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

研究 ClpC/Hsp100 型伴侣在叶绿体前蛋白输入中的功能

• 批准号: BB/J017256/1
• 负责人: Paul Jarvis
• 金额: $ 45.58万
• 依托单位: University of Leicester
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2012
• 资助国家: 英国
• 起止时间: 2012 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FJ017256%2F1
• 关键词: Investigating; function; ClpC; Hsp100; type

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.

叶绿体和线粒体是许多细胞的正常组成部分 - 它们是称为细胞器的亚细胞结构。有趣的是，这两种细胞器是从十亿多年前被其他细胞吞噬的细菌进化而来的，并且在许多方面它们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，专门负责光合作用反应（捕获阳光能量并利用其为细胞活动提供动力的过程）。由于光合作用是向生命世界输入能量的唯一重要机制，因此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发挥着重要作用。尽管叶绿体确实含有 DNA（这是它们作为自由生活的光合细菌的古老进化过程中的遗物），因此能够制造一些自己的蛋白质，但在构建完整的叶绿体所需的 3000 种左右蛋白质中，90% 以上是叶绿体中的 DNA。功能性叶绿体由细胞核内的 DNA 编码。因此，大多数叶绿体蛋白是在叶绿体外部、称为胞质溶胶的细胞基质中产生的。由于叶绿体均被双层膜或包膜包围，而蛋白质的被动运动不受其影响，因此这提出了一个重大问题。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，穿过包膜，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：一个位于外被膜中，称为 TOC（“叶绿体外被膜 Translocon”的缩写），另一个位于内被膜中，称为 TIC。该项目的重点是 TIC 机器，特别是与 TIC 复合物相关的一种名为 Hsp93 的蛋白质。这种 Hsp93 蛋白是一种 ATP 酶（即它水解 ATP 以释放能量），并且是称为“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。在此过程中，一些伴侣可以对目标蛋白施加“拉力”，以促进其从一个位置到另一个位置的传递。基于多项证据，Hsp93被认为为叶绿体蛋白质输入提供驱动力，并通过拉动那些需要输入的蛋白质来发挥作用（即，它被认为是所谓的“叶绿体蛋白质的核心部分”）蛋白质输入马达”）。因此，导入机制期间发生的大部分 ATP 消耗暂时归因于 Hsp93。然而，这些假设仍然缺乏直接证据。我们建议通过操纵完整植物中 Hsp93 蛋白的活性来直接测试这些想法，并评估此类操作对叶绿体蛋白输入效率的影响。由于叶绿体执行基本功能，并且蛋白质输入对于叶绿体发育至关重要，因此，没有功能性叶绿体蛋白质输入机制的植物无法生存（事实上，它们在胚胎阶段就死亡）也就不足为奇了。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生存，因此叶绿体蛋白的进口在全球范围内至关重要。更重要的是，由于叶绿体在许多重要经济产品（例如油和淀粉）的合成中发挥着重要作用，因此更全面地了解这些细胞器如何发育可能使我们能够提高农作物的生产力，或以其他方式操纵它们产品。

项目成果

Evolutionary, molecular and genetic analyses of Tic22 homologues in Arabidopsis thaliana chloroplasts.

• DOI: 10.1371/journal.pone.0063863
• 发表时间: 2013
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Kasmati AR;Töpel M;Khan NZ;Patel R;Ling Q;Karim S;Aronsson H;Jarvis P
• 通讯作者: Jarvis P

Methods in Molecular Biology: The isolation of plant organelles and structures, methods and protocols

分子生物学方法：植物细胞器和结构的分离、方法和方案

• 发表时间: 2017
• 作者: Flores-Perez U

Functional Analysis of the Hsp93/ClpC Chaperone at the Chloroplast Envelope

• DOI: 10.1104/pp.15.01538
• 发表时间: 2016-01-01
• 期刊: PLANT PHYSIOLOGY
• 影响因子: 7.4
• 作者: Flores-Perez, Ursula;Bedard, Jocelyn;Jarvis, Paul
• 通讯作者: Jarvis, Paul

Genetic and Physical Interaction Studies Reveal Functional Similarities between ALBINO3 and ALBINO4 in Arabidopsis.

遗传和物理相互作用研究揭示了拟南芥中 ALBINO3 和 ALBINO4 之间的功能相似性。

• DOI: 10.1104/pp.15.00376
• 发表时间: 2015
• 期刊: Plant physiology
• 影响因子: 7.4
• 作者: Trösch R