Regulation of the Yeast Hxt6 Hexose Transporter by the Rod1 α-Arrestin, the Snf1 Protein Kinase, and the Bmh2 14-3-3 Protein

[EN] Cell viability requires adaptation to changing environmental conditions. Ubiquitin-mediated endocytosis plays a crucial role in this process, because it provides a mechanism to remove transport proteins from the membrane. Arrestin-related trafficking proteins are important regulators of the endocytic pathway in yeast, facilitating selective ubiquitylation of target proteins by the E3 ubiquitin ligase, Rsp5. Specifically, Rod1 (Art4) has been reported to regulate the endocytosis of both the Hxt1, Hxt3, and Hxt6 glucose transporters and the Jen1 lactate transporter. Also, the AMP kinase homologue, Snf1, and 14-3-3 proteins have been shown to regulate Jen1 via Rod1. Here, we further characterized the role of Rod1, Snf1, and 14-3-3 in the signal transduction route involved in the endocytic regulation of the Hxt6 high affinity glucose transporter by showing that Snf1 interacts specifically with Rod1 and Rog3 (Art7), that the interaction between the Bmh2 and several arrestin-related trafficking proteins may be modulated by carbon source, and that both the 14-3-3 protein Bmh2 and the Snf1 regulatory domain interact with the arrestin-like domain containing the N-terminal half of Rod1 (amino acids 1-395). Finally, using both co-immunoprecipitation and bimolecular fluorescence complementation, we demonstrated the interaction of Rod1 with Hxt6 and showed that the localization of the Rod1-Hxt6 complex at the plasma membrane is affected by carbon source and is reduced upon overexpression of SNF1 and BMH2.Supported by a predoctoral fellowship from the Polytechnic University of Valencia.Llopis Torregrosa, V.; Ferri-Blazquez, A.; Adam-Artigues, A.; Deffontaines, E.; Van Heusden, GPH.; Yenush, L. (2016). Regulation of the Yeast Hxt6 Hexose Transporter by the Rod1 α-Arrestin, the Snf1 Protein Kinase, and the Bmh2 14-3-3 Protein. Journal of Biological Chemistry. 291(29):14973-14985. https://doi.org/10.1074/jbc.M116.733923S149731498529129Mulet, J. M., Llopis-Torregrosa, V., Primo, C., Marqués, M. C., & Yenush, L. (2013). Endocytic regulation of alkali metal transport proteins in mammals, yeast and plants. Current Genetics, 59(4), 207-230. doi:10.1007/s00294-013-0401-2Hein, C., Springael, J.-Y., Volland, C., Haguenauer-Tsapis, R., & Andre, B. (1995). NPI1, an essential yeast gene involved in induced degradation of Gap1 and Fur4 permeases, encodes the Rsp5 ubiquitin-protein ligase. Molecular Microbiology, 18(1), 77-87. doi:10.1111/j.1365-2958.1995.mmi_18010077.xBabst, M., Katzmann, D. J., Estepa-Sabal, E. J., Meerloo, T., & Emr, S. D. (2002). Escrt-III. Developmental Cell, 3(2), 271-282. doi:10.1016/s1534-5807(02)00220-4Babst, M., Katzmann, D. J., Snyder, W. B., Wendland, B., & Emr, S. D. (2002). Endosome-Associated Complex, ESCRT-II, Recruits Transport Machinery for Protein Sorting at the Multivesicular Body. Developmental Cell, 3(2), 283-289. doi:10.1016/s1534-5807(02)00219-8Katzmann, D. J., Babst, M., & Emr, S. D. (2001). Ubiquitin-Dependent Sorting into the Multivesicular Body