Georgette Bonnec

Switch from an Aquaporin to a Glycerol Channel by Two Amino Acids Substitution

The MIP (major intrinsic protein) proteins constitute a channel family of currently 150 members that have been identified in cell membranes of organisms ranging from bacteria to man. Among these proteins, two functionally distinct subgroups are characterized: aquaporins that allow specific water transfer and glycerol channels that are involved in glycerol and small neutral solutes transport. Since the flow of small molecules across cell membranes is vital for every living organism, the study of such proteins is of particular interest. For instance, aquaporins located in kidney cell membranes are responsible for reabsorption of 150 liters of water/day in adult human. To understand the molecular mechanisms of solute transport specificity, we analyzed mutant aquaporins in which highly conserved residues have been substituted by amino acids located at the same positions in glycerol channels. Here, we show that substitution of a tyrosine and a tryptophan by a proline and a leucine, respectively, in the sixth transmembrane helix of an aquaporin leads to a switch in the selectivity of the channel, from water to glycerol.

Localization of the estradiol receptor mRNA in the forebrain of the rainbow trout.

In situ hybridization was used to localize the cells that express the estradiol receptor gene (ER) in the forebrain (hypothalamus, preoptic area, telencephalon) of the rainbow trout (Oncorhynchus mykiss). Both sense and anti-sense [35S]UTP-labeled single-stranded RNA probes were generated from the estradiol binding domain of the ER cDNA. The sense probe was used to evaluate the background of the hybridization reaction. In the forebrain, specific signal appeared in three areas: the posterior hypothalamus, the preoptic area, and the ventral telencephalon. Our localization correlates with [3H]estradiol binding studies in other teleost species. In the pituitary, we observed a weak signal when compared to the signal observed in the forebrain (about ten grains/cell in the pituitary against 35 grains/cell in the posterior hypothalamus). A significant difference was also observed between the intensity of labeling per cell when different forebrain nuclei were compared. We provide here evidence for a tissue-specific regulation of the ER mRNA levels in the trout hypothalamo-pituitary axis.

Differential regulation of the estrogen receptor mRNA by estradiol in the trout hypothalamus and pituitary

In an attempt to understand the molecular mechanisms by which steroids can modulate brain functions in fish, we first localized the cells which produce estrogen receptor mRNA in the rainbow trout forebrain (Salbert et al., 1991). We now report how estradiol itself can alter the estrogen receptor mRNA content of these cells in a sterile strain of female rainbow trout. We also examined liver and pituitary levels of the estrogen receptor mRNA under the same estrogenic treatment. As revealed by slot blot and in situ hybridisations, a single injection (1.5 mg/kg) of estradiol can induce a strong increase (about five-fold) in the estrogen receptor mRNA levels in the liver, as well as a moderate increase (about two-fold) in two nuclei of the hypothalamus/preoptic area: the nucleus lateralis tuberis and the nucleus preopticus periventricularis. Conversely, no modifications of these levels were observed in the pars intermedia and the proximal pars distalis of the pituitary. Moreover, a comparison between estrogen receptor mRNA levels in the brain of sexually active female trout and in the brain of these sterile animals revealed that, in these latter, estrogen receptor mRNA levels are lower but can be increased by a single estradiol injection and reached the levels observed in mature females.

Role of C-terminal Domain and Transmembrane Helices 5 and 6 in Function and Quaternary Structure of Major Intrinsic Proteins ANALYSIS OF AQUAPORIN/GLYCEROL FACILITATOR CHIMERIC PROTEINS

We previously observed that aquaporins and glycerol facilitators exhibit different oligomeric states when studied by sedimentation on density gradients following nondenaturing detergent solubilization. To determine the domains of major intrinsic protein (MIP) family proteins involved in oligomerization, we constructed protein chimeras corresponding to the aquaporin AQPcic substituted in the loop E (including the proximal part of transmembrane domain (TM) 5) and/or the C-terminal part (including the distal part of TM 6) by the equivalent domain of the glycerol channel aquaglyceroporin (GlpF) (chimeras called AGA, AAG, and AGG). The analogous chimeras of GlpF were also constructed (chimeras GAG, GGA, and GAA). cRNA corresponding to all constructs were injected into Xenopus oocytes. AQPcic, GlpF, AAG, AGG, and GAG were targeted to plasma membranes. Water or glycerol membrane permeability measurements demonstrated that only the AAG chimera exhibited a channel function corresponding to water transport. Analysis of all proteins expressed either in oocytes or in yeast by velocity sedimentation on sucrose gradients following solubilization by 2% n-octyl glucoside indicated that only AQPcic and AAG exist in tetrameric forms. GlpF, GAG, and GAA sediment in a monomeric form, whereas GGA and AGG were found mono/dimeric. These data bring new evidence that, within the MIP family, aquaporins and GlpFs behave differently toward nondenaturing detergents. We demonstrate that the C-terminal part of AQPcic, including the distal half of TM 6, can be substituted by the equivalent domain of GlpF (AAG chimera) without modifying the transport specificity. Our results also suggest that interactions of TM 5 of one monomer with TM 1 of the adjacent monomer are crucial for aquaporin tetramer stability.

