Membrane insertion and topology of the TRanslocating chain-Associating Membrane protein (TRAM)

The translocating chain-associating membrane protein (TRAM) is a glycoprotein involved in the translocation of secreted proteins into the endoplasmic reticulum (ER) lumen and in the insertion of integral membrane proteins into the lipid bilayer. As a major step toward elucidating the structure of the functional ER translocation/insertion machinery, we have characterized the membrane integration mechanism and the transmembrane topology of TRAM using two approaches: photocross-linking and truncated C-terminal reporter tag fusions. Our data indicate that TRAM is recognized by the signal recognition particle and translocon components, and suggest a membrane topology with eight transmembrane segments, including several poorly hydrophobic segments. Furthermore, we studied the membrane insertion capacity of these poorly hydrophobic segments into the ER membrane by themselves. Finally, we confirmed the main features of the proposed membrane topology in mammalian cells expressing full-length TRAM

Charge Pair Interactions in Transmembrane Helices and Turn Propensity of the Connecting Sequence Promote Helical Hairpin Insertion

α-Helical hairpins, consisting of a pair of closely spaced transmembrane (TM) helices that are connected by a short interfacial turn, are the simplest structural motifs found in multi-spanning membrane proteins. In naturally occurring hairpins, the presence of polar residues is common and predicted to complicate membrane insertion. We postulate that the pre-packing process offsets any energetic cost of allocating polar and charged residues within the hydrophobic environment of biological membranes. Consistent with this idea, we provide here experimental evidence demonstrating that helical hairpin insertion into biological membranes can be driven by electrostatic interactions between closely separated, poorly hydrophobic sequences. Additionally, we observe that the integral hairpin can be stabilized by a short loop heavily populated by turn-promoting residues. We conclude that the combined effect of TM¿TM electrostatic interactions and tight turns plays an important role in generating the functional architecture of membrane proteins and propose that helical hairpin motifs can be acquired within the context of the Sec61 translocon at the early stages of membrane protein biosynthesis. Taken together, these data further underline the potential complexities involved in accurately predicting TM domains from primary structures

Structure-based statistical analysis of transmembrane helices

Recent advances in determination of the high-resolution structure of membrane proteins now enable analysis of the main features of amino acids in transmembrane (TM) segments in comparison with amino acids in water-soluble helices. In this work, we conducted a large-scale analysis of the prevalent locations of amino acids by using a data set of 170 structures of integral membrane proteins obtained from the MPtopo database and 930 structures of water-soluble helical proteins obtained from the protein data bank. Large hydrophobic amino acids (Leu, Val, Ile, and Phe) plus Gly were clearly prevalent in TM helices whereas polar amino acids (Glu, Lys, Asp, Arg, and Gln) were less frequent in this type of helix. The distribution of amino acids along TM helices was also examined. As expected, hydrophobic and slightly polar amino acids are commonly found in the hydrophobic core of the membrane whereas aromatic (Trp and Tyr), Pro, and the hydrophilic amino acids (Asn, His, and Gln) occur more frequently in the interface regions. Charged amino acids are also statistically prevalent outside the hydrophobic core of the membrane, and whereas acidic amino acids are frequently found at both cytoplasmic and extra-cytoplasmic interfaces, basic amino acids cluster at the cytoplasmic interface. These results strongly support the experimentally demonstrated biased distribution of positively charged amino acids (that is, the so-called the positive-inside rule) with structural data

