A functional Rim101 complex is required for proper accumulation of the Ena1 Na+-ATPase protein in response to salt stress in Saccharomyces cerevisiae

[EN] The maintenance of ionic homeostasis is essential for cell viability, thus the activity of plasma membrane ion transporters must be tightly controlled. Previous studies in Saccharomyces cerevisiae revealed that the proper trafficking of several nutrient permeases requires the E3 ubiquitin ligase Rsp5 and, in many cases, the presence of specific adaptor proteins needed for Rsp5 substrate recognition. Among these adaptor proteins are nine members of the arrestin-related trafficking adaptor (ART) family. We studied the possible role of the ART family in the regulation of monovalent cation transporters. We show here that the salt sensitivity phenotype of the rim8/art9 mutant is due to severe defects in Ena1 protein accumulation, which is not attributable to transcriptional defects. Many components of the Rim pathway are required for correct Ena1 accumulation, but not for the accumulation of other nutrient permeases. Moreover, we observe that strains lacking components of the endosomal sorting complexes required for transport (ESCRT) pathway previously described to play a role in Rim complex formation present similar defects in Ena1 accumulation. Our results show that, in response to salt stress, a functional Rim complex via specific ESCRT interactions is required for the proper accumulation of the Ena1 protein, but not induction of the ENA1 gene.This work was supported by grants BFU2011-30197-C03-03 from the Spanish Ministry of Science and Innovation (Madrid, Spain) and EUI2009-04147 [Systems Biology of Microorganisms (SysMo2) European Research Area-Network (ERA-NET)]. V. L-T. was supported by a pre-doctoral fellowship from the Polytechnic University of Valencia.Marqués, MC.; Zamarbide-Forés, S.; Pedelini, L.; Llopis Torregrosa, V.; Yenush, L. (2015). A functional Rim101 complex is required for proper accumulation of the Ena1 Na+-ATPase protein in response to salt stress in Saccharomyces cerevisiae. FEMS Yeast Research. 15(4):1-12. https://doi.org/10.1093/femsyr/fov017S112154Abriel, H., Loffing, J., Rebhun, J. F., Pratt, J. H., Schild, L., Horisberger, J.-D., … Staub, O. (1999). Defective regulation of the epithelial Na+ channel by Nedd4 in Liddle’s syndrome. Journal of Clinical Investigation, 103(5), 667-673. doi:10.1172/jci5713Alepuz, P. M., Cunningham, K. W., & Estruch, F. (1997). Glucose repression affects ion homeostasis in yeast through the regulation of the stress‐activated ENA1 gene. Molecular Microbiology, 26(1), 91-98. doi:10.1046/j.1365-2958.1997.5531917.xArino, J., Ramos, J., & Sychrova, H. (2010). Alkali Metal Cation Transport and Homeostasis in Yeasts. Microbiology and Molecular Biology Reviews, 74(1), 95-120. doi:10.1128/mmbr.00042-09Bagnat, M., Chang, A., & Simons, K. (2001). Plasma Membrane Proton ATPase Pma1p Requires Raft Association for Surface Delivery in Yeast. Molecular Biology of the Cell, 12(12), 4129-4138. doi:10.1091/mbc.12.12.4129Boysen, J. H., & Mitchell, A. P. (2006). Control of Bro1-Domain Protein Rim20 Localization by External pH, ESCRT Machinery, and the Saccharomyces cerevisiae Rim101 Pathway. Molecular Biology of the Cell, 17(3), 1344-1353. doi:10.1091/mbc.e05-10-0949Calcagno-Pizarelli, A. M., Hervas-Aguilar, A., Galindo, A., Abenza, J. F., Penalva, M. A., & Arst, H. N. (2011). Rescue of Aspergillus nidulans severely debilitating null mutations in ESCRT-0, I, II and III genes by inactivation of a salt-tolerance pathway allows examination of ESCRT gene roles in pH signalling. Journal of Cell Science, 124(23), 4064-4076. doi:10.1242/jcs.088344Crespo, J. L., Daicho, K., Ushimaru, T., & Hall, M. N. (2001). The GATA Transcription Factors GLN3 and GAT1 Link TOR to Salt Stress inSaccharomyces cerevisiae. Journal of Biological Chemistry, 276(37), 34441-34444. doi:10.1074/jbc.m103601200Galindo, A., Calcagno-Pizarelli, A. M., Arst, H. N., & Penalva, M. A. (2012). An ordered pathway for the assembly of fungal ESCRT-containing ambient pH signalling complexes at the plasma membrane. Journal of Cell Science, 125(7), 1784-1795. doi:10.1242/jcs.098897Garí, E., Piedrafita, L., Aldea, M., & Herrero, E. (1997). A Set of Vectors with a Tetracycline-Regulatable Promoter System for Modulated Gene Expression inSaccharomyces cerevisiae. Yeast, 13(9), 837-848. doi:10.1002/(sici)1097-0061(199707)13:93.0.co;2-tGaxiola, 