An RNA trapping mechanism in Alphavirus mRNA promotes ribosome stalling and translation initiation

We are indebted to Luis Menendez′s lab for helping us with acrylamide gels for sequencing and Juanjo Berlanga andMiguel Angel Rodriguez Gabriel for their support and discussions. Institutional support from the Fundacion Ramon Areces is also acknowledged. Completion of this project took approximately 3 years and the estimated cost was 10000 , excluding salaries.During translation initiation, eukaryotic initiation factor 2 (eIF2) delivers the Met-tRNA to the 40S ribosomal subunit to locate the initiation codon (AUGi) of mRNA during the scanning process. Stress-induced eIF2 phosphorylation leads to a general blockade of translation initiation and represents a key antiviral pathway in mammals. However, some viral mRNAs can initiate translation in the presence of phosphorylated eIF2 via stable RNA stem-loop structures (DLP; Downstream LooP) located in their coding sequence (CDS), which promote 43S preinitiation complex stalling on the initiation codon. We show here that during the scanning process, DLPs of Alphavirus mRNA become trapped in ES6S region (680-914 nt) of 18S rRNA that are projected from the solvent side of 40S subunit. This trapping can lock the progress of the 40S subunit on the mRNA in a way that places the upstream initiator AUGi on the P site of 40S subunit, obviating the participation of eIF2. Notably, the DLP structure is released from 18S rRNA upon 60S ribosomal subunit joining, suggesting conformational changes in ES6Ss during the initiation process. These novel findings illustrate how viral mRNA is threaded into the 40S subunit during the scanning process, exploiting the topology of the 40S subunit solvent side to enhance its translation in vertebrate hosts.S

Molecular architecture of the human GINS complex

Chromosomal DNA replication is strictly regulated through a sequence of steps that involve many macromolecular protein complexes. One of these is the GINS complex, which is required for initiation and elongation phases in eukaryotic DNA replication. The GINS complex consists of four paralogous subunits. At the G1/S transition, GINS is recruited to the origins of replication where it assembles with cell-division cycle protein (Cdc)45 and the minichromosome maintenance mutant (MCM)2–7 to form the Cdc45/Mcm2–7/GINS (CMG) complex, the presumed replicative helicase. We isolated the human GINS complex and have shown that it can bind to DNA. By using single-particle electron microscopy and three-dimensional reconstruction, we obtained a medium-resolution volume of the human GINS complex, which shows a horseshoe shape. Analysis of the protein interactions using mass spectrometry and monoclonal antibody mapping shows the subunit organization within the GINS complex. The structure and DNA-binding data suggest how GINS could interact with DNA and also its possible role in the CMG helicase complex

Structural basis for the inactivation of cytosolic DNA sensing by the vaccinia virus

Detection of cytosolic DNA is a central element of the innate immunity system against viral infection. The Ku heterodimer, a component of the NHEJ pathway of DNA repair in the nucleus, functions as DNA sensor that detects dsDNA of viruses that replicate in the cytoplasm. Vaccinia virus expresses two proteins, C4 and C16, that inactivate DNA sensing and enhance virulence. The structural basis for this is unknown. Here we determine the structure of the C16 – Ku complex using cryoEM. Ku binds dsDNA by a preformed ring but C16 sterically blocks this access route, abrogating binding to a dsDNA end and its insertion into DNA-PK, thereby averting signalling into the downstream innate immunity system. C4 replicates these activities using a domain with 54% identity to C16. Our results reveal how vaccinia virus subverts the capacity of Ku to recognize viral DNA

Structural basis for substrate specificity of heteromeric transporters of neutral amino acids

