Identifying Targets/Pathways of Chemical Probes for the Plant Endomembrane System

液泡在植物细胞中很突出，是生存所必需的。这个隔室负责储存水果和蔬菜的风味和营养所需的糖、色素、离子、蛋白质和挥发性化合物，并通过调节细胞液的pH来维持细胞的动态平衡。植物液泡分解和回收细胞成分，并参与解毒，酵母和哺乳动物溶酶体也是如此。然而，植物液泡在防御中发挥着额外的功能。液泡是内膜系统的一部分，内膜系统包括内质网、高尔基体、跨高尔基体网络、前液泡隔室、内小体和质膜。这些隔室之间的运输是通过囊泡运输进行的。除了在货物运输中的作用外，植物内膜系统对发育和信号转导也是必不可少的。拟南芥内膜系统中很大比例的基因敲除突变要么是致命的，要么由于完全或部分冗余而不提供可见的表型。然而，使用不同的化学物质来询问分子过程，为快速有效地剖析机制和基因网络提供了一种新的途径，而这种方法是基于突变的方法不可行的。已经确定了三种新的化合物，它们专门干扰膜或可溶性蛋白质到液泡的运输。Sortin1影响液泡的形态和液泡腔蛋白CPY和转化酶的运输。然而，它不影响液泡膜或其他膜蛋白的运输，也不影响高尔基体、内质网或内体的形态。相反，Gravacin干扰蛋白质向液泡膜的转运(以及至少一种质膜蛋白的转运)，但不影响管腔蛋白的输送。另一种名为050的化学物质影响通过内质网的蛋白质的定位，并诱导多个隔室的融合。药理试剂的这种组合是一个强大的资源，可以发现这些途径中贩运机制的新组成部分。现在的挑战是：通过遗传方法确定每种化合物的靶点(S)或/和途径(S)。这一奖项的学术价值在于，将使用我们的试剂识别蛋白质运输机制的新组件。此外，这组不同的化学探针将使科学家能够了解蛋白质运输到液泡的途径与细胞内其他途径之间的关系，这对传统的突变方法来说是一个挑战。更广泛地说，化学基因组学方法可以很容易地转化为任何真核系统，以及具有重要经济意义的植物和人类疾病和营养。一种真正跨学科的方法来了解内膜贩运的机制，产生了一批新的科学家。在过去的几年里，莱科尔实验室的学生和博士后已经在细胞和分子生物学、遗传学、化学和计算科学方面变得精通和经验丰富，所有这些都是确保提案成功并在劳动力中具有竞争力所必需的。该奖项的广泛影响将继续下去，除了科学发现外，还将产生新一代多样化的科学家，他们可以组成21世纪研究和教育所需的互补团队。

The vacuole is prominent in plant cells and is required for viability. This compartment is responsible for storing sugars, pigments, ions, proteins, and volatile compounds necessary for the flavor and nutrition of fruits and vegetables, and it maintains cellular homeostasis by regulating cytosolic pH. Plant vacuoles breakdown and recycle cellular components, and are involved in detoxification, as do yeast and mammalian lysosomes. However, plant vacuoles perform additional functions in defense. The vacuole is part of the endomembrane system, which includes the endoplasmic reticulum, the Golgi apparatus, the trans-Golgi network, pre-vacuolar compartments, endosomes and the plasma membrane. Transport between these compartments occurs via vesicle trafficking. Beyond a role in cargo delivery, the plant endomembrane system is essential for development and signal transduction. A large proportion of knockout mutations in the Arabidopsis endomembrane system are either lethal or provide no visible phenotype due to complete or partial redundancy. However, the use of diverse chemicals to interrogate molecular processes provides a novel avenue for rapid and effective dissection of mechanisms and gene networks in ways not feasible with mutation-based approaches. Three novel compounds have been identified that specifically disrupt the trafficking of membrane or soluble proteins to the vacuole. Sortin1 affects the morphology of the vacuole and the delivery of the vacuolar lumen proteins CPY and invertase. However, it does not affect the delivery of tonoplast or other membrane proteins or the morphology of Golgi, ER or endosomes. In contrast, Gravacin interferes with protein trafficking to the tonoplast (and the trafficking of at least one plasma membrane protein) but does not affect the delivery of lumen proteins. Another chemical, known as 050, affects the localization of proteins that traffic through the ER and induces the fusion of multiple compartments. This combination of pharmacological reagents is a powerful resource to discover new components of the trafficking machinery within these pathways. The challenge now is to: identify target(s) or/and pathway(s) of each compound by genetic approaches. The intellectual merit of this award is that novel components of the protein trafficking machinery will be identified using our reagents. Furthermore, this diverse set of chemical probes will permit scientists to understand the relationships between protein trafficking pathways to the vacuole and other pathways within the cell, a challenge for classical mutational approaches. More broadly, the chemical genomics approach can be easily translated to any eukaryotic system, as well as plants of economic importance and human disease and nutrition. A truly interdisciplinary approach to understand the mechanisms of endomembrane trafficking has produced a new cohort of scientists. During the last several years, students and postdocs in the Raikhel laboratory have become well-versed and experienced in cell and molecular biology, genetics, chemistry and computational sciences, all necessary to ensure the success of the proposal and to become competitive in the workforce. The broad impact of this award will continue by producing, in addition to scientific discoveries, a new generation of diverse scientists who can form complementary teams required for 21st century research and education.