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(作为自由生活的光合细菌的古代进化历史的遗迹)，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3000种蛋白质中，超过90%是在细胞核的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)，另一个在内被膜上被称为TIC。每台机器由几种不同的蛋白质组成，这些蛋白质相互协作，以确保进口的效率。我们正在研究一种名为拟南芥的模式植物，它在研究中具有许多优势，例如可以获得大量的突变体(每个突变体都有一个特定基因的突变)。一种这样的突变植物ppi1在TOC基因中存在缺陷，以至于叶绿体蛋白输入不能有效地工作。几年前，我们发现了另一种名为SP1的突变(这代表着‘ppi1的抑制因子’)，它显著提高了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)编码一种被称为泛素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针对它们进行降解来实现的。由于这一调控机制以前并不知道在叶绿体中起作用，我们相信我们已经取得了重要突破。我们认为SP1通过调节TOC机制来控制蛋白质的进口效率。在SP1突变体中，这种控制机制受到了一定程度的干扰，使某些TOC蛋白积累到更高的水平，从而提高了蛋白质的进口效率。我们将做实验来检验这些理论。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。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将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 (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.