Despite having similar structures, each member of the heteromeric amino acid transporter (HAT) family shows exquisite preference for the exchange of certain amino acids. Substrate specificity determines the physiological function of each HAT and their role in human diseases. However, HAT transport preference for some amino acids over others is not yet fully understood. Using cryo–electron microscopy of apo human LAT2/CD98hc and a multidisciplinary approach, we elucidate key molecular determinants governing neutral amino acid specificity in HATs. A few residues in the substrate-binding pocket determine substrate preference. Here, we describe mutations that interconvert the substrate profiles of LAT2/CD98hc, LAT1/CD98hc, and Asc1/CD98hc. In addition, a region far from the substrate-binding pocket critically influences the conformation of the substrate-binding site and substrate preference. This region accumulates mutations that alter substrate specificity and cause hearing loss and cataracts. Here, we uncover molecular mechanisms governing substrate specificity within the HAT family of neutral amino acid transporters and provide the structural bases for mutations in LAT2/CD98hc that alter substrate specificity and that are associated with several pathologies.his work was funded by “la Caixa” Foundation, Health Research grant 2020 (LCF/PR/HR20/52400017) to MP and OL, by the Spanish Ministry of Science, Innovation and Universities (MCIU/AEI) grants SAF2015-64869-R-FEDER and RTI2018-094211-B-100-FEDER to MP, and SAF2017-82632-P to OL, co-funded by the European Regional Development Fund (ERDF); the support of Catalan Government (grant 2017 SGR 961) to MP, and the support of the National Institute of Health Carlos III to CNIO; grants 31 Y2018/BIO4747 and P2018/NMT4443 from the Autonomous Region of Madrid and co-funded by the European Social Fund and the European Regional Development Fund to OL. CFR is funded by BES-2015-071348 PhD fellowship by the Spanish Ministry of Science, Innovation and Universities (MCIU/AEI). We gratefully acknowledge institutional funding from the Spanish State Research Agency of the Spanish Ministry of Science and Innovation – Programa Estatal de Fomento de la Investigación Científica y Técnica de Excelencia -Centres of Excellence “Severo Ochoa” CEX2019-000891-S and CEX2019-000913-S. IRB Barcelona is a member of the CERCA System of the Catalan Government P.B. is supported by a CIBERER contract.Peer ReviewedPostprint (author's final draft

Structural basis for substrate specificity of heteromeric transporters of neutral amino acids

Despite having similar structures, each member of the heteromeric amino acid transporter (HAT) family shows exquisite preference for the exchange of certain amino acids. Substrate specificity determines the physiological function of each HAT and their role in human diseases. However, HAT transport preference for some amino acids over others is not yet fully understood. Using cryo-electron microscopy of apo human LAT2/CD98hc and a multidisciplinary approach, we elucidate key molecular determinants governing neutral amino acid specificity in HATs. A few residues in the substrate-binding pocket determine substrate preference. Here, we describe mutations that interconvert the substrate profiles of LAT2/CD98hc, LAT1/CD98hc, and Asc1/CD98hc. In addition, a region far from the substrate-binding pocket critically influences the conformation of the substrate-binding site and substrate preference. This region accumulates mutations that alter substrate specificity and cause hearing loss and cataracts. Here, we uncover molecular mechanisms governing substrate specificity within the HAT family of neutral amino acid transporters and provide the structural bases for mutations in LAT2/CD98hc that alter substrate specificity and that are associated with several pathologies

Widespread displacement of DNA- and RNA-binding factors underlies toxicity of arginine-rich cell-penetrating peptides

Due to their capability to transport chemicals or proteins into target cells, cell-penetrating peptides (CPPs) are being developed as therapy delivery tools. However, and despite their interesting properties, arginine-rich CPPs often show toxicity for reasons that remain poorly understood. Using a (PR)n dipeptide repeat that has been linked to amyotrophic lateral sclerosis (ALS) as a model of an arginine-rich CPP, we here show that the presence of (PR)n leads to a generalized displacement of RNA- and DNA-binding proteins from chromatin and mRNA. Accordingly, any reaction involving nucleic acids, such as RNA transcription, translation, splicing and degradation, or DNA replication and repair, is impaired by the presence of the CPPs. Interestingly, the effects of (PR)n are fully mimicked by protamine, a small arginine-rich protein that displaces histones from chromatin during spermatogenesis. We propose that widespread coating of nucleic acids and consequent displacement of RNA- and DNA-binding factors from chromatin and mRNA accounts for the toxicity of arginine-rich CPPs, including those that have been recently associated with the onset of ALS.Fundación Botín, by Banco Santander through its Santander Universities Global Division and by grants from the Spanish Ministry of Science, Innovation and Universities (RTI2018-102204-B-I00, co-financed with European FEDER funds) and the European Research Council (ERC-617840) to OF; DKFZ NCT3.0 Integrative Project in Cancer Research grant (NCT3.0_2015.54 DysregPT) and SFB 1036/TP07 from the Deutsche Forschungsgemeinschaft to G.S

Regulation of RUVBL1-RUVBL2 AAA-ATPases by the nonsense-mediated mRNA decay factor DHX34, as evidenced by Cryo-EM

