Investigating the function of a ClpC/Hsp100-type chaperone in chloroplast preprotein import

叶绿体和线粒体是许多细胞的正常组成部分--它们是亚细胞结构，称为细胞器。有趣的是，这两个细胞器是从10亿多年前被其他细胞吞噬的细菌进化而来的，它们在许多方面仍然类似于自由生活的细菌。叶绿体存在于植物细胞中，含有绿色色素叶绿素，专门负责光合作用(捕捉阳光能量并利用其为细胞活动提供动力的过程)的反应。由于光合作用是向生物世界输入能量的唯一重要机制，叶绿体不仅对植物，而且对地球上的所有生命都具有不可估量的重要性。叶绿体在许多其他方面也很重要，因为它们在油脂、蛋白质和淀粉的生物合成中起着至关重要的作用。尽管叶绿体确实含有DNA(这是作为自由生活的光合细菌的古代进化历史的遗物)，因此能够制造自己的一些蛋白质，但建立一个功能齐全的叶绿体所需的大约3000种蛋白质中，超过90%是在细胞核内的DNA上编码的。因此，大多数叶绿体蛋白是在叶绿体以外的细胞基质中制造的，称为细胞质。由于每个叶绿体都被双层膜或被膜包围，不受蛋白质被动运动的影响，这是一个重要的问题。为了克服这个问题，叶绿体进化出了一种复杂的蛋白质输入装置，它使用能量(以ATP的形式)驱动蛋白质从细胞质输入，穿过被膜，进入叶绿体内部。这种蛋白质输入装置由两个分子机器组成：一个在外被膜上，称为TOC(叶绿体外被膜上转运子的缩写)，另一个在内被膜上，称为TIC。这个项目的重点是TIC机器，特别是一种名为Hsp93的蛋白质，它与TIC复合体有关。这种Hsp93蛋白是一种ATPase(即，它将ATP水解酶以释放能量)，并且是被称为“分子伴侣”的蛋白质家族的成员。这种伴侣蛋白能够与其他蛋白质结合，特别是当它们处于未折叠状态时。在这样做的过程中，一些伴侣可以对目标蛋白质施加“拉力”，以促进其从一个位置传递到另一个位置。根据几条证据，Hsp93被认为是叶绿体蛋白进口的驱动力，并通过拉动需要进口的蛋白质来发挥作用(即，它被认为是所谓的叶绿体蛋白进口马达的核心部分)。因此，在进口机制期间发生的大部分ATP消耗暂定归因于Hsp93。然而，这些假设的直接证据仍然缺乏。我们建议通过操纵完整植物中Hsp93蛋白的活性，并评估这种操纵对叶绿体蛋白进口效率的影响，直接测试这些想法。由于叶绿体执行必要的功能，而且蛋白质输入对叶绿体的发育至关重要，因此，如果了解到没有有效的叶绿体蛋白质输入机制的植物无法生存(事实上，它们在胚胎阶段死亡)，这一点也就不足为奇了。因此，叶绿体蛋白的输入是植物的一个必不可少的过程。同样，由于我们最终都依赖植物产品生存，因此在全球范围内，叶绿体蛋白的进口是必不可少的。更重要的是，由于叶绿体在许多重要的经济产物(如油和淀粉)的合成中起着重要的作用，对这些细胞器如何发育的更全面的了解可能使我们能够提高作物的生产力，或者以其他方式操纵它们的产物。

Chloroplasts and mitochondria are normal components of many cells - they are sub-cellular structures called organelles. Interestingly, these two organelles evolved from bacteria that were engulfed by other cells more than a billion years ago, and in many ways they still resemble free-living bacteria. Chloroplasts are found in plant cells, contain the green pigment chlorophyll, and are exclusively responsible for the reactions of photosynthesis (the process that captures sunlight energy and uses it to power the activities of the cell). Since photosynthesis is the only significant mechanism of energy-input into the living world, chloroplasts are of inestimable importance, not just to plants but to all life on Earth. Chloroplasts are also important in many other ways, since they play essential roles in the biosynthesis of oils, proteins and starch. Although chloroplasts do contain DNA (which is a relic from their ancient, evolutionary past as free-living photosynthetic bacteria), and are therefore able to make some of their own proteins, over 90% of the 3000 or so proteins required to build a fully functional chloroplast are encoded on DNA within the cell nucleus. The majority of chloroplast proteins are therefore made outside of the chloroplast, in the cellular matrix known as the cytosol. Since chloroplasts are each surrounded by a double membrane, or envelope, that is impervious to the passive movement of proteins, this presents a significant problem. To overcome the problem, chloroplasts have evolved a sophisticated protein import apparatus, which uses energy (in the form of ATP) to drive the import of proteins from the cytosol, across the envelope, and into the chloroplast interior. This protein import apparatus comprises two molecular machines: one in the outer envelope membrane called TOC (an abbreviation of "Translocon at the outer envelope membrane of chloroplasts"), and another in the inner envelope membrane called TIC. This project is focused on the TIC machine, and in particular on a protein called Hsp93 which is associated with the TIC complex. This Hsp93 protein is an ATPase (i.e. it hydrolyses ATP to release energy), and is a member of a family of proteins called the "molecular chaperones". Such chaperone proteins are able to bind to other proteins, particularly when they are in an unfolded state. In doing this, some chaperones can exert a "pulling force" on the target protein, to facilitate its passage from one location to another. Based on several lines of evidence, Hsp93 is thought to provide the driving force for chloroplast protein import, and to act by pulling on those proteins that need to be imported (i.e. it is believed to be a core part of the so-called "chloroplast protein import motor"). Thus, much of the ATP consumption that occurs during the import mechanism is tentatively attributed to Hsp93. However, direct proof of these hypotheses is still lacking. We propose to test these ideas directly, by manipulating the activities of the Hsp93 protein in intact plants, and assessing the consequences of such manipulations on chloroplast protein import efficiency. Because chloroplasts carry out essential functions, and because protein import is essential for chloroplast development, it should come as no surprise to learn that plants without a functional chloroplast protein import machinery are unable to survive (in fact, they die at the embryo stage). Thus, chloroplast protein import is an essential process for plants. Similarly, since we are all ultimately dependent upon plant products for survival, it follows that chloroplast protein import is essential on a global scale. What is more, since chloroplasts play a major role in the synthesis of many economically important products (such as oils and starch), a more complete understanding of how these organelles develop may enable us to enhance the productivity of crop plants, or otherwise manipulate their products.