Optimization of non-denaturing protein extraction conditions for plant PPR proteins

[EN] Pentatricopeptide repeat proteins are one of the major protein families in flowering plants, containing around 450 members. They participate in RNA editing and are related to plant growth, development and reproduction, as well as to responses to ABA and abiotic stresses. Their characteristics have been described in silico; however, relatively little is known about their biochemical properties. Different types of PPR proteins, with different tasks in RNA editing, have been suggested to interact in an editosome to complete RNA editing. Other non-PPR editing factors, such as the multiple organellar RNA editing factors and the organelle RNA recognition motif-containing protein family, for example, have also been described in plants. However, while evidence on protein interactions between non-PPR RNA editing proteins is accumulating, very few PPR protein interactions have been reported; possibly due to their high instability. In this manuscript, we aimed to optimize the conditions for non denaturing protein extraction of PPR proteins allowing in vivo protein analyses, such as interaction assays by co-immunoprecipitation. The unusually high protein degradation rate, the aggregation properties and the high pl, as well as the ATP-dependence of some PPR proteins, are key aspects to be considered when extracting PPR proteins in a non-denatured state. During extraction of PPR proteins, the use of proteasome and phosphatase inhibitors is critical. The use of the ATP-cofactor reduces considerably the degradation of PPR proteins. A short centrifugation step to discard cell debris is essential to avoid PPR precipitation; while in some cases, addition of a reductant is needed, probably caused by the pl/pH context. This work provides an easy and rapid optimized non-denaturing total protein extraction protocol from plant tissue, suitable for polypeptides of the PPR family.This work was supported by Research Foundation Flanders (FWO) (project G.0C84.14N). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.r D.V.D.S. gratefully acknowledges Ghent University and the Research Foundation Flanders (FWO) for financial support (project G.0084.14N). N.A.-C. and D.V.D.S. acknowledge Maria Helena S. Goldman for the N. benthamiana seeds, helpful suggestions regarding experimental design and data interpretation, and critical reading of the manuscript.Andrés-Colás, N.; Van Der Straeten, D. (2017). Optimization of non-denaturing protein extraction conditions for plant PPR proteins. PLoS ONE. 12(11):1-15. https://doi.org/10.1371/journal.pone.01877531151211Barkan, A., & Small, I. (2014). Pentatricopeptide Repeat Proteins in Plants. Annual Review of Plant Biology, 65(1), 415-442. doi:10.1146/annurev-arplant-050213-040159Saha, D., Prasad, A. M., & Srinivasan, R. (2007). Pentatricopeptide repeat proteins and their emerging roles in plants. Plant Physiology and Biochemistry, 45(8), 521-534. doi:10.1016/j.plaphy.2007.03.026SCHMITZLINNEWEBER, C., & SMALL, I. (2008). Pentatricopeptide repeat proteins: a socket set for organelle gene expression. Trends in Plant Science, 13(12), 663-670. doi:10.1016/j.tplants.2008.10.001De Longevialle, A. F., Meyer, E. H., Andrés, C., Taylor, N. L., Lurin, C., Millar, A. H., & Small, I. D. (2007). The Pentatricopeptide Repeat Gene OTP43 Is Required for trans-Splicing of the Mitochondrial nad1 Intron 1 in Arabidopsis thaliana. The Plant Cell, 19(10), 3256-3265. doi:10.1105/tpc.107.054841Liu, Y., He, J., Chen, Z., Ren, X., Hong, X., & Gong, Z. (2010). ABA overly-sensitive 5 (ABO5), encoding a pentatricopeptide repeat protein required for cis-splicing of mitochondrial nad2 intron 3, is involved in the abscisic acid response in Arabidopsis. The Plant Journal, 63(5), 749-765. doi:10.1111/j.1365-313x.2010.04280.xSung, T.