Protein Import Into the Complex Chloroplasts of Euglena

蛋白质导入眼虫复杂叶绿体

• 批准号: 0080345
• 负责人: Steven Schwartzbach
• 金额: $ 23.2万
• 依托单位: University of Nebraska-Lincoln
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-15 至 2001-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0080345&HistoricalAwards=false
• 关键词: Protein; Import; Into; Complex; Chloroplasts

Chloroplasts are the subcellular organelles of plants that convert light energy from the sun into biologically usable form and generate the atmospheric oxygen without which life as we know it on earth today could not exist. The vital chemical reaction catalyzed by the enzymes in the chloroplasts is the combining of carbon dioxide with water and energy to produce glucose plus molecular oxygen. The glucose is then used by the plant (and any animal that eats the plant) as an energy-rich food that can be utilized either directly or indirectly to fuel metabolism. The compartmentation of chloroplast proteins within the multiply membrane-bounded chloroplast is intrinsically critical to its function. Since most of the chloroplast proteins, including the light-harvesting enzymes, are manufactured in the cytoplasm, the cells have evolved a complex mechanism for transporting these proteins to their proper places in the chloroplast. In higher plants, the chloroplasts contain two envelope membranes, and although not all the details are worked out, there is already a good deal known about the protein translocation machineries in higher plant chloroplasts. However, in certain algal cells, the chloroplasts contain three or even four envelope membranes. Far less is understood about how proteins are translocated into these so-called complex chloroplasts. Understanding the biogenesis of complex chloroplasts has important implications for evolution as well as for our understanding of fundamental mechanisms of eukaryotic protein targetting in general. This project has as its long term goal the understanding of protein targetting and translocation to the complex chloroplasts of the unicellular alga Euglena. Eukaryotic cells target and translocate proteins to specific subcellular compartments in ways that differ from one compartment to another in their details; however, there are some general themes, such as the presence of specific targeting sequences in the protein, that are common to all these processes. Generally, chloroplast protein precursors have an N-terminal presequence, the transit peptide, containing information required for post-translational import into chloroplasts. Precursors to both stromal and thylakoid proteins contain a functionally similar transit peptide that targets the precursor to the stroma through a general import pathway. Protein translocation into the endoplasmic reticulum (ER) is usually co-translational and dependent upon an N-terminal presequence, the signal peptide. Integral membrane proteins contain hydrophobic stop transfer membrane anchor sequences that stop translocation, anchoring the protein within the membrane. Proteins destined for other intracellular compartments are transported in vesicles from the ER to the Golgi apparatus, sorted in the Golgi apparatus and packaged in transport vesicles for transfer to their final intracellular location. Euglena chloroplasts contain 3 envelope membranes. Stromal and thylakoid proteins are transported in vesicles as integral membrane proteins from the ER to the Golgi apparatus to the outermost plastid envelope membrane, rather than directly from the cytoplasm to chloroplast. All Euglena chloroplast protein presequences are 140 amino acids in length with a similar structure composed of a N-terminal signal peptide and a second hydrophobic domain 60 amino acids from the signal peptidase cleavage site. This second hydrophobic domain anchors the protein in the membrane with the presequence N-terminus inside the microsomal lumen and the C-terminus on the cytoplasmic membrane face. A Euglena precursor was imported into pea chloroplasts indicating that the Euglena presequence contains a transit peptide recognized by the higher plant import machinery. The Euglena intermediate and inner envelope should contain proteins homologous to components of the plant envelope import apparatus that interact with the transit peptide. Euglena plastid envelope proteins have not been characterized. The most novel targeting region of the Euglena presequence, the Golgi to chloroplast targeting sequence, is also unidentified. The first objective of this project is to develop procedures to isolate the Euglena chloroplast envelope and identify Euglena chloroplast envelope proteins that are part of the protein import apparatus. Advantage will be taken of the fact that two plant envelope proteins, Toc 34 and Toc159, that interact with the transit peptide are GTP binding proteins. This biochemical property will be used to isolate the Euglena Toc homologues. Peptide sequence will be obtained and used to design degenerate primers for cDNA isolation by PCR. Isolation of the cDNAs represents the first step toward characterization of the Euglena envelope import apparatus. The second objective is to develop a Euglena transformation system that can be used to identify the Golgi to chloroplast targeting domain and the chloroplast import (transit peptide) domains in the Euglena chloroplast protein precursor presequence. The Sh ble gene encoding zeocin resistance fused to the 5' and 3' untranslated regions of the Euglena LHCPII gene will be used as a positive selectable marker allowing optimization of transformation conditions by electroporation or biolistic transformation. Cells will be co-transformed with the Sh ble gene and precursor-GFP fusion protein presequence deletion constructs. Zeocin resistant cells will be screened by fluorescence microscopy for GFP expression and confocal microscopy will be used for an initial determination of intracellular localization. The precursor-GFP fusion proteins will contain the Euglena stromal polyprotein processing peptidase cleavage site so that upon chloroplast import, GFP will be released from the fusion protein providing a simple biochemical assay (western blotting) to confirm import. The third objective is to identify the ancestral function of the Euglena presequence domains by using confocal microscopy to determine the intracellular localization of precursor-GFP fusion protein presequence deletion constructs in mammalian cells.