Pathway Requires the Function of a Conserved Endosomal Protein Sorting Complex, ESCRT-I. Cell, 106(2), 145-155. doi:10.1016/s0092-8674(01)00434-2Lauwers, E., Erpapazoglou, Z., Haguenauer-Tsapis, R., & André, B. (2010). The ubiquitin code of yeast permease trafficking. Trends in Cell Biology, 20(4), 196-204. doi:10.1016/j.tcb.2010.01.004Lin, C. H., MacGurn, J. A., Chu, T., Stefan, C. J., & Emr, S. D. (2008). Arrestin-Related Ubiquitin-Ligase Adaptors Regulate Endocytosis and Protein Turnover at the Cell Surface. Cell, 135(4), 714-725. doi:10.1016/j.cell.2008.09.025Aubry, L., & Klein, G. (2013). True Arrestins and Arrestin-Fold Proteins. The Molecular Biology of Arrestins, 21-56. doi:10.1016/b978-0-12-394440-5.00002-4Hatakeyama, R., Kamiya, M., Takahara, T., & Maeda, T. (2010). Endocytosis of the Aspartic Acid/Glutamic Acid Transporter Dip5 Is Triggered by Substrate-Dependent Recruitment of the Rsp5 Ubiquitin Ligase via the Arrestin-Like Protein Aly2. Molecular and Cellular Biology, 30(24), 5598-5607. doi:10.1128/mcb.00464-10Nikko, E., Sullivan, J. A., & Pelham, H. R. B. (2008). Arrestin-like proteins mediate ubiquitination and endocytosis of the yeast metal transporter Smf1. EMBO reports, 9(12), 1216-1221. doi:10.1038/embor.2008.199Nikko, E., & Pelham, H. R. B. (2009). Arrestin-Mediated Endocytosis of Yeast Plasma Membrane Transporters. Traffic, 10(12), 1856-1867. doi:10.1111/j.1600-0854.2009.00990.xMacGurn, J. A., Hsu, P.-C., Smolka, M. B., & Emr, S. D. (2011). TORC1 Regulates Endocytosis via Npr1-Mediated Phosphoinhibition of a Ubiquitin Ligase Adaptor. Cell, 147(5), 1104-1117. doi:10.1016/j.cell.2011.09.054Merhi, A., & Andre, B. (2012). Internal Amino Acids Promote Gap1 Permease Ubiquitylation via TORC1/Npr1/14-3-3-Dependent Control of the Bul Arrestin-Like Adaptors. Molecular and Cellular Biology, 32(22), 4510-4522. doi:10.1128/mcb.00463-12O’Donnell, A. F., Huang, L., Thorner, J., & Cyert, M. S. (2013). A Calcineurin-dependent Switch Controls the Trafficking Function of α-Arrestin Aly1/Art6. Journal of Biological Chemistry, 288(33), 24063-24080. doi:10.1074/jbc.m113.478511O’Donnell, A. F., McCartney, R. R., Chandrashekarappa, D. G., Zhang, B. B., Thorner, J., & Schmidt, M. C. (2014). 2-Deoxyglucose Impairs Saccharomyces cerevisiae Growth by Stimulating Snf1-Regulated and α-Arrestin-Mediated Trafficking of Hexose Transporters 1 and 3. Molecular and Cellular Biology, 35(6), 939-955. doi:10.1128/mcb.01183-14Becuwe, M., Vieira, N., Lara, D., Gomes-Rezende, J., Soares-Cunha, C., Casal, M., … Léon, S. (2012). A molecular switch on an arrestin-like protein relays glucose signaling to transporter endocytosis. The Journal of Cell Biology, 196(2), 247-259. doi:10.1083/jcb.201109113Alvaro, C. G., Aindow, A., & Thorner, J. (2016). Differential Phosphorylation Provides a Switch to Control How α-Arrestin Rod1 Down-regulates Mating Pheromone Response inSaccharomyces cerevisiae. Genetics, 203(1), 299-317. doi:10.1534/genetics.115.186122Alvaro, C. G., O’Donnell, A. F., Prosser, D. C., Augustine, A. A., Goldman, A., Brodsky, J. L., … Thorner, J. (2014). Specific  -Arrestins Negatively Regulate