Defining the scope and components of ubiquitin-dependent chloroplast-associated protein degradation

人类人口正在迅速增长，到2050年将超过90亿。这对粮食安全构成重大挑战，对自然资源造成越来越大的压力。因此，提高作物产量的驱动力以及对次优生长条件的适应力比以往任何时候都更强。为了满足这些需求，必须发展改良作物。通过对模式植物塔勒的研究，我们最近取得了一些重大突破：我们发现了一种新的调控过程，名为“CHLORAD”，它控制着植物生长的重要方面，包括植物对干旱和盐度等环境胁迫的反应。重要的是，改变CHLORAD活性使植物更耐受这种胁迫。在这个项目中，我们将定义CHLORAD的分子靶点和机制，并在此过程中更好地理解它如何用于提供新的作物改良策略。CHLORAD（代表“叶绿体相关蛋白降解”）调节植物细胞内称为叶绿体的结构的发育和运作，叶绿体是正常的细胞成分（即，细胞器）。它们定义了植物，含有绿色色素叶绿素，负责光合作用，利用阳光能量为细胞活动和植物生长提供动力。由于光合作用是能量输入到生命世界的唯一重要机制，叶绿体不仅对植物而且对地球上的所有生命都非常重要。叶绿体在植物对胁迫的反应中也起着关键作用，因此也是工程适应性作物的理想目标。叶绿体由数千种不同的蛋白质组成，其中大部分由细胞核中的基因编码，因此在细胞器外的细胞基质（称为细胞质）中产生。由于叶绿体被双层膜“包膜”所包围，因此需要复杂的机器才能将这些蛋白质导入细胞器。这包括两个膜中的分子机器，称为TOC（“叶绿体外膜上的易位子”）和TIC。目前已知的CHLORAD作用于TOC机制，分解其组成蛋白质，以控制其他蛋白质进入细胞器（这反过来又影响细胞器的发育和运作）。CHLORAD机制的已知成分是称为SP1，SP2和CDC 48的蛋白质。第一种是SP1，它是一种“泛素E3连接酶”，可以标记不需要的蛋白质，并将它们靶向去除。SP2蛋白在叶绿体外膜中形成通道，为去除SP1标记的蛋白质提供出口途径。最后，CDC48是一个分子马达，驱动提取不需要的细胞质，然后在那里他们被分解。我们未发表的结果显示，在CHLORAD中，其他蛋白质（或“辅因子”）与CDC48一起工作。我们相信这些辅因子是将CDC 48对接到不需要的TOC蛋白上以及随后从CDC 48释放这些蛋白所必需的。在这里，我们将详细研究这些辅因子，以更好地了解不需要的叶绿体蛋白质是如何在CHLORAD中被去除的。此外，我们有额外的新结果表明，CHLORAD作用于比以前设想的更多数量的靶蛋白（即，不仅仅是TOC蛋白），包括叶绿体内部的蛋白。我们将系统地识别这些新的目标，并试图了解它们是如何被CHLORAD处理的，即使在细胞器的深处。通过了解目标的身份信息，我们还将探索CHLORAD对植物生长和发育的更广泛的生理意义。总体而言，所获得的知识将增强我们对CHLORAD的理解，并将对提高叶绿体性能的作物的开发具有重要意义。

The human population is growing rapidly and set to exceed 9bn by 2050. This presents significant challenges to food security, and places ever increasing pressure on natural resources. Thus, the drivers for increased crop yields with resilience to sub-optimal growing conditions are stronger than ever. To meet these demands it will be essential to develop improved crops. Through research on the model plant thale cress, we recently made some significant breakthroughs: We discovered a new regulatory process, named "CHLORAD", that controls vital aspects of plant growth, including plant responses to environmental stresses like drought and salinity. Significantly, modifying CHLORAD activity makes plants more tolerant of such stresses. In this project, we will define the molecular targets and mechanisms of CHLORAD, and in so doing develop a better understanding of how it can be used to deliver novel crop improvement strategies.CHLORAD (which stands for "chloroplast-associated protein degradation") regulates the development and operation of structures inside plant cells called chloroplasts, which are normal cellular constituents (i.e., organelles). They define plants, contain the green pigment chlorophyll, and are responsible for photosynthesis, harnessing sunlight energy to power the activities of the cell and the growth of the plant. As photosynthesis is the only significant mechanism of energy-input into the living world, chloroplasts are of huge importance, not just to plants but to all life on Earth. Chloroplasts also have critical roles in plant responses to stress, and so are ideal targets for engineering resilient crops.Chloroplasts are composed of thousands of different proteins, most of which are encoded by genes in the cell nucleus and so are made outside of the organelle in the cellular matrix known as the cytosol. As chloroplasts are surrounded by a double-membrane "envelope", sophisticated machinery is needed to enable the import of these proteins into the organelle. This comprises molecular machines in both membranes, called TOC (for "Translocon at the Outer membrane of Chloroplasts") and TIC. Each machine is composed of several proteins that work cooperatively.Currently, CHLORAD is known to act on the TOC machinery, breaking up its constituent proteins in order to control which other proteins are imported into the organelle (this in turn influences organelle development and operation). Known components of the CHLORAD machinery are proteins called SP1, SP2 and CDC48. The first, SP1, is a "ubiquitin E3 ligase" that labels-up unwanted proteins to target them for removal. The SP2 protein forms a channel in the chloroplast outer membrane, providing the exit route for removal of proteins labelled by SP1. Lastly, CDC48 is a molecular motor that drives extraction of the unwanted to the cytosol, where they are then broken down.Our unpublished results have revealed that additional proteins (or "cofactors") work together with CDC48 in CHLORAD. We believe that these cofactors are required for the docking of CDC48 onto the unwanted TOC proteins, and for the subsequent release of those proteins from CDC48. Here, we will study these cofactors in detail, to better understand how unwanted chloroplast proteins are removed in CHLORAD. Furthermore, we have additional new results showing that CHLORAD acts on a much larger number of target proteins than previously envisaged (i.e., not just TOC proteins), including proteins of the chloroplast interior. We will systematically identify these novel targets, and seek to understand how they are processed by CHLORAD even when deep inside the organelle. Informed by information on the identity of the targets, we will also explore the broader physiological significance of CHLORAD, for plant growth and development.Overall, the knowledge gained will enhance our understanding of CHLORAD, and will be important for the development of crops with improved chloroplast performance.