Investigating the roles of Arabidopsis STIC1 and STIC2 in chloroplast protein transport

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，这两种细胞器是从十亿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，并负责光合作用反应（捕获阳光能量并用它来制造糖的过程）。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、蛋白质和淀粉的合成中起着至关重要的作用。虽然叶绿体确实含有DNA（这是它们作为自由生活的光合细菌进化过程中的遗物），因此可以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%被编码在细胞核的DNA上。因此，大多数叶绿体蛋白质是在细胞器外的细胞质基质中形成的，即胞质溶胶。由于每个叶绿体都被一层双层膜或包膜所包围，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）驱动细胞质中蛋白质的进口。这种进口仪器包括两个分子机器：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。每台机器由多种蛋白质组成，相互配合，保证了进口效率。TIC机器的一个特点是它从叶绿体内部或基质中招募一类特殊的蛋白质，称为“伴侣蛋白”。这些基质伴侣就像马达一样，利用ATP的能量来驱动蛋白质的输入。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）。其中一个这样的突变植物tic40在TIC基因上有缺陷，使得叶绿体蛋白的输入不能有效地工作。几年前，我们发现了其他名为stic1和stic2的突变（stic代表“tic40的抑制因子”），它们显著提高了tic40的蛋白质输入效率。最近，我们发现了stic突变影响哪些基因（以及哪些蛋白质）：STIC1属于一个已知的蛋白质转运因子家族，该家族以前不认为在叶绿体包膜中起作用，而STIC2与一组功能未知的细菌蛋白质有关。有趣的是，我们已经证明了STIC2可以与STIC1结合，也可以与基质伴侣和TIC机器结合。因此，我们认为STIC2可能是上述进口电机的新组件。它也可能有助于引导新输入的蛋白质从TIC装置到达它们的最终目的地，这可能是STIC1发挥作用的地方。我们将做实验来检验这些理论。由于叶绿体执行基本功能，并且由于蛋白质输入对叶绿体发育至关重要，因此没有叶绿体蛋白质输入机制的植物无法生存（事实上，它们在胚胎时死亡）并不奇怪。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。由于叶绿体在许多重要经济产品（如油、淀粉）的合成中起着重要作用，因此更好地了解这些细胞器的发育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 over 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 responsible for the reactions of photosynthesis (the process that captures sunlight energy and uses it to make sugars). As 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, as they play essential roles in the synthesis of oils, proteins and starch. Although chloroplasts do contain DNA (a relic from their evolutionary past as free-living photosynthetic bacteria), and so can make some of their own proteins, >90% of the 3000 proteins needed to build a fully-functional chloroplast are encoded on DNA in the cell nucleus. Thus, most chloroplast proteins are made outside of the organelle in the cellular matrix known as the cytosol. As 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 evolved a sophisticated protein import apparatus, which uses energy (in the form of ATP) to drive the import of proteins from the cytosol. This import apparatus comprises two molecular machines: one in the outer envelope membrane called TOC (an abbreviation of "Translocon at the outer envelope of chloroplasts"), and another in the inner envelope membrane called TIC. Each machine is made up of several proteins which cooperate to ensure the efficiency of import. One of the features of the TIC machine is that it recruits a special class of proteins from the chloroplast interior, or stroma, called "chaperones". These stromal chaperones act like a motor as they use the energy from ATP to drive protein 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, tic40, has a defect in a TIC gene such that chloroplast protein import does not work efficiently. Several years ago we identified other mutations called stic1 and stic2 (stic stands for "suppressor of tic40") which significantly improve protein import efficiency in tic40. Recently, we discovered which genes (and therefore which proteins) the stic mutations affect: STIC1 belongs to a family of well-known protein transport factors that were not previously thought to act in the chloroplast envelope, while STIC2 is related to a group of bacterial proteins of unknown function. Interestingly, we have shown that STIC2 can bind to STIC1, as well to stromal chaperones and the TIC machine. Thus, we believe that STIC2 may be a new component of the aforementioned import motor. It may also help to guide newly-imported proteins from the TIC apparatus to their final destination, which is perhaps where STIC1 plays its role. We will do experiments to test these theories. As chloroplasts carry out essential functions, and because protein import is essential for chloroplast development, it is not surprising that plants without a functional chloroplast protein import machinery are unable to survive (in fact, they die as embryos). Similarly, as we are all ultimately dependent upon plant products for survival, it follows that chloroplast protein import is essential on a global scale. As chloroplasts play major roles in the synthesis of many economically important products (e.g., oils, starch), a better understanding of how these organelles develop will enable us to enhance the productivity of crop plants or otherwise manipulate their products.