SP1 and ubiquitin-mediated control of chloroplast protein 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。尽管叶绿体确实含有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种蛋白质中，有90%以上是在细胞核的DNA上编码的。因此，大多数叶绿体蛋白质都是在叶绿体外的细胞质基质中生成的。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。每台机器由几种不同的蛋白质组成，这些蛋白质相互配合以确保进口效率。我们研究的是一种叫做拟南芥的模式植物，它有很多研究优势，比如大量的突变体（每一个都有一个特定基因的突变）。其中一种突变植物ppi1在TOC基因上有缺陷，导致叶绿体蛋白的输入不能有效地工作。几年前，我们发现了另一种称为sp1的突变（这代表“ppi1的抑制因子”），它显着提高了ppi1的蛋白质输入效率。最近，我们发现sp1中的缺陷基因（sp1基因）编码一种称为“泛素E3连接酶”的调节蛋白。这些方法通常是通过标记不需要的蛋白质并将其降解。由于以前不知道这种控制机制在叶绿体中起作用，我们相信我们已经取得了重要的突破。我们认为SP1通过调节TOC机制的数量来控制蛋白质的进口效率。在sp1突变体中，这种控制机制受到一定程度的干扰，使某些TOC蛋白积累到更高的水平，从而提高了蛋白质进口效率。我们将做实验来检验这些理论。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。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，因此对这些细胞器如何发育的更全面的了解将使我们能够提高作物的生产力，或以其他方式操纵其产品。

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 (a relic from their ancient, evolutionary past as free-living photosynthetic bacteria), and so are able to make some of their own proteins, over 90% of the 3000 proteins needed to build a fully functional chloroplast are encoded on DNA in the cell nucleus. Most 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. Each machine is made up of several different proteins which cooperate to ensure the efficiency of import. We work on a model plant called Arabidopsis that has many advantages for research, such as an availability of numerous mutants (each one with a mutation in a specific gene). One such mutant plant, ppi1, has a defect in a TOC gene such that chloroplast protein import does not work efficiently. Several years ago, we identified another mutation called sp1 (this stands for 'suppressor of ppi1'), which significantly improves protein import efficiency in ppi1. Very recently, we discovered that the defective gene in sp1 (the SP1 gene) encodes a type of regulatory protein called a 'ubiquitin E3 ligase'. These usually work by labelling-up unwanted proteins and targeting them for degradation. Because this control mechanism was not previously known to operate in chloroplasts, we believe that we have made an important breakthrough. We think that SP1 controls protein import efficiency by regulating the amount of the TOC machinery. In the sp1 mutant, this control mechanism is disturbed somewhat, allowing certain TOC proteins to accumulate to a higher level, thereby improving protein import efficiency. We will do experiments to test these theories. 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. Because chloroplasts play major roles in the synthesis of many economically important products (such as oils and starch), a more complete understanding of how these organelles develop will enable us to enhance the productivity of crop plants, or otherwise manipulate their products.