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.xGiaever, G., Chu, A. M., Ni, L., Connelly, C., Riles, L., Véronneau, S., … André, B. (2002). Functional profiling of the Saccharomyces cerevisiae genome. Nature, 418(6896), 387-391. doi:10.1038/nature00935Hayashi, M., Fukuzawa, T., Sorimachi, H., & Maeda, T. (2005). Constitutive Activation of the pH-Responsive Rim101 Pathway in Yeast Mutants Defective in Late Steps of the MVB/ESCRT Pathway. Molecular and Cellular Biology, 25(21), 9478-9490. doi:10.1128/mcb.25.21.9478-9490.2005Herrador, A., Herranz, S., Lara, D., & Vincent, O. (2009). Recruitment of the ESCRT Machinery to a Putative Seven-Transmembrane-Domain Receptor Is Mediated by an Arrestin-Related Protein. Molecular and Cellular Biology, 30(4), 897-907. doi:10.1128/mcb.00132-09Herranz, S., Rodriguez, J. M., Bussink, H.-J., Sanchez-Ferrero, J. C., Arst, H. N., Penalva, M. A., & Vincent, O. (2005). Arrestin-related proteins mediate pH signaling in fungi. Proceedings of the National Academy of Sciences, 102(34), 12141-12146. doi:10.1073/pnas.0504776102Horák, J. (2003). The role of ubiquitin in down-regulation and intracellular sorting of membrane proteins: insights from yeast. Biochimica et Biophysica Acta (BBA) - Biomembranes, 1614(2), 139-155. doi:10.1016/s0005-2736(03)00195-0Ito, T., Chiba, T., Ozawa, R., Yoshida, M., Hattori, M., & Sakaki, Y. (2001). A comprehensive two-hybrid analysis to explore the yeast protein interactome. Proceedings of the National Academy of Sciences, 98(8), 4569-4574. doi:10.1073/pnas.061034498Lamb, T. M., & Mitchell, A. P. (2003). The Transcription Factor Rim101p Governs Ion Tolerance and Cell Differentiation by Direct Repression of the Regulatory Genes NRG1 and SMP1 in Saccharomyces cerevisiae. Molecular and Cellular Biology, 23(2), 677-686. doi:10.1128/mcb.23.2.677-686.2003Lamb, T. M., Xu, W., Diamond, A., & Mitchell, A. P. (2000). Alkaline Response Genes ofSaccharomyces cerevisiaeand Their Relationship to theRIM101Pathway. Journal of Biological Chemistry, 276(3), 1850-1856. doi:10.1074/jbc.m008381200Lauwers, 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.2010.01.004Léon, S., & Haguenauer-Tsapis, R. (2009). Ubiquitin ligase adaptors: Regulators of ubiquitylation and endocytosis of plasma membrane proteins. Experimental Cell Research, 315(9), 1574-1583. doi:10.1016/j.yexcr.2008.11.014Liu, Y. (2006). Quality control of a mutant plasma membrane ATPase: ubiquitylation prevents cell-surface stability. Journal of Cell Science, 119(2), 360-369. doi:10.1242/jcs.02749Longtine, 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-uMacGurn, 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-060210-093619Maeda, T. (2012). The signaling mechanism of ambient pH sensing and adaptation in yeast and fungi. FEBS Journal, 279(8), 1407-1413. doi:10.1111/j.1742-4658.2012.08548.xMärquez, J., & Serrano, R. (1996). Multiple transduction pathways regulate the sodium-extrusion gene PMR2/ENA1 during salt stress in yeast. FEBS Letters, 382(1-2), 89-92. doi:10.1016/0014-5793(96)00157-3Mulet, J. M., Leube, M. P., Kron, S. J., Rios, G., Fink, G. R., & Serrano, R. (1999). A Novel Mechanism of Ion Homeostasis and Salt Tolerance in Yeast: the Hal4 and Hal5 Protein Kinases Modulate the Trk1-Trk2 Potassium Transporter. Molecular and Cellular Biology, 19(5), 3328-3337. doi:10.1128/mcb.19.5.3328Mulet, 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/s00294-013-0401-2Obara, K., & Kihara, A. (2014). Signaling Events of the Rim101 Pathway Occur at the Plasma Membrane in a Ubiquitination-Dependent Manner. Molecular and Cellular Biology, 34(18), 3525-3534. doi:10.1128/mcb.00408-14Perez-Valle, J., Jenkins, H., Merchan, S., Montiel, V., Ramos, J., Sharma, S., … Yenush, L. (2007). Key Role for Intracellular K+ and Protein Kinases Sat4/Hal4 and Hal5 in the Plasma Membrane Stabilization of Yeast Nutrient Transporters. Molecular and Cellular Biology, 27(16), 5725-5736. doi:10.1128/mcb.01375-06Pérez-Valle, J., Rothe, J., Primo, C., Martínez Pastor, M., Ariño, J., Pascual-Ahuir, A., … Yenush, L. (2010). Hal4 and Hal5 Protein Kinases Are Required for General Control of Carbon and Nitrogen Uptake and Metabolism. Eukaryotic Cell, 9(12), 1881-1890. doi:10.1128/ec.00184-10Platara, M., Ruiz, A., Serrano, R., Palomino, A., Moreno, F., & Ariño, J. (2006). The Transcriptional Response of the Yeast Na+-ATPaseENA1Gene to Alkaline Stress Involves Three Main Signaling Pathways. Journal of Biological Chemistry, 281(48), 