Saccharomyces cerevisiae Pheromone Response by Down-Modulating the G-Protein-Coupled Receptor Ste2. Molecular and Cellular Biology, 34(14), 2660-2681. doi:10.1128/mcb.00230-14Shinoda, J., & Kikuchi, Y. (2007). Rod1, an arrestin-related protein, is phosphorylated by Snf1-kinase in Saccharomyces cerevisiae. Biochemical and Biophysical Research Communications, 364(2), 258-263. doi:10.1016/j.bbrc.2007.09.134Ichimura, T., Yamamura, H., Sasamoto, K., Tominaga, Y., Taoka, M., Kakiuchi, K., … Isobe, T. (2005). 14-3-3 Proteins Modulate the Expression of Epithelial Na+Channels by Phosphorylation-dependent Interaction with Nedd4-2 Ubiquitin Ligase. Journal of Biological Chemistry, 280(13), 13187-13194. doi:10.1074/jbc.m412884200Bhalla, V., Daidié, D., Li, H., Pao, A. C., LaGrange, L. P., Wang, J., … Pearce, D. (2005). Serum- and Glucocorticoid-Regulated Kinase 1 Regulates Ubiquitin Ligase Neural Precursor Cell-Expressed, Developmentally Down-Regulated Protein 4-2 by Inducing Interaction with 14-3-3. Molecular Endocrinology, 19(12), 3073-3084. doi:10.1210/me.2005-0193Jiang, R., & Carlson, M. (1996). Glucose regulates protein interactions within the yeast SNF1 protein kinase complex. Genes & Development, 10(24), 3105-3115. doi:10.1101/gad.10.24.3105Proszynski, T. J., Klemm, R. W., Gravert, M., Hsu, P. P., Gloor, Y., Wagner, J., … Walch-Solimena, C. (2005). A genome-wide visual screen reveals a role for sphingolipids and ergosterol in cell surface delivery in yeast. Proceedings of the National Academy of Sciences, 102(50), 17981-17986. doi:10.1073/pnas.0509107102MacGurn, J. A., Hsu, P.-C., & Emr, S. D. (2012). Ubiquitin and Membrane Protein Turnover: From Cradle to Grave. Annual Review of Biochemistry, 81(1), 231-259. doi:10.1146/annurev-biochem-060210-093619Becuwe, M. , and Léon, S. (2014) Integrated control of transporter endocytosis and recycling by the arrestin-related protein Rod1 and the ubiquitin ligase Rsp5. Elife 3, 03307Alvarez, C. E. (2008). On the origins of arrestin and rhodopsin. BMC Evolutionary Biology, 8(1), 222. doi:10.1186/1471-2148-8-222Chutkow, W. A., Patwari, P., Yoshioka, J., & Lee, R. T. (2007). Thioredoxin-interacting Protein (Txnip) Is a Critical Regulator of Hepatic Glucose Production. Journal of Biological Chemistry, 283(4), 2397-2406. doi:10.1074/jbc.m708169200Sheth, S. S., Castellani, L. W., Chari, S., Wagg, C., Thipphavong, C. K., Bodnar, J. S., … Lusis, A. J. (2004). Thioredoxin-interacting protein deficiency disrupts the fasting-feeding metabolic transition. Journal of Lipid Research, 46(1), 123-134. doi:10.1194/jlr.m400341-jlr200Bodnar, J. S., Chatterjee, A., Castellani, L. W., Ross, D. A., Ohmen, J., Cavalcoli, J., … Lusis, A. J. (2001). Positional cloning of the combined hyperlipidemia gene Hyplip1. Nature Genetics, 30(1), 110-116. doi:10.1038/ng811Parikh, H., Carlsson, E., Chutkow, W. A., Johansson, L. E., Storgaard, H., Poulsen, P., … Mootha, V. K. (2007). TXNIP Regulates Peripheral Glucose Metabolism in Humans. PLoS Medicine, 4(5), e158. doi:10.1371/journal.pmed.0040158Yoshioka, J., Imahashi, K., Gabel, S. A., Chutkow, W. A., Burds, A. A., Gannon, J., … Lee, R. T. (2007). Targeted