Analysis of Protein Import into Chloroplasts Isolated from Stressed Plants
Analysis of Protein Import into Chloroplasts Isolated from Stressed Plants
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DOI：
10.3791/54717
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发表时间：
2016-11-01
影响因子：
1.2
通讯作者：
Jarvis, Paul
中科院分区：
文献类型：
--
作者：
Ling, Qihua;Jarvis, Paul
叶绿体是细胞器，在植物中具有许多重要作用，不仅包括光合作用，还包括许多其他代谢和信号功能。此外，叶绿体对于植物对各种非生物胁迫的响应是至关重要的，例如盐分和渗透胁迫。一个叶绿体可能含有多达3000种不同的蛋白质，其中一些蛋白质是由它自己的基因组编码的。然而，大多数叶绿体蛋白是在细胞核中编码并在胞质中合成的，这些蛋白需要通过叶绿体被膜上的转座子输入到叶绿体中。最近的研究表明，胁迫可以主动调节叶绿体蛋白的输入。为了在生物化学上研究应激条件下蛋白质进口的这种调节，我们开发了这里描述的方法，作为一种快速而直接的方法，可以在任何实验室轻松实现。在这种方法中，植物在正常条件下生长，然后在液体培养中暴露在胁迫条件下。收集植物材料，然后通过均质释放叶绿体。粗匀浆通过密度梯度离心法分离，从而能够分离完整的叶绿体。通过计数评估叶绿体产量，并在显微镜下检查叶绿体完整性。在蛋白质进口试验中，将纯化的叶绿体与S-35标记的体外翻译前体蛋白孵育，并进行时程实验，以比较胁迫条件下不同基因型之间的进口速率。我们给出了用这种方法产生的数据，这些数据表明，在渗透胁迫条件下，调节突变体向叶绿体输入蛋白质的速率发生了特定的变化。
Chloroplasts are organelles with many vital roles in plants, which include not only photosynthesis but numerous other metabolic and signaling functions. Furthermore, chloroplasts are critical for plant responses to various abiotic stresses, such as salinity and osmotic stresses. A chloroplast may contain up to similar to 3,000 different proteins, some of which are encoded by its own genome. However, the majority of chloroplast proteins are encoded in the nucleus and synthesized in the cytosol, and these proteins need to be imported into the chloroplast through translocons at the chloroplast envelope membranes. Recent studies have shown that the chloroplast protein import can be actively regulated by stress. To biochemically investigate such regulation of protein import under stress conditions, we developed the method described here as a quick and straightforward procedure that can easily be achieved in any laboratory. In this method, plants are grown under normal conditions and then exposed to stress conditions in liquid culture. Plant material is collected, and chloroplasts are then released by homogenization. The crude homogenate is separated by density gradient centrifugation, enabling isolation of the intact chloroplasts. Chloroplast yield is assessed by counting, and chloroplast intactness is checked under a microscope. For the protein import assays, purified chloroplasts are incubated with S-35 radiolabeled in vitro translated precursor proteins, and time-course experiments are conducted to enable comparisons of import rates between genotypes under stress conditions. We present data generated using this method which show that the rate of protein import into chloroplasts from a regulatory mutant is specifically altered under osmotic stress conditions.