated water channel in saccharomyces cerevisiaePublication . Meyrial, V.; Laizé, Vincent; Gobin, R.; Ripoche, P.; Hohmann, S.; Tacnet, F.The Saccharomyces cerevisiae strain Σ1278b possesses two putative aquaporins, Aqy1-1p and Aqy2-1p. Previous work demonstrated that Aqy1-1p functions as a water channel in Xenopus oocyte. However, no function could be attributed to Aqy2-1p in this system. Specific antibodies were used to follow the expression of Aqy1-1p and Aqy2-1p in the yeast. Aqy1-1p was never detected whatever the growth phase and culture conditions tested. In contrast, Aqy2-1p was detected only during the exponential growth phase in rich medium containing glucose. Aqy2-1p expression was repressed by hyper-osmotic culture conditions. Both immunocytochemistry and biochemical subcellular fractionation demonstrated that Aqy2-1p is located on the endoplasmic reticulum (ER) as well as on the plasma membrane. In microsomal vesicles enriched in ER, a water channel activity due to Aqy2-1p was detected by stopped-flow analysis. Our results show that the expression of aquaporins is tightly controlled. The physiological relevance of aquaporin-mediated water transport in yeast is discussed.
- Functional expression of the human CHIP28 water channel in a yeast secretory mutantPublication . Laizé, Vincent; Rousselet, G.; Verbavatz, J. M.; Berthonaud, V.; Gobin, R.; Roudier, N.; Abrami, L.; Ripoche, P.; Tacnet, F.The temperature-sensitive Saccharomyces cerevisiae mutant strain NY17, deficient in the secretory pathway (sec6-4 mutation), is used for the heterologous expression of the human CHIP28 water channel. After a heat-shock, the protein is present in partially purified post-golgi secretory vesicles, lmmunodetection and water transport studies, directly made on the vesicles, showed that CHIP28 is highly expressed and active in the yeast membranes.
- Molecular and functional study of AQY1 from Saccharomyces cerevisiae: role of the C-terminal domainPublication . Laizé, Vincent; Gobin, R.; Rousselet, G.; Badier, C.; Hohmann, S.; Ripoche, P.; Tacnet, F.The yeast YPR192w gene, which encodes a protein (Aqy1p) with strong homology to aquaporins (AQPs), was cloned from nine S. cerevisiae strains. The osmotic water permeability coefficient (Pf) of X. laevis oocytes expressing the gene cloned from the S1278b strain (AQY1-1) was 5.7 times higher than the Pf of oocytes expressing the gene cloned from other strains (AQY1-2). Aqy1-1p, initially cloned without its C-terminus (Aqy1-1DCp), mediated an ;3 times higher water permeability than the full-length protein. This corresponds to a 3-fold higher protein density in the oocyte plasma membrane, as shown by freeze-fracture electron microscopy. Pf measurements in yeast spheroplasts confirmed the presence of functional water channels in S1278b and a pharmacological study indicated that this strain contains at least a second functional aquaporin.
- Polymorphism of Saccharomyces cerevisiae aquaporinsPublication . Laizé, Vincent; Tacnet, F.; Ripoche, P.; Hohmann, S.Aquaporin water channels facilitate the transmembrane diffusion of water and higher organisms possess a large number of isoforms. The genome of the yeast Saccharomyce cerevisiae contains two highly similar aquaporin genes, AQY1 and AQY2. AQY1 has been shown to encode a functional water channel but only in certain laboratory strains. Here we show that the AQY2 gene is interrupted by an 11 bp deletion in 23 of the 27 laboratory strains tested, with the exception of strains from the S1278b background, which also exhibit a functional Aqy1p. However, although the AQY2 gene from S1278b is highly homologous to functional aquaporins, we did not observe Aqy2p mediated water transport in Xenopus oocytes. A survey of 52 yeast strains revealed that all industrial and wild yeasts carry the allele encoding a functional Aqy1p, while none of these strains appear to have a functional Aqy2p. We conclude that natural and industrial conditions provide selective pressure to maintain AQY1 but apparently not AQY2.
- Purification and functional reconstitution of the human CHIP28 water channel expressed in Saccharomyces cerevisiaePublication . Laizé, Vincent; Ripoche, P.; Tacnet, F.The yeast Saccharomyces cerevisiae was used for these proteins are of primary importance to appreciate heterologous expression of the human CHIP28 water their physiological role in living organisms. Aquaporin-1 channel (Aquaporin-1). A nine-amino- Recently, CHIP28 was expressed functionally in acid epitope of the influenza hemagglutinin protein yeast secretory vesicles in our laboratory (3). This work (HA epitope), recognized by the monoclonal antibody indicated that wild-type or mutant aquaporins could 12CA5, was chosen to tag CHIP28 at its N-terminus. be produced easily in this new heterologous expression Epitope-tagged CHIP28 was purified from yeast ex- system for functional analyses. Such studies are fretracts by immunochromatography on protein A/ quently limited in the other known expression systems, 12CA5-coupled beads, after KI extraction and deter- mainly due to maturation or sorting defects (4). It begent solubilization, then concentrated by anion ex- came possible to produce large amounts of aquaporins change chromatography. Purified protein was recon- from yeast cultures for biochemical and biophysical stituted in proteoliposomes and was shown to function studies. Here, we describe a simple method to purify as a water channel by stopped-flow spectrophotome- try. This study demonstrates that the yeast has the yeast-expressed CHIP28, after addition of an epitope capacity to produce functional aquaporins at levels tag at the protein N-terminus. The purification procesufficient for biochemical and biophysical analyses.