36632-36642. doi:10.1074/jbc.m606483200Proft, M., & Serrano, R. (1999). Repressors and Upstream Repressing Sequences of the Stress-RegulatedENA1Gene inSaccharomyces cerevisiae: bZIP Protein Sko1p Confers HOG-Dependent Osmotic Regulation. Molecular and Cellular Biology, 19(1), 537-546. doi:10.1128/mcb.19.1.537Rienzo, A., Pascual-Ahuir, A., & Proft, M. (2012). The use of a real-time luciferase assay to quantify gene expression dynamics in the living yeast cell. Yeast, 29(6), 219-231. doi:10.1002/yea.2905Rotin, D., & Staub, O. (2010). Role of the ubiquitin system in regulating ion transport. Pflügers Archiv - European Journal of Physiology, 461(1), 1-21. doi:10.1007/s00424-010-0893-2Ruiz, A., & Ariño, J. (2007). Function and Regulation of theSaccharomyces cerevisiae ENASodium ATPase System. Eukaryotic Cell, 6(12), 2175-2183. doi:10.1128/ec.00337-07Schild, L., Lu, Y., Gautschi, I., Schneeberger, E., Lifton, R. P., & Rossier, B. C. (1996). Identification of a PY motif in the epithelial Na channel subunits as a target sequence for mutations causing channel activation found in Liddle syndrome. The EMBO Journal, 15(10), 2381-2387. doi:10.1002/j.1460-2075.1996.tb00594.xSerrano, R., Ruiz, A., Bernal, D., Chambers, J. R., & Ariño, J. (2002). The transcriptional response to alkaline pH in Saccharomyces cerevisiae: evidence for calcium-mediated signalling. Molecular Microbiology, 46(5), 1319-1333. doi:10.1046/j.1365-2958.2002.03246.xStaub, O., Dho, S., Henry, P., Correa, J., Ishikawa, T., McGlade, J., & Rotin, D. (1996). WW domains of Nedd4 bind to the proline-rich PY motifs in the epithelial Na+ channel deleted in Liddle’s syndrome. The EMBO Journal, 15(10), 2371-2380. doi:10.1002/j.1460-2075.1996.tb00593.xSubramanian, S., Woolford, C. A., Desai, J. V., Lanni, F., & Mitchell, A. P. (2012). cis - and trans -Acting Localization Determinants of pH Response Regulator Rim13 in Saccharomyces cerevisiae. Eukaryotic Cell, 11(10), 1201-1209. doi:10.1128/ec.00158-12Tréton, B., Blanchin-Roland, S., Lambert, M., Lépingle, A., & Gaillardin, C. (2000). Ambient pH signalling in ascomycetous yeasts involves homologues of the Aspergillus nidulans genes palF and palH. Molecular and General Genetics MGG, 263(3), 505-513. doi:10.1007/s004380051195Vidan, S., & Mitchell, A. P. (1997). Stimulation of yeast meiotic gene expression by the glucose-repressible protein kinase Rim15p. Molecular and Cellular Biology, 17(5), 2688-2697. doi:10.1128/mcb.17.5.2688Wadskog, I., Forsmark, A., Rossi, G., Konopka, C., Öyen, M., Goksör, M., … Adler, L. (2006). The Yeast Tumor Suppressor Homologue Sro7p Is Required for Targeting of the Sodium Pumping ATPase to the Cell Surface. Molecular Biology of the Cell, 17(12), 4988-5003. doi:10.1091/mbc.e05-08-0798Wang, G., Yang, J., & Huibregtse, J. M. (1999). Functional Domains of the Rsp5 Ubiquitin-Protein Ligase. Molecular and Cellular Biology, 19(1), 342-352. doi:10.1128/mcb.19.1.342Xu, W., & Mitchell, A. P. (2001). Yeast PalA/AIP1/Alix Homolog Rim20p Associates with a PEST-Like Region and Is Required for Its Proteolytic Cleavage. Journal of Bacteriology, 183(23), 6917-6923. doi:10.1128/jb.183.23.6917-6923.2001Xu, W., Smith, F. J., Subaran, R., & Mitchell, A. P. (2004). Multivesicular Body-ESCRT Components Function in pH Response Regulation in Saccharomyces cerevisiae and Candida albicans. Molecular Biology of the Cell, 15(12), 5528-5537. doi:10.1091/mbc.e04-08-0666Yang, B., & Kumar, S. (2009). Nedd4 and Nedd4-2: closely related ubiquitin-protein ligases with distinct physiological functions. Cell Death & Differentiation, 17(1), 68-77. doi:10.1038/cdd.2009.84Yoshikawa, K., Tanaka, T., Furusawa, C., Nagahisa, K., Hirasawa, T., & Shimizu, H. (2009). Comprehensive phenotypic analysis for identification of genes affecting growth under ethanol stress inSaccharomyces cerevisiae. FEMS Yeast Research, 9(1), 32-44. doi:10.1111/j.1567-1364.2008.00456.xZahrádka, J., & Sychrová, H. (2012). Plasma-membrane hyperpolarization diminishes the cation efflux via Nha1 antiporter and Ena ATPase under potassium-limiting conditions. FEMS Yeast Research, 12(4), 439-446. doi:10.1111/j.1567-1364.2012.00793.xZhao, J., Lin, W., Ma, X., Lu, Q., Ma, X., Bian, G., & Jiang, L. (2010). The protein kinase Hal5p is the high-copy suppressor of lithium-sensitive mutations of genes involved in the sporulation and meiosis as well as the ergosterol biosynthesis in Saccharomyces cerevisiae. Genomics, 95(5), 290-298. doi:10.1016/j.ygeno.2010.02.01

