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——它们是被称为细胞器的亚细胞结构。有趣的是，这两种细胞器是从10亿多年前被其他细胞吞噬的细菌进化而来的，在许多方面它们仍然像自由生活的细菌。叶绿体存在于植物细胞中，含有绿色色素叶绿素，并专门负责光合作用反应（捕获阳光能量并利用它为细胞活动提供动力的过程）。由于光合作用是生物世界唯一重要的能量输入机制，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、蛋白质和淀粉的生物合成中起着至关重要的作用。虽然叶绿体确实含有DNA （DNA是它们作为自由生活的光合细菌进化而来的古老遗迹），因此能够制造一些自己的蛋白质，但构建一个功能齐全的叶绿体所需的3000多种蛋白质中，有90%以上是在细胞核内的DNA上编码的。因此，大多数叶绿体蛋白质是在叶绿体外的细胞质基质中产生的。由于叶绿体都被一层双层膜或包膜包围着，这对蛋白质的被动运动是不渗透的，这就提出了一个重要的问题。为了克服这个问题，叶绿体进化出了一种复杂的蛋白质进口装置，它利用能量（以ATP的形式）驱动蛋白质从细胞质中进口，穿过包膜，进入叶绿体内部。这种蛋白质输入装置包括两个分子机器：一个在外包膜上称为TOC（“叶绿体外包膜上的Translocon”的缩写），另一个在内包膜上称为TIC。这个项目的重点是TIC机器，特别是一种叫做Hsp93的蛋白质，它与TIC复合物有关。这种Hsp93蛋白是一种ATP酶（即它水解ATP以释放能量），是一种被称为“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结合，特别是当它们处于未折叠状态时。在这个过程中，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移动到另一个位置。基于多项证据，Hsp93被认为为叶绿体蛋白进口提供了驱动力，并通过拉动那些需要进口的蛋白来起作用（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，在输入机制中发生的大部分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，并评估这种操作对叶绿体蛋白进口效率的影响，直接验证这些想法。由于叶绿体具有基本的功能，而蛋白质的输入对叶绿体的发育至关重要，因此，如果没有叶绿体蛋白质输入机制的植物无法存活（事实上，它们在胚胎阶段就死亡了），这应该不足为奇。因此，叶绿体蛋白的输入是植物的一个重要过程。同样，由于我们最终都依赖植物产品来生存，因此叶绿体蛋白质的进口在全球范围内是必不可少的。更重要的是，由于叶绿体在许多经济上重要产品（如油和淀粉）的合成中起着重要作用，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品。

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.