-Y., Tseng, C.-C., & Hsieh, M.-H. (2010). The SLO1 PPR protein is required for RNA editing at multiple sites with similar upstream sequences in Arabidopsis mitochondria. The Plant Journal, 63(3), 499-511. doi:10.1111/j.1365-313x.2010.04258.xZhu, Q., Dugardeyn, J., Zhang, C., Takenaka, M., Kühn, K., Craddock, C., … Van Der Straeten, D. (2012). SLO2, a mitochondrial pentatricopeptide repeat protein affecting several RNA editing sites, is required for energy metabolism. The Plant Journal, 71(5), 836-849. doi:10.1111/j.1365-313x.2012.05036.xZhu, Q., Dugardeyn, J., Zhang, C., Mühlenbock, P., Eastmond, P. J., Valcke, R., … Van Der Straeten, D. (2014). The Arabidopsis thaliana RNA Editing Factor SLO2, which Affects the Mitochondrial Electron Transport Chain, Participates in Multiple Stress and Hormone Responses. Molecular Plant, 7(2), 290-310. doi:10.1093/mp/sst102Colcombet, J., Lopez-Obando, M., Heurtevin, L., Bernard, C., Martin, K., Berthomé, R., & Lurin, C. (2013). Systematic study of subcellular localization of Arabidopsis PPR proteins confirms a massive targeting to organelles. RNA Biology, 10(9), 1557-1575. doi:10.4161/rna.26128Ichinose, M., & Sugita, M. (2016). RNA Editing and Its Molecular Mechanism in Plant Organelles. Genes, 8(1), 5. doi:10.3390/genes8010005Sun, T., Bentolila, S., & Hanson, M. R. (2016). The Unexpected Diversity of Plant Organelle RNA Editosomes. Trends in Plant Science, 21(11), 962-973. doi:10.1016/j.tplants.2016.07.005Yagi, Y., Tachikawa, M., Noguchi, H., Satoh, S., Obokata, J., & Nakamura, T. (2013). Pentatricopeptide repeat proteins involved in plant organellar RNA editing. RNA Biology, 10(9), 1419-1425. doi:10.4161/rna.24908Lurin, C., Andrés, C., Aubourg, S., Bellaoui, M., Bitton, F., Bruyère, C., … Small, I. (2004). Genome-Wide Analysis of Arabidopsis Pentatricopeptide Repeat Proteins Reveals Their Essential Role in Organelle Biogenesis. The Plant Cell, 16(8), 2089-2103. doi:10.1105/tpc.104.022236Bentolila, S., Heller, W. P., Sun, T., Babina, A. M., Friso, G., van Wijk, K. J., & Hanson, M. R. (2012). RIP1, a member of an Arabidopsis protein family, interacts with the protein RARE1 and broadly affects RNA editing. Proceedings of the National Academy of Sciences, 109(22), E1453-E1461. doi:10.1073/pnas.1121465109Glass, F., Härtel, B., Zehrmann, A., Verbitskiy, D., & Takenaka, M. (2015). MEF13 Requires MORF3 and MORF8 for RNA Editing at Eight Targets in Mitochondrial mRNAs in Arabidopsis thaliana. Molecular Plant, 8(10), 1466-1477. doi:10.1016/j.molp.2015.05.008Härtel, B., Zehrmann, A., Verbitskiy, D., van der Merwe, J. A., Brennicke, A., & Takenaka, M. (2013). MEF10 is required for RNA editing at nad2-842 in mitochondria of Arabidopsis thaliana and interacts with MORF8. Plant Molecular Biology, 81(4-5), 337-346. doi:10.1007/s11103-012-0003-2Hu, J., Wang, K., Huang, W., Liu, G., Gao, Y., Wang, J., … Zhu, Y. (2012). The Rice Pentatricopeptide Repeat Protein RF5 Restores