An Update on the Intracellular and Intercellular Trafficking of Carmoviruses

[EN] Despite harboring the smallest genomes among plant RNA viruses, carmoviruses have emerged as an ideal model system for studying essential steps of the viral cycle including intracellular and intercellular trafficking. Two small movement proteins, formerly known as double gene block proteins (DGBp1 and DGBp2), have been involved in the movement throughout the plant of some members of carmovirus genera. DGBp1 RNA-binding capability was indispensable for cell-to-cell movement indicating that viral genomes must interact with DGBp1 to be transported. Further investigation on Melon necrotic spot virus (MNSV) DGBp1 subcellular localization and dynamics also supported this idea as this protein showed an actin-dependent movement along microfilaments and accumulated at the cellular periphery. Regarding DGBp2, subcellular localization studies showed that MNSV and Pelargonium flower break virus DGBp2s were inserted into the endoplasmic reticulum (ER) membrane but only MNSV DGBp2 trafficked to plasmodesmata (PD) via the Golgi apparatus through a COPII-dependent pathway. DGBp2 function is still unknown but its localization at PD was a requisite for an efficient cell-to-cell movement. It is also known that MNSV infection can induce a dramatic reorganization of mitochondria resulting in anomalous organelles containing viral RNAs. These putative viral factories were frequently found associated with the ER near the PD leading to the possibility that MNSV movement and replication could be spatially linked. Here, we update the current knowledge of the plant endomembrane system involvement in carmovirus intra-and intercellular movement and the tentative model proposed for MNSV transport within plant cells.This work was funded by grant BIO2014-54862-R from the Spanish Direccion General de Investigacion Cientifica y Tecnica (DGICYT) and the Prometeo Program GV2014/010 from the Generalitat Valenciana.Navarro Bohigues, JA.; Pallás Benet, V. (2017). An Update on the Intracellular and Intercellular Trafficking of Carmoviruses. Frontiers in Plant Science. 8:1-7. https://doi.org/10.3389/fpls.2017.01801S178Adams, M. J., Lefkowitz, E. J., King, A. M. Q., Harrach, B., Harrison, R. L., Knowles, N. J., … Davison, A. J. (2016). Ratification vote on taxonomic proposals to the International Committee on Taxonomy of Viruses (2016). Archives of Virology, 161(10), 2921-2949. doi:10.1007/s00705-016-2977-6Blake, J. A., Lee, K. W., Morris, T. J., & Elthon, T. E. (2007). Effects of turnip crinkle virus infection on the structure and function of mitochondria and expression of stress proteins in turnips. Physiologia Plantarum, 129(4), 698-706. doi:10.1111/j.1399-3054.2006.00852.xBlanco-Pérez, M., Pérez-Cañamás, M., Ruiz, L., & Hernández, C. (2016). Efficient Translation of Pelargonium line pattern virus RNAs Relies on a TED-Like 3´-Translational Enhancer that Communicates with the Corresponding 5´-Region through a Long-Distance RNA-RNA Interaction. PLOS ONE, 11(4), e0152593. doi:10.1371/journal.pone.0152593Brandizzi, F., Frangne, N., Marc-Martin, S., Hawes, C., Neuhaus, J.-M., & Paris, N. (2002). The Destination for Single-Pass Membrane Proteins Is Influenced Markedly by the Length of the Hydrophobic Domain. The Plant Cell, 14(5), 1077-1092. doi:10.1105/tpc.000620Carrington, J. C., Heaton, L. A., Zuidema, D., Hillman, B. I., & Morris, T. J. (1989). The genome structure of turnip crinkle virus. Virology, 170(1), 219-226. doi:10.1016/0042-6822(89)90369-3Chandra-Shekara, A. C., Navarre, D., Kachroo, A., Kang, H.