Probing Plant Endomembrane Pathways with Sortin 1 and Tagged Triazines

与其他真核生物相比，拟南芥含有大量的基因，预计参与运输过程。与酵母不同，许多运输基因对植物是必需的，并且内膜系统组分中的大部分敲除突变是致命的。拟南芥内膜系统的分析表明，除了适当的货物交付，它是必不可少的植物发育和信号转导方面。为了获得对内膜系统的亚细胞相互作用的新见解，研究这些必需基因的功能至关重要。化学基因组学（英语：Chemical genomics）是利用小分子来改变或破坏特定蛋白质的功能。相比之下，经典遗传学利用突变来破坏基因功能。化学基因组学的概念是蛋白质结构或可以通过化学结合改变。与基因相比，蛋白质的结构极其复杂，因此潜在的效应物数量巨大。在实践层面上，化学基因组学需要筛选不同的化学库，以获得感兴趣的过程或基因产物的效应物。这种方法克服了突变体致死性的局限性，因为化学品可以以时空的方式应用，是可调的，其影响通常是可逆的。化学基因组学提供了一种替代经典遗传学的方法，经典遗传学通常因无法识别与内膜运输等基本功能相关的功能丧失突变而受到阻碍。在模式植物拟南芥中进行的化学基因组学筛选鉴定了一种诱导羧肽酶Y（CPY）分泌的化学物质（分选素1），CPY是一种通常通过N-末端前肽（NTPP）途径靶向液泡的蛋白质。Sortin 1在幼苗中引起液泡生物发生和根发育的可逆缺陷。它导致C-末端前肽（CTPP）标记蛋白的异常分泌，但不导致通过绕过高尔基体的前体囊泡靶向的蛋白质的异常分泌。 本项目中进行的实验利用生物化学、蛋白质组学、细胞生物学和组合化学来研究分选蛋白1对蛋白质靶向途径的影响。该化合物的分子靶点将通过超敏和耐药突变体筛选进行鉴定;其中两个将进行详细表征。一种适用于非模式生物的方法也将被追求，其中标记的三嗪化合物库将被筛选的新的化学物质，干扰液泡靶向。这些化学品也可以被固定用于基于亲和性的快速靶纯化。该项目的主要目标是确定和分析贩运途径的新组成部分及其与相关信号转导和发育网络的相互作用。它还将为研究任何感兴趣的重要途径提供一套新方法。待鉴定的生物活性化学物质将用于探测保守的过程，例如在其他生物体中的内膜运输。在拟南芥中鉴定的靶标可用于非模式物种以改善农艺性状并增加作物价值。参与该项目的年轻科学家将在跨学科化学基因组学研究领域拥有广泛的综合经验，并将接受良好的培训，为独立的学术生涯做好准备。

Compared to other eukaryotes, Arabidopsis contains a large number of genes predicted to be involved in trafficking processes. Unlike yeast, many trafficking genes are essential for plants and a large proportion of knockout mutations in components of the endomembrane system are lethal. Analysis of the Arabidopsis endomembrane system has shown that beyond proper delivery of cargoes, it is essential for aspects of plant development and signal transduction. To gain new insights into the sub-cellular interactions of the endomembrane system, it is critical to examine the function(s) of these essential genes. Chemical genomics (i.e. genomics-scale chemical genetics) is the use of small molecules to modify or disrupt the function of specific proteins. By contrast, classical genetics utilizes mutations to disrupt gene function. The concept of chemical genomics is that protein structure or can be altered by chemical binding. Given the immense structural complexities of proteins compared to genes, the number of potential effectors is vast. At the practical level, chemical genomics entails screening diverse chemical libraries for effectors of a process or a gene product of interest. This approach overcomes the limitations of mutant lethality because chemicals can be applied in a spaciotemporal manner, are tunable and their effects are frequently reversible. Chemical genomics provides an alternative to classical genetics, which is often hampered by an inability to identify loss-of-function mutations associated with essential functions such as endomembrane trafficking. A chemical-genomics screen in the model plant Arabidopsis thaliana identified a chemical (Sortin 1) that induces secretion of carboxypeptidase Y (CPY), a protein normally targeted to vacuoles via the N-terminal propeptide (NTPP) pathway. Sortin1 causes reversible defects in vacuole biogenesis and root development in seedlings. It leads to aberrant secretion of a C-terminal propeptide (CTPP) marker protein but not of a protein that is targeted via precursor vesicles that bypass the Golgi. Experiments to be performed in this project utilize biochemistry, proteomics, cell biology, and combinatorial chemistry to investigate Sortin 1 effects on protein targeting pathway(s). The molecular targets of the compound will be identified via hypersensitive and resistant mutant screens; two will be characterized in detail. An approach applicable to nonmodel organisms will also be pursued in which a tagged triazine library of compounds will be screened for new chemicals that interfere with vacuole targeting. These chemicals can also be immobilized for rapid affinity based target purification. The major goal of this project is to identify and analyze new components of trafficking pathways and their interaction with related signal transduction and developmental networks. It will also provide a suite of new approaches for investigating any essential pathway of interest. The bioactive chemicals to be identified will be useful for probing conserved processes such as endomembrane trafficking in other organisms. Targets identified in Arabidopsis may be used in non-model species to improve agronomic traits and increase crop value. Young scientists involved in this project will have a broad integrated experience within interdisciplinary chemical genomics research and will be well trained and prepared for independent academic careers.