Loss of the Intellectual Disability and Autism Gene Cc2d1a and Its Homolog Cc2d1b Differentially Affect Spatial Memory, Anxiety, and Hyperactivity

Hundreds of genes are mutated in non-syndromic intellectual disability (ID) and autism spectrum disorder (ASD), with each gene often involved in only a handful of cases. Such heterogeneity can be daunting, but rare recessive loss of function (LOF) mutations can be a good starting point to provide insight into the mechanisms of neurodevelopmental disease. Biallelic LOF mutations in the signaling scaffold CC2D1Acause a rare form of autosomal recessive ID, sometimes associated with ASD and seizures. In parallel, we recently reported that Cc2d1a-deficient mice present with cognitive and social deficits, hyperactivity and anxiety. In Drosophila, loss of the only ortholog of Cc2d1a, lgd, is embryonically lethal, while in vertebrates, Cc2d1a has a homolog Cc2d1b which appears to be compensating, indicating that Cc2d1a and Cc2d1b have a redundant function in humans and mice. Here, we generate an allelic series of Cc2d1a and Cc2d1b LOF to determine the relative role of these genes during behavioral development. We generated Cc2d1b knockout (KO), Cc2d1a/1b double heterozygous and double KO mice, then performed behavioral studies to analyze learning and memory, social interactions, anxiety, and hyperactivity. We found that Cc2d1a and Cc2d1b have partially overlapping roles. Overall, loss of Cc2d1b is less severe than loss of Cc2d1a, only leading to cognitive deficits, while Cc2d1a/1b double heterozygous animals are similar to Cc2d1a-deficient mice. These results will help us better understand the deficits in individuals with CC2D1A mutations, suggesting that recessive CC2D1B mutations and trans-heterozygous CC2D1A and CC2D1B mutations could also contribute to the genetics of ID

Maternal imprinting on cognition markers of wild type and transgenic Alzheimer's disease model mice

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Slow oscillatory activity and levodopa-induced dyskinesias in Parkinson’s disease

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The laminin-keratin link shields the nucleus from mechanical deformation and signalling

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Peptide Models - XXIV: An ab Initio Study on N-formyl-l-prolinamide With Trans Peptide Bond. The Existence or Non-existence of Alpha(l) And Epsilon(l) Conformations

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Cancer-associated mutations of the adenosine A2A receptor have diverse influences on ligand binding and receptor functions

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