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that degrades aberrant mRNAs and also regulates the expression of a wide range of physiological transcripts. RUVBL1 and RUVBL2 AAA-ATPases form an hetero-hexameric ring that is part of several macromolecular complexes such as INO80, SWR1, and R2TP. Interestingly, RUVBL1-RUVBL2 ATPase activity is required for NMD activation by an unknown mechanism. Here, we show that DHX34, an RNA helicase regulating NMD initiation, directly interacts with RUVBL1-RUVBL2 in vitro and in cells. Cryo-EM reveals that DHX34 induces extensive changes in the N-termini of every RUVBL2 subunit in the complex, stabilizing a conformation that does not bind nucleotide and thereby down-regulates ATP hydrolysis of the complex. Using ATPase-deficient mutants, we find that DHX34 acts exclusively on the RUVBL2 subunits. We propose a model, where DHX34 acts to couple RUVBL1-RUVBL2 ATPase activity to the assembly of factors required to initiate the NMD response.Spanish Ministry of Science and Innovation SAF2017-82632-P Andres Lopez-Perrote Carlos F Rodriguez Marina Serna Oscar Llorca. Autonomous Government of Madrid Y2018/BIO4747 Ana Gonzalez-Corpas Oscar Llorca. Autonomous Government of Madrid P2018/NMT4443 Ana Gonzalez-Corpas Oscar Llorca MRC Core funding Javier F Caceres Spanish Ministry of Science and Innovation BES-2015-071348 Carlos F Rodriguez The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.S

Molecular architecture of a multifunctional MCM complex

DNA replication is strictly regulated through a sequence of steps that involve many macromolecular protein complexes. One of them is the replicative helicase, which is required for initiation and elongation phases. A MCM helicase found as a prophage in the genome of Bacillus cereus is fused with a primase domain constituting an integrative arrangement of two essential activities for replication. We have isolated this helicase–primase complex (BcMCM) showing that it can bind DNA and displays not only helicase and primase but also DNA polymerase activity. Using single-particle electron microscopy and 3D reconstruction, we obtained structures of BcMCM using ATPγS or ADP in the absence and presence of DNA. The complex depicts the typical hexameric ring shape. The dissection of the unwinding mechanism using site-directed mutagenesis in the Walker A, Walker B, arginine finger and the helicase channels, suggests that the BcMCM complex unwinds DNA following the extrusion model similarly to the E1 helicase from papillomavirus

Calcium-dependent oligomerization of CAR proteins at cell membrane modulates ABA signaling