Fertility in Hong-Lian Cytoplasmic Male-Sterile Lines via a Complex with the Glycine-Rich Protein GRP162. The Plant Cell, 24(1), 109-122. doi:10.1105/tpc.111.093211Sun, T., Germain, A., Giloteaux, L., Hammani, K., Barkan, A., Hanson, M. R., & Bentolila, S. (2013). An RNA recognition motif-containing protein is required for plastid RNA editing in Arabidopsis and maize. Proceedings of the National Academy of Sciences, 110(12), E1169-E1178. doi:10.1073/pnas.1220162110Sun, T., Shi, X., Friso, G., Van Wijk, K., Bentolila, S., & Hanson, M. R. (2015). A Zinc Finger Motif-Containing Protein Is Essential for Chloroplast RNA Editing. PLOS Genetics, 11(3), e1005028. doi:10.1371/journal.pgen.1005028Takenaka, M., Zehrmann, A., Verbitskiy, D., Kugelmann, M., Hartel, B., & Brennicke, A. (2012). Multiple organellar RNA editing factor (MORF) family proteins are required for RNA editing in mitochondria and plastids of plants. Proceedings of the National Academy of Sciences, 109(13), 5104-5109. doi:10.1073/pnas.1202452109Boussardon, C., Salone, V., Avon, A., Berthomé, R., Hammani, K., Okuda, K., … Lurin, C. (2012). Two Interacting Proteins Are Necessary for the Editing of the NdhD-1 Site in Arabidopsis Plastids. The Plant Cell, 24(9), 3684-3694. doi:10.1105/tpc.112.099507Coquille, S., Filipovska, A., Chia, T., Rajappa, L., Lingford, J. P., Razif, M. F. M., … Rackham, O. (2014). An artificial PPR scaffold for programmable RNA recognition. Nature Communications, 5(1). doi:10.1038/ncomms6729Uyttewaal, M., Arnal, N., Quadrado, M., Martin-Canadell, A., Vrielynck, N., Hiard, S., … Mireau, H. (2008). Characterization of Raphanus sativus Pentatricopeptide Repeat Proteins Encoded by the Fertility Restorer Locus for Ogura Cytoplasmic Male Sterility. The Plant Cell, 20(12), 3331-3345. doi:10.1105/tpc.107.057208Kobayashi, T., Yagi, Y., & Nakamura, T. (2016). Development of Genome Engineering Tools from Plant-Specific PPR Proteins Using Animal Cultured Cells. Chromosome and Genomic Engineering in Plants, 147-155. doi:10.1007/978-1-4939-4931-1_11Kun, W., Feng, G., Renshan, Z., Shaoqing, L., & Yingguo, Z. (2010). Expression, Purification, and Secondary Structure Prediction of Pentatricopeptide Repeat Protein RF1A from Rice. Plant Molecular Biology Reporter, 29(3), 739-744. doi:10.1007/s11105-010-0260-7Yin, P., Li, Q., Yan, C., Liu, Y., Liu, J., Yu, F., … Yan, N. (2013). Structural basis for the modular recognition of single-stranded RNA by PPR proteins. Nature, 504(7478), 168-171. doi:10.1038/nature12651Law, Y.-S., Zhang, R., Guan, X., Cheng, S., Sun, F., Duncan, O., … Lim, B. L. (2015). Phosphorylation and Dephosphorylation of the Presequence of Precursor MULTIPLE ORGANELLAR RNA EDITING FACTOR3 during Import into Mitochondria from Arabidopsis. Plant Physiology, 169(2), 1344-1355. doi:10.1104/pp.15.01115Hershko, A., & Ciechanover, A. (1998). THE UBIQUITIN SYSTEM. Annual Review of Biochemistry, 67(1), 425-479. doi:10.1146/annurev.biochem.67.1.425Hegeman, C. E., Hayes, M. L., & Hanson, M. R. (2005). Substrate and cofactor requirements for RNA editing of chloroplast transcripts in Arabidopsis in vitro. The Plant Journal, 42(1), 124-132. doi:10.1111/j.1365-313x.2005.02360.xHooper, C. M., Castleden, I. R., Tanz, S. K., Aryamanesh, N., & Millar, A. H. (2016). SUBA4: the interactive data analysis centre for Arabidopsis subcellular protein locations. Nucleic Acids Research, 45(D1), D1064-D1074. doi:10.1093/nar/gkw1041Jaedicke, K., Lichtenthäler, A. L., Meyberg, R., Zeidler, M., & Hughes, J. (2012). A phytochrome–phototropin light signaling complex at the plasma membrane. Proceedings of the National Academy of Sciences, 109(30), 12231-12236. doi:10.1073/pnas.1120203109Singh, R., Lee, M.