-G., Klessig, D., & Kachroo, P. (2004). Signaling requirements and role of salicylic acid in HRT- and rrt-mediated resistance to turnip crinkle virus in Arabidopsis. The Plant Journal, 40(5), 647-659. doi:10.1111/j.1365-313x.2004.02241.xCohen, Y., Gisel, A., & Zambryski, P. C. (2000). Cell-to-Cell and Systemic Movement of Recombinant Green Fluorescent Protein-Tagged Turnip Crinkle Viruses. Virology, 273(2), 258-266. doi:10.1006/viro.2000.0441Cohen, Y., Qu, F., Gisel, A., Morris, T. J., & Zambryski, P. C. (2000). Nuclear Localization of Turnip Crinkle Virus Movement Protein p8. Virology, 273(2), 276-285. doi:10.1006/viro.2000.0440Gao, F., Kasprzak, W., Stupina, V. A., Shapiro, B. A., & Simon, A. E. (2012). A Ribosome-Binding, 3′ Translational Enhancer Has a T-Shaped Structure and Engages in a Long-Distance RNA-RNA Interaction. Journal of Virology, 86(18), 9828-9842. doi:10.1128/jvi.00677-12García-Castillo, S., Sánchez-Pina, M. A., & Pallás, V. (2003). Spatio-temporal analysis of the RNAs, coat and movement (p7) proteins of Carnation mottle virus in Chenopodium quinoa plants. Journal of General Virology, 84(3), 745-749. doi:10.1099/vir.0.18715-0Genovés, A., Navarro, J. A., & Pallás, V. (2006). Functional analysis of the five melon necrotic spot virus genome-encoded proteins. Journal of General Virology, 87(8), 2371-2380. doi:10.1099/vir.0.81793-0Genovés, A., Navarro, J. A., & Pallás, V. (2009). A self-interacting carmovirus movement protein plays a role in binding of viral RNA during the cell-to-cell movement and shows an actin cytoskeleton dependent location in cell periphery. Virology, 395(1), 133-142. doi:10.1016/j.virol.2009.08.042Genoves, A., Pallas, V., & Navarro, J. A. (2011). Contribution of Topology Determinants of a Viral Movement Protein to Its Membrane Association, Intracellular Traffic, and Viral Cell-to-Cell Movement. Journal of Virology, 85(15), 7797-7809. doi:10.1128/jvi.02465-10Gómez-Aix, C., García-García, M., Aranda, M. A., & Sánchez-Pina, M. A. (2015). Melon necrotic spot virus Replication Occurs in Association with Altered Mitochondria. Molecular Plant-Microbe Interactions®, 28(4), 387-397. doi:10.1094/mpmi-09-14-0274-rGrangeon, R., Jiang, J., & Laliberté, J.-F. (2012). Host endomembrane recruitment for plant RNA virus replication. Current Opinion in Virology, 2(6), 683-690. doi:10.1016/j.coviro.2012.10.003Grangeon, R., Jiang, J., Wan, J., Agbeci, M., Zheng, H., & Laliberté, J.-F. (2013). 6K2-induced vesicles can move cell to cell during turnip mosaic virus infection. Frontiers in Microbiology, 4. doi:10.3389/fmicb.2013.00351Guilley, H., Carrington, J. C., Balàzs, E., Jonard, G., Richards, K., & Morris, T. J. (1985). Nucleotide sequence and genome organization of carnation mottle virus RNA. Nucleic Acids Research, 13(18), 6663-6677. doi:10.1093/nar/13.18.6663Hacker, D. L., Petty, I. T. D., Wei, N., & Morris, T. J. (1992). Turnip crinkle virus genes required for RNA replication and virus movement. Virology, 186(1), 1-8. doi:10.1016/0042-6822(92)90055-tHerrera-Vásquez, J. A., Córdoba-Sellés, M. C., Cebrián, M. C., Alfaro-Fernández, A., & Jordá, C. (2009). Seed transmission ofMelon necrotic spot virusand efficacy of seed-disinfection treatments. Plant Pathology, 58(3), 436-442. doi:10.1111/j.1365-3059.2008.01985.xJiang, J., & Laliberté, J.-F. (2016). Membrane Association for Plant Virus Replication and Movement. Current Research Topics in Plant Virology, 67-85. doi:10.1007/978-3-319-32919-2_3Kaido, M., Tsuno, Y., Mise, K., & Okuno, T. (2009). Endoplasmic reticulum targeting of the Red clover necrotic mosaic virus movement protein is associated with the replication of viral RNA1 but not that of RNA2. Virology, 395(2), 232-242. doi:10.1016/j.virol.2009.09.022Kawakami, S., Watanabe, Y., & Beachy, R. N. (2004). Tobacco mosaic virus infection spreads cell to cell as intact replication complexes. Proceedings of the National Academy of Sciences, 101(16), 6291-6296. doi:10.1073/pnas.0401221101Krczal, G. (1995). Transmission of Pelargonium Flower Break Virus (PFBV) in Irrigation Systems and by Thrips. Plant Disease, 79(2), 163. doi:10.1094/pd-79-0163Lerch-Bader, M., Lundin, C., Kim, H., Nilsson, I., & von Heijne, G. (2008). Contribution of positively charged flanking residues to the insertion of transmembrane helices into the endoplasmic reticulum. Proceedings of the National Academy of Sciences, 105(11), 4127-4132. doi:10.1073/pnas.0711580105Lesemann, D.