Chloroplast Protein Import in Chlamydomonas reinhardtii

9406540大多数叶绿体和线粒体蛋白质由核基因编码，在细胞质中合成，并在翻译后输入。光合作用细胞必须能够准确地将蛋白质从线粒体和叶绿体中分离出来。靶向特异性是由转运肽介导的，这些转运肽存在于胞浆前体上，并在进入细胞器后不久或期间被移除。在进口过程中与转运肽相互作用的蛋白质可能区分叶绿体和线粒体前体。本项目旨在将莱茵衣藻的分子遗传学和经典遗传学应用于有关叶绿体蛋白输入的问题。稳定的核突变体由于缺乏叶绿体ATPase(CF1-伽马亚基)或叶绿体中的叶绿体蛋白而不能进行光合作用，已经被分离出来，并可以利用相应的野生型结构基因转化为光合作用能力。为了确定衣藻叶绿体蛋白导入所需的转运肽的区域，推测的导入结构域将受到广泛的缺失和定点突变。突变的基因将被重新引入相应的零突变体中，以确定它是否能够补充非光合作用表型。同样的突变将在同源的体外叶绿体蛋白进口试验中进行测试。结果应该给出体内和体外进口所需的转运肽的详细特征图。然后，将建立一个基因选择，以确定能够补偿转运肽突变的基因外抑制子。为了最大限度地减少被恢复的逆转株和转运肽连锁抑制子突变的数量，将使用携带两个特定转运肽突变副本的菌株来分离抑制子，一个副本与塑料蓝蛋白基因关联，另一个与CF1-伽马基因关联。DEP在起始菌株结束时，需要积累一种或两种蛋白质来恢复光合作用。在任何一种情况下，这两种基因产物的积累都将通过免疫印迹分析进行筛选。与原始转运肽无关的抑制子将是最有意义的，因为它们可能识别与转运肽相互作用的蛋白质输入途径的组件。将从候选抑制子中制备叶绿体，并在体外测试它们输入带有相应转运肽突变的前体的能力。克隆在这些抑制子菌株中改变的基因应该确定在进口过程中与转运肽相互作用的蛋白质。这项工作将提供对转运肽功能需求的洞察，并将识别在叶绿体输入过程中与转运肽相互作用的蛋白质。除了少数例外，地球上的生命完全依赖于光合作用，光合作用是利用来自太阳的光能将大气中的二氧化碳和水转化为糖和氧气的过程。在真核植物中，叶绿体是光合作用的亚细胞位置，因此对地球上的生命至关重要。叶绿体是植物细胞内的半自主细胞器，其中光合作用机制与细胞质的其余部分被一组两个同心排列的细胞膜隔开。由于组成光合作用机制的大部分蛋白质是在细胞质中合成的，因此需要一种机制来将这些蛋白质穿过这两层膜带入叶绿体。这种机制必须是选择性的，因为细胞中还有其他膜结合的细胞器，如线粒体和过氧化物体，它们具有不同的功能，执行这些功能所需的蛋白质含量也不同，它们的蛋白质也是从细胞质输入的。这个项目代表了一种非常巧妙的方法来解决两个密切相关的问题：1，在某些蛋白质上，将这些蛋白质特异性地送往叶绿体的“信号”是什么？以及2，叶绿体中哪些蛋白质参与了对该信号的选择性识别，并参与了运往叶绿体的细胞质蛋白的输入？如果成功，该项目将在理解叶绿体生物发生和亚细胞靶向的特异性方面取得重大进展。将获得的信息在农业生物技术中具有明显的潜在用途。***

9406540 Kindle Most chloroplast and mitochondrial proteins are encoded by nuclear genes, synthesized in the cytosol, and imported post translationally. Photosynthetic cells must be able to accurately sort proteins between mitochondria and chloroplasts. Targeting specificity is mediated by transit peptides which are present on cytosolic precursors and removed during or shortly after import into the organelle. Proteins that interact with the transit peptide during the import process presumably discriminate between chloroplast and mitochondrial precursors. This project seeks to apply the molecular and classical genetics of Chlamydomonas reinhardtii to questions concening chloroplast protein import. Stable nuclear mutants that are non-photosynthetic because they lack plastocyanin or the gamma-subunit of the chloroplast ATPase (CF1-gamma) have been isolated and can be transformed to photosynthetic competence using the corresponding wild-type structural gene. To define the region of the transit peptide that is required for chloroplast protein import in Chlamydomonas, the putative import domain will be subject to extensive deletion and site specific mutagenesis. The mutated gene will be reintroduced into the corresponding null mutant to determine whether it is able to complement the non-photosynthetic phenotype. The same mutations will be tested in a homologous in vitro chloroplast protein import assay. The results should give a detailed picture of the features of the transit peptide that are required for in vivo and in vitro import. A genetic selection will then be set up to identify extragenic supressors that are able to compensate for the transit peptide mutation. To minimize the number of revertants and transit peptide-linked suppressor mutations that are recovered, suppressors will be isolated using strains that carry two copies of the specfiic transit peptide mutation, one linked to the plastocyanin gene, the other linked to the CF1-gamma gene. Dep ending on the starting strain, accumulation of one or both proteins will be required to restore photosynthesis. In either case, accumulation of both gene products will be screened by immunoblot analysis. Suppressors that are unlinked to the original transit peptides will be of most interest since they may identify components of the protein import pathway that interact with the transit peptide. Chloroplasts will be prepared from candidate suppressors and tested in vitro for their ability to import precursors with the corresponding transit peptide mutation. The cloning of genes that are altered in such suppressor strains should define proteins that interact with the transit peptide during import. This work will provide insight into the requirements for transit peptide function and will identify proteins that interact with the transit peptide during chloroplast import. %%% With few exceptions, life on earth is completely dependent on photosynthesis, the process whereby light energy from the sun is used to convert atmospheric carbon dioxide plus water into sugar and oxygen. Chloroplasts are the subcellular site of photosynthesis in eukaryotic plants, and as such are critical to life on earth. Chloroplasts are semi-autonomous organelles within the plant cell, in which the photosynthetic machinery is separated from the rest of the cytoplasm by a set of two concentrically arranged cellular membranes. Since most of the proteins which comprise the photosynthetic machinery are synthesized in the cytoplasm, a mechanism is needed to bring those proteins across these two membranes into the chloroplast. The mechanism must be selective, since there are other membrane-bounded organelles in the cell, such as mitochondria and peroxisomes, which have different functions, and different protein contents in order to carry out those functions, and whose proteins are also imported from the cytosol. This project represents a very clever approach to two closely related question s: 1, what is the "signal" on certain proteins which destines those proteins specifically to the chloroplast?; and 2, what are the proteins in the chloroplast which are involved in the selective recognition of that signal and import of cytosolic proteins destined for the chloroplasts? If successful, the project will provide major advances in the understanding of chloroplast biogenesis and specificity of subcellular targeting. The information that will be obtained has obvious potential utility in agricultural biotechnology. ***