[EN] Regulation of ion transport in plants is essential for cell function. Abiotic stress unbalances cell ion homeostasis, and plants tend to readjust it, regulating membrane transporters and channels. The plant hormone abscisic acid (ABA) and the second messenger Ca2+ are central in such processes, as they are involved in the regulation of protein kinases and phosphatases that control ion transport activity in response to environmental stimuli. The identification and characterization of the molecular mechanisms underlying the effect of ABA and Ca2+ signaling pathways on membrane function are central and could provide opportunities for crop improvement. The C2-domain ABA-related (CAR) family of small proteins is involved in the Ca2+-dependent recruitment of the pyrabactin resistance 1/PYR1like (PYR/PYL) ABA receptors to the membrane. However, to fully understand CAR function, it is necessary to define a molecular mechanism that integrates Ca2+ sensing, membrane interaction, and the recognition of the PYR/PYL interacting partners. We present structural and biochemical data showing that CARs are peripheral membrane proteins that functionally cluster on the membrane and generate strong positive membrane curvature in a Ca2+-dependent manner. These features represent a mechanism for the generation, stabilization, and/or specific recognition of membrane discontinuities. Such structures may act as signaling platforms involved in the recruitment of PYR/PYL receptors and other signaling components involved in cell responses to stress.A.A. and J.A.M. thank the European Syncrotron Radiation Facility and EMBL for access to the synchrotron radiation source. This work was funded by Ministerio de Economia y Competitividad (MINECO) Grants BFU2014-59796-R (to A.A.), BFU2011-28184-C02 (to M.J.S.-B.), and BIO2014-52537-R (to P.L.R.) and Comunidad de Madrid Grant S2010/BMD-2457 (to A.A and M.M.). M.J.S.-B. is supported by Ramon y Cajal Contract RYC-2008-03449 from MINECO and M.D. by a fellowship from Senacyt-Ifarhu. Access to the High Throughput Crystallization facility at European Molecular Biology Laboratory (EMBL) Grenoble was supported by the European Community's Seventh Framework Programme through the Protein Production Platform project (P-CUBE) Grant 227764.Diaz, M.; Sanchez-Barrena, MJ.; Gonzalez Rubio, JM.; Rodríguez Solovey, LN.; Fernández, D.; Antoni-Alandes, R.; Yunta, C.... (2016). Calcium-dependent oligomerization of CAR proteins at cell membrane modulates ABA signaling. Proceedings of the National Academy of Sciences. 113(3):E396-E405. https://doi.org/10.1073/pnas.1512779113SE396E4051133Serrano, R., & Rodriguez-Navarro, A. (2001). Ion homeostasis during salt stress in plants. Current Opinion in Cell Biology, 13(4), 399-404. doi:10.1016/s0955-0674(00)00227-1Bassil, E., & Blumwald, E. (2014). The ins and outs of intracellular ion homeostasis: NHX-type cation/H + transporters. Current Opinion in Plant Biology, 22, 1-6. doi:10.1016/j.pbi.2014.08.002Batistič, O., & Kudla, J. (2012). Analysis of calcium signaling pathways in plants. Biochimica et Biophysica Acta (BBA) - General Subjects, 1820(8), 1283-1293. doi:10.1016/j.bbagen.2011.10.012Cutler, S. R., Rodriguez, P. L., Finkelstein, R. R., & Abrams, S. R. (2010). Abscisic Acid: Emergence of a Core Signaling Network. Annual Review of Plant Biology, 61(1), 651-679. doi:10.1146/annurev-arplant-042809-112122McAinsh, M. R., Brownlee, C., & Hetherington, A. M. (1990). Abscisic acid-induced elevation of guard cell cytosolic Ca2+ precedes stomatal closure. Nature, 343(6254), 186-188. doi:10.1038/343186a0Maierhofer, T., Diekmann, M., Offenborn, J. N., Lind, C., Bauer, H., Hashimoto, K., … Hedrich, R. (2014). Site- and kinase-specific phosphorylation-mediated activation of SLAC1, a guard cell anion channel stimulated by abscisic acid. Science Signaling, 7(342), ra86-ra86. doi:10.1126/scisignal.2005703Allen, G. J., Kwak, J. M., Chu, S. P., Llopis, J., Tsien, R. Y., Harper, J. F., & Schroeder, J. I. (1999). Cameleon calcium indicator reports cytoplasmic calcium dynamics in Arabidopsis guard cells. The Plant Journal, 19(6), 735-747. doi:10.1046/j.1365-313x.1999.00574.xLee, S. C., Lan, W.