-E., & Adam, G. (1994). ELECTRON MICROSCOPICAL AND SEROLOGICAL STUDIES ON FOUR ISOMETRICAL PELARGONIUM VIRUSES. Acta Horticulturae, (377), 41-54. doi:10.17660/actahortic.1994.377.3Li, W., Qu, F., & Morris, T. J. (1998). Cell-to-Cell Movement of Turnip Crinkle Virus Is Controlled by Two Small Open Reading Frames That Functionin trans. Virology, 244(2), 405-416. doi:10.1006/viro.1998.9125Liu, C., & Nelson, R. S. (2013). The cell biology of Tobacco mosaic virus replication and movement. Frontiers in Plant Science, 4. doi:10.3389/fpls.2013.00012Marcos, J. F., Vilar, M., Pérez-Payá, E., & Pallás, V. (1999). In VivoDetection, RNA-Binding Properties and Characterization of the RNA-Binding Domain of the p7 Putative Movement Protein from Carnation Mottle Carmovirus (CarMV). Virology, 255(2), 354-365. doi:10.1006/viro.1998.9596Martínez-Gil, L., Johnson, A. E., & Mingarro, I. (2010). Membrane Insertion and Biogenesis of the Turnip Crinkle Virus p9 Movement Protein. Journal of Virology, 84(11), 5520-5527. doi:10.1128/jvi.00125-10Martínez-Gil, L., Saurí, A., Vilar, M., Pallás, V., & Mingarro, I. (2007). Membrane insertion and topology of the p7B movement protein of Melon Necrotic Spot Virus (MNSV). Virology, 367(2), 348-357. doi:10.1016/j.virol.2007.06.006Martínez-Turiño, S., & Hernández, C. (2009). Inhibition of RNA silencing by the coat protein of Pelargonium flower break virus: distinctions from closely related suppressors. Journal of General Virology, 90(2), 519-525. doi:10.1099/vir.0.006098-0Martínez-Turiño, S., & Hernández, C. (2011). A membrane-associated movement protein of Pelargonium flower break virus shows RNA-binding activity and contains a biologically relevant leucine zipper-like motif. Virology, 413(2), 310-319. doi:10.1016/j.virol.2011.03.001Martínez-Turiño, S., & Hernández, C. (2012). Analysis of the subcellular targeting of the smaller replicase protein of Pelargonium flower break virus. Virus Research, 163(2), 580-591. doi:10.1016/j.virusres.2011.12.011Mello, A. F. S., Clark, A. J., & Perry, K. L. (2009). Capsid protein of cowpea chlorotic mottle virus is a determinant for vector transmission by a beetle. Journal of General Virology, 91(2), 545-551. doi:10.1099/vir.0.016402-0Miras, M., Sempere, R. N., Kraft, J. J., Miller, W. A., Aranda, M. A., & Truniger, V. (2013). Interfamilial recombination between viruses led to acquisition of a novel translation-enhancing RNA element that allows resistance breaking. New Phytologist, 202(1), 233-246. doi:10.1111/nph.12650Mochizuki, T., Hirai, K., Kanda, A., Ohnishi, J., Ohki, T., & Tsuda, S. (2009). Induction of necrosis via mitochondrial targeting of Melon necrotic spot virus replication protein p29 by its second transmembrane domain. Virology, 390(2), 239-249. doi:10.1016/j.virol.2009.05.012Morozov, S. Y., & Solovyev, A. G. (2003). Triple gene block: modular design of a multifunctional machine for plant virus movement. Journal of General Virology, 84(6), 1351-1366. doi:10.1099/vir.0.18922-0Mueller, S. J., & Reski, R. (2015). Mitochondrial Dynamics and the ER: The Plant Perspective. Frontiers in Cell and Developmental Biology, 3. doi:10.3389/fcell.2015.00078Navarro, J. A., Genovés, A., Climent, J., Saurí, A., Martínez-Gil, L., Mingarro, I., & Pallás, V. (2006). RNA-binding properties and membrane insertion of Melon