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?…
Declarações
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 |
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Citar este artigo
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).