生理活性类囊体的分离及其在能量依赖性蛋白转运检测中的应用
Summary
本文提出了用于叶绿体双精氨酸易位 (cpTat)、分泌 (cpSec1) 和信号识别粒子 (cpSRP) 通路的生理活性类囊体和蛋白质转运测定的高产量隔离的协议。
Abstract
叶绿体是绿色植物中的细胞器, 负责执行许多必要的代谢通路, 尤其是光合作用。在叶绿体内, 类囊体膜系统将所有的光合颜料、反应中心配合物和大多数电子载体都安置在一起, 并负责光依赖性 ATP 的合成。90% 以上的叶绿体蛋白被编码在细胞核中, 翻译成细胞质, 随后导入叶绿体。进一步蛋白质运输入或横跨囊状体膜利用四个易位途径之一。在这里, 我们描述了一个高产的方法, 以分离的运输主管类囊体从豌豆 (豌豆大蒜), 连同运输化验通过三能源依赖 cpTat, cpSec1 和 cpSRP 介导的途径。这些方法使实验与囊体蛋白定位, 传输能量学, 和蛋白质易位的机制跨生物膜。
Introduction
几乎所有负责适当叶绿体功能的蛋白质机械都必须从细胞质1移位。在叶绿体信封, 蛋白质基质是通过 translocon 的外层膜 (TOC) 和 translocon 的内膜 (TIC)2进口。进一步靶向囊体膜和流明发生通过双精氨酸易位 (cpTat)3, 分泌 (cpSec1)4, 信号识别粒子 (cpSRP)5和自发插入通路6.一种对生理活性叶绿体和类囊体膜进行高产分离的方法, 是测量易位事件的热力学和动力学的必要条件, 了解各途径中不同的传输机制, 并将特定的蛋白质基板感兴趣的六个不同的隔间的叶绿体。
分离的膜从叶绿体提供更好的实验控制环境因素 (如盐和基质浓度, ATP/GTP 的存在, 和 pH 条件), 影响测量的运输热力学和动力学。由于同样的原因, 这种体外环境有利于对易位机械细节的探索。此外, 虽然叶绿体蛋白定位的预测软件改善了7,8,体外传输化验提供了一个更快的方法, 以证实显微镜下的荧光化验,需要基因编码的荧光标记, 植物转化和/或特定的抗体。在这里, 我们提出了从豌豆 (豌豆大蒜) 隔离叶绿体和囊体的协议, 以及为每个能量依赖性囊体易位通路优化的运输化验。
Protocol
1. 初始材料 在400毫升蒸馏水中浸泡大约55克豌豆3小时, 然后在一个塑料托盘 (35 厘米 x 20 厘米 x 6 厘米) 在覆盖着薄薄的蛭石层的土壤中播种。 将豌豆的托盘生长在20摄氏度以下12/12 小时/暗 (50 µE/米2s) 周期9至15天。 根据首选方法制备蛋白质基质。注: 我们已经准备了蛋白质基质使用多种方法, 包括 1) 从纯化质粒体外转录, 然后用小麦胚芽提取物或兔网织裂解…
Representative Results
为了测量成功运输的基体的数量, 有一个或多个 “百分比输入” 车道是有用的。对于下面所示的数据, 10% 的最终运输反应没有类囊体使用。这种 “百分比输入” 也有助于可视化前体基底的大小。所述百分比表示已知的、定义的用于比较所述运输基板的基板量, 并可根据需要使用最初制备的蛋白质进行缩放。另外, 建议在 0.75 mm 聚丙烯酰胺凝胶的单一车道上加载少于4µg 的智利当…
Discussion
叶绿体和囊体分离
过度破损会导致叶绿体分离不良, 而在梯度分离后, 不良的囊体产生。最好通过确保所有物质在十五年代的混合和脉动之前被浸没, 直到完全匀质之前, 轻轻地融汇收获的组织。如有必要, 在每回合中使用较少组织的多轮混合。
冷藏所有与收获组织接触的材料可以帮助分离的叶绿体保持2小时的活动。这是重要的是保持在冰上的叶绿体在黑暗?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
这份手稿是由化学科学、地球科学和生物科学司资助的, 408 美国能源部基础能源科学办公室通过赠款 DE-SC0017035
Materials
| Pisum sativum seeds | Seedway LLC, Hall, NY | 8686 – Little Marvel | |
| Miracloth | Calbiochem, Gibbstown, NJ | 475855-1 | |
| 80% Acetone | Sigma, Saint Louis, MO | 67-64-1 | |
| Blender with sharpened blades | Waring Commercial | BB155S | |
| Polytron 10-35 | Fischer Sci | 13-874-617 | |
| Percoll | Sigma, Saint Louis, MO | GE17-0891-01 | |
| Beckman J2-MC with JA 20 rotor | Beckman-Coulter | 8043-30-1180 | |
| Sorvall RC-5B with HB-4 rotor | Sorvall | 8327-30-1016 | |
| 100 mM dithiothreitol (DTT) in 1xIB | Sigma, Saint Louis, MO | 12/3/83 | Can be frozen in aliquots for future use |
| 200 mM MgATP in 1xIB | Sigma, Saint Louis, MO | 74804-12-9 | Can be frozen in aliquots for future use |
| Thermolysin in 1xIB (2mg/mL) | Sigma, Saint Louis, MO | 9073-78-3 | Can be frozen in aliquots for future use |
| HEPES | Sigma, Saint Louis, MO | H3375 | |
| K-Tricine | Sigma, Saint Louis, MO | T0377 | |
| Sorbitol | Sigma, Saint Louis, MO | 50-70-4 | |
| Magnesium Chloride | Sigma, Saint Louis, MO | 7791-18-6 | |
| Manganese Chloride | Sigma, Saint Louis, MO | 13446-34-9 | |
| EDTA | Sigma, Saint Louis, MO | 60-00-4 | |
| BSA | Sigma, Saint Louis, MO | 9048-46-8 | |
| Tris | Sigma, Saint Louis, MO | 77-86-1 | |
| SDS | Sigma, Saint Louis, MO | 151-21-3 | |
| Glycerol | Sigma, Saint Louis, MO | 56-81-5 | |
| Bromophenol Blue | Sigma, Saint Louis, MO | 115-39-9 | |
| B-Mercaptoethanol | Sigma, Saint Louis, MO | 60-24-2 |
Referencias
- Ellis, R. Chloroplast protein synthesis: principles and problems. Sub-cellular biochemistry. 9, 237 (1983).