necrotic spot virus (MNSV) double gene block movement proteins. Virology, 356(1-2), 57-67. doi:10.1016/j.virol.2006.07.040Nieto, C., Morales, M., Orjeda, G., Clepet, C., Monfort, A., Sturbois, B., … Bendahmane, A. (2006). AneIF4Eallele confers resistance to an uncapped and non-polyadenylated RNA virus in melon. The Plant Journal, 48(3), 452-462. doi:10.1111/j.1365-313x.2006.02885.xOhki, T., Akita, F., Mochizuki, T., Kanda, A., Sasaya, T., & Tsuda, S. (2010). The protruding domain of the coat protein of Melon necrotic spot virus is involved in compatibility with and transmission by the fungal vector Olpidium bornovanus. Virology, 402(1), 129-134. doi:10.1016/j.virol.2010.03.020Panavas, T., Hawkins, C. M., Panaviene, Z., & Nagy, P. D. (2005). The role of the p33:p33/p92 interaction domain in RNA replication and intracellular localization of p33 and p92 proteins of Cucumber necrosis tombusvirus. Virology, 338(1), 81-95. doi:10.1016/j.virol.2005.04.025Powers, J. G., Sit, T. L., Qu, F., Morris, T. J., Kim, K.-H., & Lommel, S. A. (2008). A Versatile Assay for the Identification of RNA Silencing Suppressors Based on Complementation of Viral Movement. Molecular Plant-Microbe Interactions®, 21(7), 879-890. doi:10.1094/mpmi-21-7-0879Qu, F., Ren, T., & Morris, T. J. (2003). The Coat Protein of Turnip Crinkle Virus Suppresses Posttranscriptional Gene Silencing at an Early Initiation Step. Journal of Virology, 77(1), 511-522. doi:10.1128/jvi.77.1.511-522.2003Riviere, C. J., & Rochon, D. M. (1990). Nucleotide sequence and genomic organization of melon necrotic spot virus. Journal of General Virology, 71(9), 1887-1896. doi:10.1099/0022-1317-71-9-1887Romero-Brey, I., & Bartenschlager, R. (2014). Membranous Replication Factories Induced by Plus-Strand RNA Viruses. Viruses, 6(7), 2826-2857. doi:10.3390/v6072826Russo, M., & Martelli, G. P. (1982). Ultrastructure of turnip crinkle- and saguaro cactus virus-infected tissues. Virology, 118(1), 109-116. doi:10.1016/0042-6822(82)90324-5Saurí, A., Saksena, S., Salgado, J., Johnson, A. E., & Mingarro, I. (2005). Double-spanning Plant Viral Movement Protein Integration into the Endoplasmic Reticulum Membrane Is Signal Recognition Particle-dependent, Translocon-mediated, and Concerted. Journal of Biological Chemistry, 280(27), 25907-25912. doi:10.1074/jbc.m412476200Serra-Soriano, M., Antonio Navarro, J., & Pallás, V. (2016). Dissecting the multifunctional role of the N-terminal domain of theMelon necrotic spot viruscoat protein in RNA packaging, viral movement and interference with antiviral plant defence. Molecular Plant Pathology, 18(6), 837-849. doi:10.1111/mpp.12448Serra-Soriano, M., Pallás, V., & Navarro, J. A. (2014). A model for transport of a viral membrane protein through the early secretory pathway: minimal sequence and endoplasmic reticulum lateral mobility requirements. The Plant Journal, 77(6), 863-879. doi:10.1111/tpj.12435Shi, Y., Ryabov, E. V., van Wezel, R., Li, C., Jin, M., Wang, W., … Hong, Y. (2009). Suppression of local RNA silencing is not sufficient to promote cell-to-cell movement ofTurnip crinkle virusinNicotiana benthamiana. Plant Signaling & Behavior, 4(1), 15-22. doi:10.4161/psb.4.1.7573Teakle, D. S. (1980). FUNGI. Vectors of Plant Pathogens, 417-438. doi:10.1016/b978-0-12-326450-3.50021-8Thomas, C. L., Leh, V., Lederer, C., & Maule, A. J. (2003). Turnip crinkle virus coat protein mediates suppression of RNA silencing in nicotiana benthamiana. Virology, 306(1), 33-41. doi:10.1016/s0042-6822(02)00018-1Tilsner, J., Linnik, O., Louveaux, M., Roberts, I. M., Chapman, S. N., & Oparka, K. J. (2013). 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Functional Studies on the IBD Susceptibility Gene IL23R Implicate Reduced Receptor Function in the Protective Genetic Variant R381Q