Deletion of Thioredoxin-Interacting Protein Regulates Cardiac Dysfunction in Response to Pressure Overload. Circulation Research, 101(12), 1328-1338. doi:10.1161/circresaha.106.160515Andres, A. M., Ratliff, E. P., Sachithanantham, S., & Hui, S. T. (2011). Diminished AMPK signaling response to fasting in thioredoxin-interacting protein knockout mice. FEBS Letters, 585(8), 1223-1230. doi:10.1016/j.febslet.2011.03.042Wu, N., Zheng, B., Shaywitz, A., Dagon, Y., Tower, C., Bellinger, G., … Cantley, L. C. (2013). AMPK-Dependent Degradation of TXNIP upon Energy Stress Leads to Enhanced Glucose Uptake via GLUT1. Molecular Cell, 49(6), 1167-1175. doi:10.1016/j.molcel.2013.01.035Patwari, P., Chutkow, W. A., Cummings, K., Verstraeten, V. L. R. M., Lammerding, J., Schreiter, E. R., & Lee, R. T. (2009). Thioredoxin-independent Regulation of Metabolism by the α-Arrestin Proteins. Journal of Biological Chemistry, 284(37), 24996-25003. doi:10.1074/jbc.m109.018093Paumi, C. M., Menendez, J., Arnoldo, A., Engels, K., Iyer, K. R., Thaminy, S., … Stagljar, I. (2007). Mapping Protein-Protein Interactions for the Yeast ABC Transporter Ycf1p by Integrated Split-Ubiquitin Membrane Yeast Two-Hybrid Analysis. Molecular Cell, 26(1), 15-25. doi:10.1016/j.molcel.2007.03.011Zonneveld, B. J. M. (1986). Cheap and simple yeast media. Journal of Microbiological Methods, 4(5-6), 287-291. doi:10.1016/0167-7012(86)90040-0Mayordomo, I., Regelmann, J., Horak, J., & Sanz, P. (2003). Saccharomyces cerevisiae 14-3-3 proteins Bmh1 and Bmh2 participate in the process of catabolite inactivation of maltose permease. FEBS Letters, 544(1-3), 160-164. doi:10.1016/s0014-5793(03)00498-8Zahrádka, J., van Heusden, G. P. H., & Sychrová, H. (2012). Yeast 14-3-3 proteins participate in the regulation of cell cation homeostasis via interaction with Nha1 alkali-metal-cation/proton antiporter. Biochimica et Biophysica Acta (BBA) - General Subjects, 1820(7), 849-858. doi:10.1016/j.bbagen.2012.03.013Sung, M.-K., & Huh, W.-K. (2007). Bimolecular fluorescence complementation analysis system forin vivo detection of protein–protein interaction inSaccharomyces cerevisiae. Yeast, 24(9), 767-775. doi:10.1002/yea.1504Longtine, M. S., Mckenzie III, A., Demarini, D. J., Shah, N. G., Wach, A., Brachat, A., … Pringle, J. R. (1998). Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast, 14(10), 953-961. doi:10.1002/(sici)1097-0061(199807)14:103.0.co;2-uBurd, C. G., & Emr, S. D. (1998). Phosphatidylinositol(3)-Phosphate Signaling Mediated by Specific Binding to RING FYVE Domains. Molecular Cell, 2(1), 157-162. doi:10.1016/s1097-2765(00)80125-2Yenush, L., Merchan, S., Holmes, J., & Serrano, R. (2005). pH-Responsive, Posttranslational Regulation of the Trk1 Potassium Transporter by the Type 1-Related Ppz1 Phosphatase. Molecular and Cellular Biology, 25(19), 8683-8692. doi:10.1128/mcb.25.19.8683-8692.2005Gaxiola, R., de Larrinoa, I. F., Villalba, J. M., & Serrano, R. (1992). A novel and conserved salt-induced protein is an important determinant of salt tolerance in yeast. The EMBO Journal, 11(9), 3157-3164. doi:10.1002/j.1460-2075.1992.tb05392.