- Li, H. -. m., Chiu, C. -. C. Protein transport into chloroplasts. Annual review of plant biology. 61, (2010).
- Cline, K., Ettinger, W., Theg, S. M. Protein-specific energy requirements for protein transport across or into thylakoid membranes. Two lumenal proteins are transported in the absence of ATP. Journal of Biological Chemistry. 267 (4), 2688-2696 (1992).
- Skalitzky, C. A., et al. Plastids contain a second sec translocase system with essential functions. Plant physiology. 155 (1), 354-369 (2011).
- Dabney-Smith, C., Storm, A. . Plastid Biology. , 271-289 (2014).
- Kim, S. J., Jansson, S., Hoffman, N. E., Robinson, C., Mant, A. Distinct "assisted" and "spontaneous" mechanisms for the insertion of polytopic chlorophyll-binding proteins into the thylakoid membrane. Journal of Biological Chemistry. 274 (8), 4715-4721 (1999).
- Emanuelsson, O., Nielsen, H., Von Heijne, G. C. h. l. o. r. o. P. ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites. Protein Science. 8 (5), 978-984 (1999).
- Emanuelsson, O., Brunak, S., Von Heijne, G., Nielsen, H. Locating proteins in the cell using TargetP, SignalP and related tools. Nature protocols. 2 (4), 953 (2007).
- Ling, Q., Jarvis, R. Analysis of protein import into chloroplasts isolated from stressed plants. Journal of Visualized Experiments. (117), e54717 (2016).
- Lo, S. M., Theg, S. M. . Photosynthesis Research Protocols. , 139-157 (2011).
- Vernon, L. P. Spectrophotometric determination of chlorophylls and pheophytins in plant extracts. Analytical Chemistry. 32 (9), 1144-1150 (1960).
- Knott, T. G., Robinson, C. The secA inhibitor, azide, reversibly blocks the translocation of a subset of proteins across the chloroplast thylakoid membrane. Journal of Biological Chemistry. 269 (11), 7843-7846 (1994).
- Yuan, J., Henry, R., McCaffery, M., Cline, K. SecA homolog in protein transport within chloroplasts: evidence for endosymbiont-derived sorting. Science. 266 (5186), 796-798 (1994).
- Nohara, T., Nakai, M., Goto, A., Endo, T. Isolation and characterization of the cDNA for pea chloroplast SecA Evolutionary conservation of the bacterial-type SecA-dependent protein transport within chloroplasts. FEBS letters. 364 (3), 305-308 (1995).
- Endow, J. K., Singhal, R., Fernandez, D. E., Inoue, K. Chaperone-assisted post-translational transport of plastidic type I signal peptidase 1. Journal of Biological Chemistry. 290 (48), 28778-28791 (2015).
- Luirink, J., Sinning, I. SRP-mediated protein targeting: structure and function revisited. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research. 1694 (1-3), 17-35 (2004).
- Yuan, J., Henry, R., Cline, K. Stromal factor plays an essential role in protein integration into thylakoids that cannot be replaced by unfolding or by heat shock protein. Hsp70. Proceedings of the National Academy of Sciences. 90 (18), 8552-8556 (1993).
- Tjalsma, H., van Dijl, J. M. Proteomics-based consensus prediction of protein retention in a bacterial membrane. Proteomics. 5 (17), 4472-4482 (2005).
- Widdick, D. A., Eijlander, R. T., van Dijl, J. M., Kuipers, O. P., Palmer, T. A Facile Reporter System for the Experimental Identification of Twin-Arginine Translocation (Tat) Signal Peptides from All Kingdoms of Life. Journal of Molecular Biology. 375 (3), 595-603 (2008).
- Yuan, J., Cline, K. Plastocyanin and the 33-kDa subunit of the oxygen-evolving complex are transported into thylakoids with similar requirements as predicted from pathway specificity. Journal of Biological Chemistry. 269 (28), 18463-18467 (1994).
- Kirchhoff, H., Borinski, M., Lenhert, S., Chi, L., Büchel, C. Transversal and lateral exciton energy transfer in grana thylakoids of spinach. Bioquímica. 43 (45), 14508-14516 (2004).
- Frielingsdorf, S., Jakob, M., Klösgen, R. B. A stromal pool of TatA promotes Tat-dependent protein transport across the thylakoid membrane. Journal of Biological Chemistry. 283 (49), 33838-33845 (2008).
- Tu, C. -. J., Schuenemann, D., Hoffman, N. E. Chloroplast FtsY, chloroplast signal recognition particle, and GTP are required to reconstitute the soluble phase of light-harvesting chlorophyll protein transport into thylakoid membranes. Journal of Biological Chemistry. 274 (38), 27219-27224 (1999).
Citar este artículo
Asher, A., Ganesan, I., Klasek, L., Theg, S. M. Isolation of Physiologically Active Thylakoids and Their Use in Energy-Dependent Protein Transport Assays. J. Vis. Exp. (139), e58393, doi:10.3791/58393 (2018).