-O., Lee, J.-E., Choi, J., Park, J. H., Kim, E. H., … Jwa, N.-S. (2012). Rice Mitogen-Activated Protein Kinase Interactome Analysis Using the Yeast Two-Hybrid System. Plant Physiology, 160(1), 477-487. doi:10.1104/pp.112.200071Irigoyen, M. L., Iniesto, E., Rodriguez, L., Puga, M. I., Yanagawa, Y., Pick, E., … Rubio, V. (2014). Targeted Degradation of Abscisic Acid Receptors Is Mediated by the Ubiquitin Ligase Substrate Adaptor DDA1 in Arabidopsis. The Plant Cell, 26(2), 712-728. doi:10.1105/tpc.113.122234Magori, S., & Citovsky, V. (2011). Agrobacterium Counteracts Host-Induced Degradation of Its Effector F-Box Protein. Science Signaling, 4(195), ra69-ra69. doi:10.1126/scisignal.2002124Zhou, X., Graumann, K., Evans, D. E., & Meier, I. (2012). Novel plant SUN–KASH bridges are involved in RanGAP anchoring and nuclear shape determination. Journal of Cell Biology, 196(2), 203-211. doi:10.1083/jcb.201108098Bracha‐Drori, K., Shichrur, K., Katz, A., Oliva, M., Angelovici, R., Yalovsky, S., & Ohad, N. (2004). Detection of protein–protein interactions in plants using bimolecular fluorescence complementation. The Plant Journal, 40(3), 419-427. doi:10.1111/j.1365-313x.2004.02206.xKrasileva, K. V., Dahlbeck, D., & Staskawicz, B. J. (2010). Activation of an Arabidopsis Resistance Protein Is Specified by the in Planta Association of Its Leucine-Rich Repeat Domain with the Cognate Oomycete Effector. The Plant Cell, 22(7), 2444-2458. doi:10.1105/tpc.110.075358Klenova, E. (2002). Immunoprecipitation techniques for the analysis of transcription factor complexes. Methods, 26(3), 254-259. doi:10.1016/s1046-2023(02)00029-4Manavski, N., Guyon, V., Meurer, J., Wienand, U., & Brettschneider, R. (2012). An Essential Pentatricopeptide Repeat Protein Facilitates 5′ Maturation and Translation Initiation of rps3 mRNA in Maize Mitochondria. The Plant Cell, 24(7), 3087-3105. doi:10.1105/tpc.112.099051Andrés-Colás, N., Zhu, Q., Takenaka, M., De Rybel, B., Weijers, D., & Van Der Straeten, D. (2017). Multiple PPR protein interactions are involved in the RNA editing system in Arabidopsis mitochondria and plastids. Proceedings of the National Academy of Sciences, 114(33), 8883-8888. doi:10.1073/pnas.1705815114Karimi, M., Inzé, D., & Depicker, A. (2002). GATEWAY™ vectors for Agrobacterium-mediated plant transformation. Trends in Plant Science, 7(5), 193-195. doi:10.1016/s1360-1385(02)02251-3NAKAGAWA, T., SUZUKI, T., MURATA, S., NAKAMURA, S., HINO, T., MAEO, K., … ISHIGURO, S. (2007). Improved Gateway Binary Vectors: High-Performance Vectors for Creation of Fusion Constructs in Transgenic Analysis of Plants. Bioscience, Biotechnology, and Biochemistry, 71(8), 2095-2100. doi:10.1271/bbb.70216Voinnet, O., Rivas, S., Mestre, P., & Baulcombe, D. (2003). Retracted: An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. The Plant Journal, 33(5), 949-956. doi:10.1046/j.1365-313x.2003.01676.xPukac, L. A., Carter, J. E., Morrison, K. S., & Karnovsky, M. J. (1997). Enhancement of Diaminobenzidine Colorimetric Signal in Immunoblotting. BioTechniques, 23(3), 385-388. doi:10.2144/97233bm0