叶绿体是植物的亚细胞细胞器，它将来自太阳的光能转化为生物可用的形式，并产生大气中的氧气，没有氧气，我们今天所知道的地球上的生命就不可能存在。由叶绿体中的酶催化的重要化学反应是二氧化碳与水和能量结合产生葡萄糖和分子氧。然后，葡萄糖被植物（以及任何以植物为食的动物）用作一种能量丰富的食物，可以直接或间接地用于促进新陈代谢。叶绿体蛋白在多膜结合的叶绿体内的区隔对其功能具有内在的关键作用。由于大多数叶绿体蛋白质，包括光收集酶，都是在细胞质中制造的，细胞已经进化出一种复杂的机制，将这些蛋白质运送到叶绿体中的适当位置。在高等植物中，叶绿体包含两层包膜，尽管并不是所有的细节都被解决了，但对高等植物叶绿体中的蛋白质转运机制已经有了很多了解。然而，在某些藻类细胞中，叶绿体包含三个甚至四个包膜。对于蛋白质是如何转运到这些所谓的复杂叶绿体中，人们了解得还少得多。了解复杂叶绿体的生物发生对进化以及我们对真核蛋白靶向的基本机制的理解具有重要意义。该项目的长期目标是了解蛋白质靶向和转运到单细胞藻类绿藻的复杂叶绿体。真核细胞将蛋白质靶向并转运到特定的亚细胞区室，其方式在细节上不同于另一个区室；然而，有一些普遍的主题，如蛋白质中特定靶向序列的存在，是所有这些过程的共同之处。通常，叶绿体蛋白前体有一个n端前体序列，转运肽，包含翻译后进入叶绿体所需的信息。基质蛋白和类囊体蛋白的前体都含有一种功能相似的转运肽，通过一般的进口途径靶向基质的前体。蛋白质转运到内质网（ER）通常是共翻译的，依赖于n端前序，即信号肽。整体膜蛋白含有疏水停止转移膜锚序列，停止易位，将蛋白质锚定在膜内。前往其他细胞内区室的蛋白质通过囊泡从内质网运输到高尔基体，在高尔基体中进行分类，并包装在运输囊泡中转移到其最终的细胞内位置。绿藻叶绿体含有3个包膜。基质蛋白和类囊体蛋白作为整体膜蛋白在囊泡中从内质网转运到高尔基体再到最外层的质体包膜，而不是直接从细胞质转运到叶绿体。所有绿藻叶绿体蛋白序列长度均为140个氨基酸，结构相似，由一个n端信号肽和距离信号肽酶切割位点60个氨基酸的第二个疏水结构域组成。第二个疏水结构域将蛋白质固定在膜上，其前序n端位于微粒体腔内，c端位于细胞质膜表面。鹰嘴豆前体被导入到豌豆叶绿体中，表明鹰嘴豆前体含有被高等植物导入机制识别的转运肽。绿藻中间膜和内膜应含有与植物膜导入装置相互作用的组分同源的蛋白质。绿藻质体包膜蛋白尚未被表征。绿藻前序列中最新颖的靶向区域——高尔基体到叶绿体的靶向序列也尚未确定。本项目的第一个目标是建立分离绿球藻叶绿体包膜的方法，并鉴定绿球藻叶绿体包膜蛋白，这是蛋白质输入装置的一部分。与转运肽相互作用的两种植物包膜蛋白toc34和Toc159是GTP结合蛋白，这一事实将被利用。这一生化特性将用于分离绿藻Toc同源物。获得多肽序列并用于设计PCR分离cDNA的简并引物。cdna的分离是鉴定绿藻包膜导入装置的第一步。第二个目标是开发一种绿藻转化系统，该系统可用于鉴定绿藻叶绿体蛋白前体序列中的高尔基体到叶绿体靶向结构域和叶绿体输入（转运肽）结构域。将编码zeocin抗性的shble基因融合到Euglena LHCPII基因的5‘和3’非翻译区，将作为一个阳性选择标记，允许通过电孔或生物转化优化转化条件。细胞将与shble基因和前体- gfp融合蛋白前序缺失构建体共转化。Zeocin耐药细胞将通过荧光显微镜筛选GFP表达，共聚焦显微镜将用于细胞内定位的初步确定。前体-绿色荧光蛋白融合蛋白将包含绿藻间质多蛋白加工肽酶切割位点，因此在叶绿体输入时，绿色荧光蛋白将从融合蛋白中释放出来，提供简单的生化试验（western blotting）来确认输入。第三个目标是通过共聚焦显微镜确定哺乳动物细胞中前体- gfp融合蛋白前序列缺失构建体的细胞内定位，确定Euglena前序列结构域的祖先功能。