-Z., Kim, B.-G., Li, L., Cheong, Y. H., Pandey, G. K., … Luan, S. (2007). A protein phosphorylation/dephosphorylation network regulates a plant potassium channel. Proceedings of the National Academy of Sciences, 104(40), 15959-15964. doi:10.1073/pnas.0707912104Sánchez-Barrena, M., Martínez-Ripoll, M., & Albert, A. (2013). Structural Biology of a Major Signaling Network that Regulates Plant Abiotic Stress: The CBL-CIPK Mediated Pathway. International Journal of Molecular Sciences, 14(3), 5734-5749. doi:10.3390/ijms14035734Quan, R., Lin, H., Mendoza, I., Zhang, Y., Cao, W., Yang, Y., … Guo, Y. (2007). SCABP8/CBL10, a Putative Calcium Sensor, Interacts with the Protein Kinase SOS2 to Protect Arabidopsis Shoots from Salt Stress. The Plant Cell, 19(4), 1415-1431. doi:10.1105/tpc.106.042291Ma, Y., Szostkiewicz, I., Korte, A., Moes, D., Yang, Y., Christmann, A., & Grill, E. (2009). Regulators of PP2C Phosphatase Activity Function as Abscisic Acid Sensors. Science. doi:10.1126/science.1172408Park, S.-Y., Fung, P., Nishimura, N., Jensen, D. R., Fujii, H., Zhao, Y., … Cutler, S. R. (2009). Abscisic Acid Inhibits Type 2C Protein Phosphatases via the PYR/PYL Family of START Proteins. Science. doi:10.1126/science.1173041Santiago, J., Rodrigues, A., Saez, A., Rubio, S., Antoni, R., Dupeux, F., … Rodriguez, P. L. (2009). Modulation of drought resistance by the abscisic acid receptor PYL5 through inhibition of clade A PP2Cs. The Plant Journal, 60(4), 575-588. doi:10.1111/j.1365-313x.2009.03981.xNishimura, N., Sarkeshik, A., Nito, K., Park, S.-Y., Wang, A., Carvalho, P. C., … Schroeder, J. I. (2009). PYR/PYL/RCAR family members are major in-vivo ABI1 protein phosphatase 2C-interacting proteins in Arabidopsis. The Plant Journal, 61(2), 290-299. doi:10.1111/j.1365-313x.2009.04054.xWang, P., Xue, L., Batelli, G., Lee, S., Hou, Y.-J., Van Oosten, M. J., … Zhu, J.-K. (2013). Quantitative phosphoproteomics identifies SnRK2 protein kinase substrates and reveals the effectors of abscisic acid action. Proceedings of the National Academy of Sciences, 110(27), 11205-11210. doi:10.1073/pnas.1308974110Umezawa, T., Sugiyama, N., Takahashi, F., Anderson, J. C., Ishihama, Y., Peck, S. C., & Shinozaki, K. (2013). Genetics and Phosphoproteomics Reveal a Protein Phosphorylation Network in the Abscisic Acid Signaling Pathway in Arabidopsis thaliana. Science Signaling, 6(270), rs8-rs8. doi:10.1126/scisignal.2003509Kollist, H., Nuhkat, M., & Roelfsema, M. R. G. (2014). Closing gaps: linking elements that control stomatal movement. New Phytologist, 203(1), 44-62. doi:10.1111/nph.12832Lind, C., Dreyer, I., López-Sanjurjo, E. J., von Meyer, K., Ishizaki, K., Kohchi, T., … Hedrich, R. (2015). Stomatal Guard Cells Co-opted an Ancient ABA-Dependent Desiccation Survival System to Regulate Stomatal Closure. Current Biology, 25(7), 928-935. doi:10.1016/j.cub.2015.01.067Geiger, D., Scherzer, S., Mumm, P., Stange, A., Marten, I., Bauer, H., … Hedrich, R. (2009). Activity of guard cell anion channel SLAC1 is controlled by drought-stress signaling kinase-phosphatase pair. Proceedings of the National Academy of Sciences, 106(50), 21425-21430. doi:10.1073/pnas.0912021106Imes, D., Mumm, P., Böhm, J., Al-Rasheid, K. A. S., Marten, I., Geiger, D., & Hedrich, R. (2013). Open stomata 1 (OST1) kinase controls R-type anion channel QUAC1 in Arabidopsis guard cells. The Plant Journal, 74(3), 372-382. doi:10.1111/tpj.12133Ishitani, M., Liu, J., Halfter, U., Kim, C.-S., Shi, W., & Zhu, J.-K. (2000). SOS3 Function in Plant Salt Tolerance Requires N-Myristoylation and Calcium Binding. The Plant Cell, 12(9), 1667-1677. doi:10.1105/tpc.12.9.1667Grefen, C., & Blatt, M. R. (2012). Do Calcineurin B-Like Proteins Interact Independently of the Serine Threonine Kinase CIPK23 with the K+ Channel AKT1? Lessons Learned from a Ménage à Trois. Plant Physiology, 159(3), 915-919. doi:10.1104/pp.112.198051Qiu, Q.