Mitochondrial translocation of oxidized cofilin induces caspase-independent necrotic-like programmed cell death of T cells

Oxidative stress leads to T-cell hyporesponsiveness or death. The actin-binding protein cofilin is oxidized during oxidative stress, which provokes a stiff actin cytoskeleton and T-cell hyporesponsiveness. Here, we show that long-term oxidative stress leads to translocation of cofilin into the mitochondria and necrotic-like programmed cell death (PCD) in human T cells. Notably, cofilin mutants that functionally mimic oxidation by a single mutation at oxidation-sensitive cysteins (Cys-39 or Cys-80) predominately localize within the mitochondria. The expression of these mutants alone ultimately leads to necrotic-like PCD in T cells. Accordingly, cofilin knockdown partially protects T cells from the fatal effects of long-term oxidative stress. Thus, we introduce the oxidation and mitochondrial localization of cofilin as the checkpoint for necrotic-like PCD upon oxidative stress as it occurs, for example, in tumor environments

VvNPR1.1 est l’orthologue d’AtNPR1 et sa surexpression provoque l’activation constitutive des gènes PR et la résistance à Erysiphe necator chez Vitis vinifera

La compréhension des bases moléculaires des mécanismes de résistance de la Vigne aux agresseurs biotiques constitue un prérequis à la recherche de moyens de lutte alternatifs aux pesticides. Chez Arabidopsis, NPR1 (Non expressor of PR genes 1) joue un rôle clé dans la voie de signalisation régulée par l’acide salicylique et responsable de la mise en place de la résistance aux agents pathogènes biotrophes et de la résistance systémique acquise (SAR). Nous avons identifié deux gènes homologues d’AtNPR1 chez la Vigne : VvNPR1.1 et VvNPR1.2. La caractérisation fonctionnelle de ces deux gènes montre que la surexpression de VvNPR1.1 dans le mutant npr1-2 d’Arabidopsis permet, contrairement `a VvNPR1.2, de restaurer l’expression de PR1 après traitement par du SA ou inoculation bactérienne, ainsi que la résistance à Pseudomonas syringae pv. maculicola, un agent pathogène virulent. VvNPR1.1 apparaît donc comme l’orthologue fonctionnel d’AtNPR1, alors que VvNPR1.2 assure vraisemblablement une fonction différente. La surexpression stable de VvNPR1.1 en fusion avec la GFP a également pu être réalisée chez V. vinifera cv. Chardonnay, grâce à une technique de transformation par A. tumefaciens de cals embryogènes de Vigne. Les résultats obtenus sur les plantules transformées montrent une localisation constitutive de VvNPR1-GFP dans le noyau, ainsi qu’une expression élevée des protéines PR en l’absence d’infection. De plus, les vignes surexprimant VvNPR1-GFP montrent clairement une augmentation de la r´esistance vis-à-vis de l’infection par Erysiphe necator, l’agent de l’oïdium. La forte conservation de séquence des gènes VvNPR1 chez les Vitaceae ainsi que l’ensemble de ces résultats souligne l’importance de la voie régulée par le SA et NPR1 pour la résistance aux agents pathogènes biotrophes chez la Vigne

Knowledge and attitudes of primary healthcare patients regarding population-based screening for colorectal cancer

<p>Abstract</p> <p>Background</p> <p>The aim of this study was to assess the extent of knowledge of primary health care (PHC) patients about colorectal cancer (CRC), their attitudes toward population-based screening for this disease and gender differences in these respects.</p> <p>Methods</p> <p>A questionnaire-based survey of PHC patients in the Balearic Islands and some districts of the metropolitan area of Barcelona was conducted. Individuals between 50 and 69 years of age with no history of CRC were interviewed at their PHC centers.</p> <p>Results</p> <p>We analyzed the results of 625 questionnaires, 58% of which were completed by women. Most patients believed that cancer diagnosis before symptom onset improved the chance of survival. More women than men knew the main symptoms of CRC. A total of 88.8% of patients reported that they would perform the fecal occult blood test (FOBT) for CRC screening if so requested by PHC doctors or nurses. If the FOBT was positive and a colonoscopy was offered, 84.9% of participants indicated that they would undergo the procedure, and no significant difference by gender was apparent. Fear of having cancer was the main reason for performance of an FOBT, and also for not performing the FOBT, especially in women. Fear of pain was the main reason for not wishing to undergo colonoscopy. Factors associated with reluctance to perform the FOBT were: <b><it>(i) </it></b>the idea that that many forms of cancer can be prevented by exercise and, <b><it>(ii) </it></b>a reluctance to undergo colonoscopy if an FOBT was positive. Factors associated with reluctance to undergo colonoscopy were: <b><it>(i) </it></b>residence in Barcelona, <b><it>(ii) </it></b>ignorance of the fact that early diagnosis of CRC is associated with better prognosis, <b><it>(iii) </it></b>no previous history of colonoscopy, and <b><it>(iv) </it></b>no intention to perform the FOBT for CRC screening.</p> <p>Conclusion</p> <p>We identified gaps in knowledge about CRC and prevention thereof in PHC patients from the Balearic Islands and the Barcelona region of Spain. If fears about CRC screening, and CRC per se, are addressed, and if it is emphasized that CRC is preventable, participation in CRC screening programs may improve.</p