Patterns of protein synthesis during Xenopus oocyte maturation differ according to the type of stimulation

We examined the qualitative patterns of protein synthesis in fully grown prophase-blocked oocytes of Xenopus laevis and after meiosis reinitiation accompanying maturation of the oocytes. Newly synthesized proteins labelled with [35S]methionine were run on isoelectric focusing gels and further separated in the second dimension on SDS-polyacrylamide slab gels. Three types of maturation inducer were compared: progesterone, considered as the natural inducer of Xenopus oocyte maturation, hCG (human chorionic gonadotropin) and insulin. Three polypeptides with apparent molecular masses of 37 kDa (pI 4.7-4.8), 78 kDa (pI 4.7) and 138 kDa (pI 4.6-4.7) were found to be always synthesized in all three types of stimulation, while the synthesis of a fourth one (molecular mass 116 kDa, pI 4.7) was arrested during oocyte maturation. Moreover, when the follicular cells surrounding the oocytes were part of the stimulating pathway, which is the case during hCG-induced maturation, an additional polypeptide was synthesized by the oocytes (molecular mass 106 kDa, pI 6.0-6.2). This polypeptide was not synthesized during progesterone- or insulin-induced oocyte maturation, two types of stimulation which do not require the presence of the follicular cells. The biological significance of the hCG-induced polypeptide, not necessary for oocyte maturation, is discussed. On the other hand, the four other modifications in protein synthesis taking place during all three types of maturation-inducing stimulation appear to be necessary for oocyte maturation, since oocytes which failed to mature in response to stimulation always missed one or several of these four polypeptides.

ROLE OF RAF AND MOS IN THE ACQUISITION OF COMPETENCE OF XENOPUS OOCYTES TO UNDERGO INSULIN‐INDUCED MEIOTIC MATURATION

‘URA 671 CNRS, Station Zuologique. Observatoire OcCanologique. 06230 VillefiancheMer We reported earlier that phospholipase C (PLC) and phosphomositide kinase (PI kinase) involved in the turnover of polyphosphoinositides (PPI) may control mitosis in sea urchin eggs (Ciapa et al. (1994), Nature 368, 875-878). Besides PLC second messengers, Pi 3-kinase generates 3phosphorylated inositol lipids which have been proposed to affect a wide variety of cellular processes in other cellular systems (Carpenter, C. L.. Cantley, L. C. (1996), Current Opinion in Cell Biology 8, 153158). We have investigated whether PI 3-kinase could play a role in regulating the sea urchin early embryonic development. We have immunoprecipited from egg extracts a protein of YSkDa w~tb an anti-PI 3 kinase antibody. We found that proteins immunoprecipited with anti-phosphotyrosine antibodies displayed a PI kinase activity sensitive to wortmannih, a specific inhibitor of PI 3kmase. These results suggest that Pi 3-kinase is present in sea urchin eggs. We observed that treatment of eggs with wortmannin did not affect fertilization but led to arrest -of the cell cycle after chromosome condensation and nuclear envelope breakdown bave occured. Although protein and DNA synthesis were not affected, Maturation Promoting.Factor (MPF) activation was inhibited. We found that fertilization induced a transient stimulation of a mitogen-activated protein (MAP) kinase activity at the time of mitosis and during the succeeding mitdtic cycles of sea urchin eggs: We observed that wortmannin delayed MAP kinase stimulation and inhibited its inactivation normally observed when the eggsdivide. It is accepted that high levels of MAP kinase activity are responsible for metaphase arrest of oocytes of various species inch&ins Xenonus and mouse (Sag&a, N. (1996),Tr&ds in Cell B&logy-6, 22-25). Th& fits~with our observation that MAP kinase activity increased when eggs entered mitosis and was maintained at a-high level in eggs arrested by-wottmannin. How MAP kinase and MPF interact during mitosisis still unclear. Our data show that, in wortmannin treated egns, MAP kinase can be activated and remained stimulated without a full activation of MPF, suggesting that MAP kinase may be controlled during the cell cycle bv comuonents other than MPF. Bv regulating MAP kit&e activity, PI 3-k&se &ght be an important element in the control of metaphase arrest in oocytes. MAPK, cychne B et p34”“’ during the acquisition of meiotic maturation competence in goat ooqtes. Thierrv DEDIEU, Isabelle HUE, Emilie LEDAN, Laurence GALL, INRA Unite Biologie de la Fecondation 78352 Jouy-en-Josas cMex