-S., Guo, Y., Dietrich, M. A., Schumaker, K. S., & Zhu, J.-K. (2002). Regulation of SOS1, a plasma membrane Na+/H+ exchanger in Arabidopsis thaliana, by SOS2 and SOS3. Proceedings of the National Academy of Sciences, 99(12), 8436-8441. doi:10.1073/pnas.122224699Quintero, F. J., Martinez-Atienza, J., Villalta, I., Jiang, X., Kim, W.-Y., Ali, Z., … Pardo, J. M. (2011). Activation of the plasma membrane Na/H antiporter Salt-Overly-Sensitive 1 (SOS1) by phosphorylation of an auto-inhibitory C-terminal domain. Proceedings of the National Academy of Sciences, 108(6), 2611-2616. doi:10.1073/pnas.1018921108Núñez-Ramírez, R., Sánchez-Barrena, M. J., Villalta, I., Vega, J. F., Pardo, J. M., Quintero, F. J., … Albert, A. (2012). Structural Insights on the Plant Salt-Overly-Sensitive 1 (SOS1) Na+/H+ Antiporter. Journal of Molecular Biology, 424(5), 283-294. doi:10.1016/j.jmb.2012.09.015Ohta, M., Guo, Y., Halfter, U., & Zhu, J.-K. (2003). A novel domain in the protein kinase SOS2 mediates interaction with the protein phosphatase 2C ABI2. Proceedings of the National Academy of Sciences, 100(20), 11771-11776. doi:10.1073/pnas.2034853100Xu, J., Li, H.-D., Chen, L.-Q., Wang, Y., Liu, L.-L., He, L., & Wu, W.-H. (2006). A Protein Kinase, Interacting with Two Calcineurin B-like Proteins, Regulates K+ Transporter AKT1 in Arabidopsis. Cell, 125(7), 1347-1360. doi:10.1016/j.cell.2006.06.011Geiger, D., Scherzer, S., Mumm, P., Marten, I., Ache, P., Matschi, S., … Hedrich, R. (2010). Guard cell anion channel SLAC1 is regulated by CDPK protein kinases with distinct Ca2+affinities. Proceedings of the National Academy of Sciences, 107(17), 8023-8028. doi:10.1073/pnas.0912030107Brandt, B., Brodsky, D. E., Xue, S., Negi, J., Iba, K., Kangasjarvi, J., … Schroeder, J. I. (2012). Reconstitution of abscisic acid activation of SLAC1 anion channel by CPK6 and OST1 kinases and branched ABI1 PP2C phosphatase action. Proceedings of the National Academy of Sciences, 109(26), 10593-10598. doi:10.1073/pnas.1116590109Rodriguez, L., Gonzalez-Guzman, M., Diaz, M., Rodrigues, A., Izquierdo-Garcia, A. C., Peirats-Llobet, M., … Rodriguez, P. L. (2014). C2-Domain Abscisic Acid-Related Proteins Mediate the Interaction of PYR/PYL/RCAR Abscisic Acid Receptors with the Plasma Membrane and Regulate Abscisic Acid Sensitivity in Arabidopsis. The Plant Cell, 26(12), 4802-4820. doi:10.1105/tpc.114.129973Martens, S., & McMahon, H. T. (2008). Mechanisms of membrane fusion: disparate players and common principles. Nature Reviews Molecular Cell Biology, 9(7), 543-556. doi:10.1038/nrm2417Martens, S., Kozlov, M. M., & McMahon, H. T. (2007). How Synaptotagmin Promotes Membrane Fusion. Science, 316(5828), 1205-1208. doi:10.1126/science.1142614Jahn, R., Lang, T., & Südhof, T. C. (2003). Membrane Fusion. Cell, 112(4), 519-533. doi:10.1016/s0092-8674(03)00112-0Sutter, J.-U., Sieben, C., Hartel, A., Eisenach, C., Thiel, G., & Blatt, M. R. (2007). Abscisic Acid Triggers the Endocytosis of the Arabidopsis KAT1 K+ Channel and Its Recycling to the Plasma Membrane. Current Biology, 17(16), 1396-1402. doi:10.1016/j.cub.2007.07.020Bueso, E., Rodriguez, L., Lorenzo-Orts, L., Gonzalez-Guzman, M., Sayas, E., Muñoz-Bertomeu, J., … Rodriguez, P. L. (2014). The single-subunit RING-type E3 ubiquitin ligase RSL1 targets PYL4 and PYR1 ABA receptors in plasma membrane to modulate abscisic acid signaling. The Plant Journal, 80(6), 1057-1071. doi:10.1111/tpj.12708Larsen, J. B., Jensen, M. B., Bhatia, V. K., Pedersen, S. L., Bjørnholm, T., Iversen, L., … Stamou, D. (2015). Membrane curvature enables N-Ras lipid anchor sorting to liquid-ordered membrane phases. Nature Chemical Biology, 11(3), 192-194. doi:10.1038/nchembio.1733Demir, F., Horntrich, C., Blachutzik, J. O., Scherzer, S., Reinders, Y., Kierszniowska, S., … Kreuzer, I. (2013). Arabidopsis nanodomain-delimited ABA signaling pathway regulates the anion channel SLAH3. Proceedings of the National Academy