The design of the wide field monitor for LOFT

LOFT (Large Observatory For x-ray Timing) is one of the ESA M3 missions selected within the Cosmic Vision program in 2011 to carry out an assessment phase study and compete for a launch opportunity in 2022-2024. The phase-A studies of all M3 missions were completed at the end of 2013. LOFT is designed to carry on-board two instruments with sensitivity in the 2-50 keV range: a 10 m 2 class Large Area Detector (LAD) with a <1{\deg} collimated FoV and a wide field monitor (WFM) making use of coded masks and providing an instantaneous coverage of more than 1/3 of the sky. The prime goal of the WFM will be to detect transient sources to be observed by the LAD. However, thanks to its unique combination of a wide field of view (FoV) and energy resolution (better than 500 eV), the WFM will be also an excellent monitoring instrument to study the long term variability of many classes of X-ray sources. The WFM consists of 10 independent and identical coded mask cameras arranged in 5 pairs to provide the desired sky coverage. We provide here an overview of the instrument design, configuration, and capabilities of the LOFT WFM. The compact and modular design of the WFM could easily make the instrument concept adaptable for other missions.Comment: Proc. SPIE 9144, Space Telescopes and Instrumentation 2014: Ultraviolet to Gamma Ray, 91442

Progress from ASDEX Upgrade experiments in preparing the physics basis of ITER operation and DEMO scenario development

An overview of recent results obtained at the tokamak ASDEX Upgrade (AUG) is given. A work flow for predictive profile modelling of AUG discharges was established which is able to reproduce experimental H-mode plasma profiles based on engineering parameters only. In the plasma center, theoretical predictions on plasma current redistribution by a dynamo effect were confirmed experimentally. For core transport, the stabilizing effect of fast ion distributions on turbulent transport is shown to be important to explain the core isotope effect and improves the description of hollow low-Z impurity profiles. The L-H power threshold of hydrogen plasmas is not affected by small helium admixtures and it increases continuously from the deuterium to the hydrogen level when the hydrogen concentration is raised from 0 to 100%. One focus of recent campaigns was the search for a fusion relevant integrated plasma scenario without large edge localised modes (ELMs). Results from six different ELM-free confinement regimes are compared with respect to reactor relevance: ELM suppression by magnetic perturbation coils could be attributed to toroidally asymmetric turbulent fluctuations in the vicinity of the separatrix. Stable improved confinement mode plasma phases with a detached inner divertor were obtained using a feedback control of the plasma β. The enhanced D α H-mode regime was extended to higher heating power by feedback controlled radiative cooling with argon. The quasi-coherent exhaust regime was developed into an integrated scenario at high heating power and energy confinement, with a detached divertor and without large ELMs. Small ELMs close to the separatrix lead to peeling-ballooning stability and quasi continuous power exhaust. Helium beam density fluctuation measurements confirm that transport close to the separatrix is important to achieve the different ELM-free regimes. Based on separatrix plasma parameters and interchange-drift-Alfvén turbulence, an analytic model was derived that reproduces the experimentally found important operational boundaries of the density limit and between L- and H-mode confinement. Feedback control for the X-point radiator (XPR) position was established as an important element for divertor detachment control. Stable and detached ELM-free phases with H-mode confinement quality were obtained when the XPR was moved 10 cm above the X-point. Investigations of the plasma in the future flexible snow-flake divertor of AUG by means of first SOLPS-ITER simulations with drifts activated predict beneficial detachment properties and the activation of an additional strike point by the drifts

The Large Observatory for x-ray timing

The Large Observatory For x-ray Timing (LOFT) was studied within ESA M3 Cosmic Vision framework and participated in the final down-selection for a launch slot in 2022-2024. Thanks to the unprecedented combination of effective area and spectral resolution of its main instrument, LOFT will study the behaviour of matter under extreme conditions, such as the strong gravitational field in the innermost regions of accretion flows close to black holes and neutron stars, and the supra-nuclear densities in the interior of neutron stars. The science payload is based on a Large Area Detector (LAD, 10 m2 effective area, 2-30 keV, 240 eV spectral resolution, 1° collimated field of view) and a WideField Monitor (WFM, 2-50 keV, 4 steradian field of view, 1 arcmin source location accuracy, 300 eV spectral resolution). The WFM is equipped with an on-board system for bright events (e.g. GRB) localization. The trigger time and position of these events are broadcast to the ground within 30 s from discovery. In this paper we present the status of the mission at the end of its Phase A study