Genome-wide association studies (GWAS) in several populations have demonstrated significant association of the IL23R gene with IBD (Crohn's disease (CD) and ulcerative colitis (UC)) and psoriasis, suggesting that perturbation of the IL-23 signaling pathway is relevant to the pathophysiology of these diseases. One particular variant, R381Q (rs11209026), confers strong protection against development of CD. We investigated the effects of this variant in primary T cells from healthy donors carrying IL23RR381 and IL23RQ381 haplotypes. Using a proprietary anti-IL23R antibody, ELISA, flow cytometry, phosphoflow and real-time RT-PCR methods, we examined IL23R expression and STAT3 phosphorylation and activation in response to IL-23. IL23RQ381 was associated with reduced STAT3 phosphorylation upon stimulation with IL-23 and decreased number of IL-23 responsive T-cells. We also observed slightly reduced levels of proinflammatory cytokine secretion in IL23RQ381 positive donors. Our study shows conclusively that IL23RQ381 is a loss-of-function allele, further strengthening the implication from GWAS results that the IL-23 pathway is pathogenic in human disease. This data provides an explanation for the protective role of R381Q in CD and may lead to the development of improved therapeutics for autoimmune disorders like CD

Repair or destruction: an intimate liaison between ubiquitin ligases and molecular chaperones in proteostasis

Cellular differentiation, developmental processes, and environmental factors challenge the integrity of the proteome in every eukaryotic cell. The maintenance of protein homeostasis, or proteostasis, involves folding and degradation of damaged proteins, and is essential for cellular function, organismal growth, and viability [1, 2]. Misfolded proteins that cannot be refolded by chaperone machineries are degraded by specialized proteolytic systems. A major degradation pathway regulating cellular proteostasis is the ubiquitin/proteasome-system (UPS), which regulates turnover of damaged proteins that accumulate upon stress and during aging. Despite the large number of structurally unrelated substrates, ubiquitin conjugation is remarkably selective. Substrate selectivity is mainly provided by the group of E3 enzymes. Several observations indicate that numerous E3 ubiquitin ligases intimately collaborate with molecular chaperones to maintain the cellular proteome. In this Review, we provide an overview of specialized quality control E3 ligases playing a critical role in the degradation of damaged proteins. The process of substrate recognition and turnover, the type of chaperones they team up with, and the potential pathogeneses associated with their malfunction will be further discusse

Using Structural Information of Peptides, Derived from NMR Spectroscopy, in Pharmaceutical Chemistry

The significance of information gained from the solution structure of peptides for pharmaceutical research is demonstrated with two examples: the neuropeptide Y (NPY) hormone system and a undecapeptide designed for use as a chemical sensor. In the case of NPY, the structure of the homodimer and the mode of membrane association was determined. Thereby, it was discovered that the membrane/NPY interface is formed by the same hydrophobic residues that constitute the homodimer interface. Furthermore, in the membrane-bound state, the C-terminal helix is stabilized, which is of special functional importance for the C-terminal tetrapeptide. Receptor-subtype specificity of NPY mutants may be explained through pre-orientation of residues relative to the different membrane compartments. In the case of the undecapeptide designed for use in a chemical sensor, structural information from NMR helped us to design and optimize a peptide whose unligated form is unstructured in solution but adopts a unique helical fold upon addition of sulfate. The sulfate binding pocket is formed by the N-terminal first turn