Probing Plant Endomembrane Pathways with Sortin 1 and Tagged Triazines

与其他真核生物相比，拟南芥含有大量参与运输过程的基因。与酵母不同，许多运输基因对植物来说是必不可少的，而且内膜系统成分中很大比例的敲除突变是致命的。对拟南芥内膜系统的分析表明，除了适当的货物运输外，它对植物发育和信号转导方面也是必不可少的。为了对内膜系统的亚细胞相互作用有新的认识，关键是要研究这些必需基因的功能(S)。化学基因组学(即基因组学规模的化学遗传学)是利用小分子来修饰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而改变。考虑到与基因相比，蛋白质的结构非常复杂，潜在的效应器的数量是巨大的。在实践层面上，化学基因组学需要为感兴趣的过程或基因产物的效应者筛选不同的化学库。这种方法克服了突变致命性的局限性，因为化学物质可以以时空的方式使用，是可调节的，它们的影响经常是可逆的。化学基因组学为经典遗传学提供了另一种选择，经典遗传学经常受到无法识别与基本功能(如膜转运)相关的功能丧失突变的阻碍。在模式植物拟南芥中进行的化学基因组学筛选发现了一种化学物质(Sortin 1)，它可以诱导羧基肽酶Y(CPY)的分泌，这是一种通常通过N末端前肽(NTPP)途径定位于液泡的蛋白质。Sortin1对幼苗液泡的发生和根的发育造成可逆的缺陷。它会导致C末端前肽(CTPP)标记蛋白的异常分泌，但不会导致通过绕过高尔基体的前体小泡靶向的蛋白的异常分泌。本项目将利用生物化学、蛋白质组学、细胞生物学和组合化学来研究Sortin 1对蛋白质靶向途径的影响(S)。该化合物的分子靶标将通过超敏和抗性突变筛选来鉴定；其中两个将进行详细表征。还将采用一种适用于非模式生物的方法，即对标记的三嗪化合物库进行筛选，以寻找干扰液泡靶向的新化学物质。这些化学物质也可以被固定化，用于基于亲和力的快速靶向纯化。该项目的主要目标是查明和分析贩运路径的新组成部分及其与相关信号转导和发展网络的相互作用。它还将提供一套新的方法来研究任何重要的感兴趣的途径。待鉴定的生物活性化学物质将有助于探测保守的过程，如其他生物的内膜运输。在拟南芥中确定的靶标可用于非模式物种，以改善农艺性状和增加作物价值。参与该项目的年轻科学家将在跨学科的化学基因组研究中拥有广泛的综合经验，并将受到良好的培训，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.