of Sciences, 110(20), 8296-8301. doi:10.1073/pnas.1211667110Guerrero-Valero, M., Ferrer-Orta, C., Querol-Audi, J., Marin-Vicente, C., Fita, I., Gomez-Fernandez, J. C., … Corbalan-Garcia, S. (2009). Structural and mechanistic insights into the association of PKC -C2 domain to PtdIns(4,5)P2. Proceedings of the National Academy of Sciences, 106(16), 6603-6607. doi:10.1073/pnas.0813099106Guillen, J., Ferrer-Orta, C., Buxaderas, M., Perez-Sanchez, D., Guerrero-Valero, M., Luengo-Gil, G., … Corbalan-Garcia, S. (2013). Structural insights into the Ca2+ and PI(4,5)P2 binding modes of the C2 domains of rabphilin 3A and synaptotagmin 1. Proceedings of the National Academy of Sciences, 110(51), 20503-20508. doi:10.1073/pnas.1316179110Corbalan-Garcia, S., & Gómez-Fernández, J. C. (2014). Signaling through C2 domains: More than one lipid target. Biochimica et Biophysica Acta (BBA) - Biomembranes, 1838(6), 1536-1547. doi:10.1016/j.bbamem.2014.01.008CHO, W., & STAHELIN, R. (2006). Membrane binding and subcellular targeting of C2 domains. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 1761(8), 838-849. doi:10.1016/j.bbalip.2006.06.014Verdaguer, N., Corbalan-Garcia, S., Ochoa, W. F., Fita, I., & Gómez-Fernández, J. C. (1999). Ca2+ bridges the C2 membrane-binding domain of protein kinase Cα directly to phosphatidylserine. The EMBO Journal, 18(22), 6329-6338. doi:10.1093/emboj/18.22.6329Honigmann, A., van den Bogaart, G., Iraheta, E., Risselada, H. J., Milovanovic, D., Mueller, V., … Jahn, R. (2013). Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment. Nature Structural & Molecular Biology, 20(6), 679-686. doi:10.1038/nsmb.2570Ausili, A., Corbalán-García, S., Gómez-Fernández, J. C., & Marsh, D. (2011). Membrane docking of the C2 domain from protein kinase Cα as seen by polarized ATR-IR. The role of PIP2. Biochimica et Biophysica Acta (BBA) - Biomembranes, 1808(3), 684-695. doi:10.1016/j.bbamem.2010.11.035Hermoso, J. A., Lagartera, L., González, A., Stelter, M., García, P., Martínez-Ripoll, M., … Menéndez, M. (2005). Insights into pneumococcal pathogenesis from the crystal structure of the modular teichoic acid phosphorylcholine esterase Pce. Nature Structural & Molecular Biology, 12(6), 533-538. doi:10.1038/nsmb940Thompson, D., Pepys, M. B., & Wood, S. P. (1999). The physiological structure of human C-reactive protein and its complex with phosphocholine. Structure, 7(2), 169-177. doi:10.1016/s0969-2126(99)80023-9Ochoa, W. F., Garcia-Garcia, J., Fita, I., Corbalan-Garcia, S., Verdaguer, N., & Gomez-Fernandez, J. C. (2001). Structure of the C2 domain from novel protein kinase Cϵ. A membrane binding model for Ca2+-independent C2 domains. Journal of Molecular Biology, 311(4), 837-849. doi:10.1006/jmbi.2001.4910Fuson, K., Rice, A., Mahling, R., Snow, A., Nayak, K., Shanbhogue, P., … Sutton, R. B. (2014). Alternate Splicing of Dysferlin C2A Confers Ca2+-Dependent and Ca2+-Independent Binding for Membrane Repair. Structure, 22(1), 104-115. doi:10.1016/j.str.2013.10.001Radhakrishnan, A., Stein, A., Jahn, R., & Fasshauer, D. (2009). The Ca2+Affinity of Synaptotagmin 1 Is Markedly Increased by a Specific Interaction of Its C2B Domain with Phosphatidylinositol 4,5-Bisphosphate. Journal of Biological Chemistry, 284(38), 25749-25760. doi:10.1074/jbc.m109.042499Schapire, A. L., Voigt, B., Jasik, J., Rosado, A., Lopez-Cobollo, R., Menzel, D., … Botella, M. A. (2008). Arabidopsis Synaptotagmin 1 Is Required for the Maintenance of Plasma Membrane Integrity and Cell Viability. The Plant Cell, 20(12), 3374-3388. doi:10.1105/tpc.108.063859Wang, J., Bello, O., Auclair, S. M., Wang, J., Coleman, J., Pincet, F., … Rothman, J. E. (2014). Calcium sensitive ring-like oligomers formed by synaptotagmin. Proceedings of the National Academy of Sciences, 111(38), 13966-13971. doi:10.1073/pnas.1415849111Jaenicke, R., & Rudolph, R. (1986). [12]Refolding and association of oligomeric proteins. Enzyme Structure Part L, 218-250. doi:10.1016/0076-6879(86)31043-7Goni, G. M., Epifano, C., Boskovic, J., Camacho-Artacho, M., Zhou, J., Bronowska, A., … Lietha, D. (2014). Phosphatidylinositol 4,5-bisphosphate triggers activation of focal adhesion kinase by inducing clustering and conformational changes. Proceedings of the National Academy of Sciences, 111(31), E3177-E3186. doi:10.1073/pnas.1317022111Wilkie, A. O. (1994). The molecular basis of genetic dominance. Journal of Medical Genetics, 31(2), 89-98. doi:10.1136/jmg.31.2.89Saez, A., Apostolova, N., Gonzalez-Guzman, M., Gonzalez-Garcia, M. P., Nicolas, C., Lorenzo, O., & Rodriguez, P. L. (2003). Gain-of-function and loss-of-function phenotypes of the protein phosphatase 2CHAB1reveal its role as a negative regulator of abscisic acid signalling. The Plant Journal, 37(3), 354-369. doi:10.1046/j.1365-313x.2003.01966.xSimons, K., & Gerl, M. J. (2010). Revitalizing membrane rafts: new tools and insights. Nature Reviews Molecular Cell Biology, 11(10), 688-699. doi:10.1038/nrm2977McMahon, H. T., & Boucrot, E. (2015). Membrane curvature at a glance. Journal of Cell Science, 128(6), 1065-1070. doi:10.1242/jcs.114454Tapken, W., & Murphy, A. S. (2015). Membrane nanodomains in plants: capturing form, function, and movement. Journal of Experimental Botany, 66(6), 1573-1586. doi:10.1093/jxb/erv054Lingwood, D., & Simons, K. (2007). Detergent resistance as a tool in membrane research. Nature Protocols, 2(9), 2159-2165. doi:10.1038/nprot.2007.294Schuck, P. (2000). Size-Distribution Analysis of Macromolecules by Sedimentation Velocity Ultracentrifugation and Lamm Equation Modeling. Biophysical Journal, 78(3), 1606-1619. doi:10.1016/s0006-3495(00)76713-0Bensmihen, S., To, A., Lambert, G., Kroj, T., Giraudat, J., & Parcy, F. (2004). Analysis of an activated ABI5 allele using a new selection method for transgenic Arabidopsis seeds. FEBS Letters, 561(1-3), 127-131. doi:10.1016/s0014-5793(04)00148-6Deblaere, R., Bytebier, B., De Greve, H., Deboeck, F., Schell, J., Van Montagu, M., & Leemans, J. (1985). Efficient octopine Ti plasmid-derived vectors forAgrobacterium-mediated gene transfer to plants. Nucleic Acids Research, 13(13), 4777-4788. doi:10.1093/nar/13.13.4777Clough, S. J., & Bent, A. F. (1998). Floral dip: a simplified method forAgrobacterium-mediated transformation ofArabidopsis thaliana. The Plant Journal, 16(6), 735-743. doi:10.1046/j.1365-313x.1998.00343.xHua, J. (2001). Plant growth homeostasis is controlled by the Arabidopsis BON1 and BAP1 genes. Genes & Development, 15(17), 2263-2272. doi:10.1101/gad.918101Kabsch, W. (2010). XDS. Acta Crystallographica Section D Biological Crystallography, 66(2), 125-132. doi:10.1107/s0907444909047337Winn, M. D., Ballard, C. C., Cowtan, K. D., Dodson, E. J., Emsley, P., Evans, P. R., … Wilson, K. S. (2011). Overview of theCCP4 suite and current developments. Acta Crystallographica Section D Biological Crystallography, 67(4), 235-242. doi:10.1107/s0907444910045749Adams, P. D., Afonine, P. V., Bunkóczi, G., Chen, V. B., Davis, I. W., Echols, N., … Zwart, P. H. (2010). PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallographica Section D Biological Crystallography, 66(2), 213-221. doi:10.1107/s0907444909052925Emsley, P., & Cowtan, K. (2004). Coot: model-building tools for molecular graphics. Acta Crystallographica Section D Biological Crystallography, 60(12), 2126-2132. doi:10.1107/s0907444904019158Dimasi, N., Flot, D., Dupeux, F., & Márquez, J. A. (2007). Expression, crystallization and X-ray data collection from microcrystals of the extracellular domain of the human inhibitory receptor expressed on myeloid cells IREM-1. Acta Crystallographica Section F Structural Biology and Crystallization Communications, 63(3), 204-208. doi:10.1107/s1744309107004903Emsley, P., Lohkamp, B., Scott, W. G., & Cowtan, K. (2010). Features and development ofCoot. Acta Crystallographica Section D Biological Crystallography, 66(4), 486-501. doi:10.1107/s0907444910007493DeLano (2002) The PyMOL Molecular Graphics System, 1.5.0.4 (DeLano Scientific, San Carlos, CA