The Role of 54CP in Chloroplast Protein Targeting

54CP 在叶绿体蛋白靶向中的作用

• 批准号: 9507745
• 负责人: Neil Hoffman
• 金额: $ 14万
• 依托单位: Carnegie Institution of Washington
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-01 至 1997-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9507745&HistoricalAwards=false
• 关键词: Role; 54CP; Chloroplast; Protein; Targeting

MCB-9507745 Hoffman One of the major unresolved problems of chloroplast protein transport is the mechanism by which proteins are integrated into chloroplast membranes. As most of the proteins that function in the light reactions of photosynthesis are embedded in the thylakoid membrane, the process by which proteins are inserted into this membrane is of great importance. The investigators have recently discovered that a chloroplast protein, 54CP,is essential for the integration of the major light harvesting protein (LHCP) into thylakoid membranes in vitro. The main objective of the present proposal is to test the hypothesis that 54CP plays a fundamental role in intrachloroplast protein targeting by examining the function of the protein in vivo. To accomplish this objective, plants are being created that are reduced in 54CP content or that express mutant forms of 54CP that confer a dominant negative phenotype. Such plants will allow the investigators to study the role of 54CP in protein targeting in vivo. Defects in protein targeting will be assessed by measuring leaf chlorophyll content, analyzing the polypeptide composition of the chloroplast membranes, determining the expression of chloroplast heat shock proteins, and by conducting a sensitive transport assay. Leaves will be in vivo labelled with 35S -Met and the distribution of specific thylakoid proteins in soluble and membrane fractions will be determined by immunoprecipitation. These experiments should reveal whether the targeting of LHCP is strictly dependent on a 54CP pathway and whether other chloroplast proteins require 54CP for targeting. These experiments might also reveal whether alternate transport pathways can compensate for defects in the 54CP pathway. A second objective is to elucidate details of the GTP hydrolysis cycle required for 54CP function. Several types of experiments are planned in this regard. First, mature 54CP and forms mutated in the guanine nucleotide binding domain will be purified and the nucleotide binding and hydrolysis activities measured. Second, these proteins will be preloaded into isolated chloroplasts and used to study LHCP trafficking in organello. If the proteins inhibit transport, further biochemical studies will be conducted to ascertain the site of action. The mutant forms of the protein might form stable complexes with other components of the 54CP dependent targeting pathway and facilitate their isolation. The proposed studies should advance our knowledge of the biogenesis of thylakoid membranes. A protein related to 54CP is found in the cytoplasm of all eukaryotes and plays a role in directing proteins to the secretory system. Similar proteins have also been found in prokaryotes though a function has not been clearly established. Thus the proposed studies should reveal insight into the ways in which 54p has evolved and functions in different organisms and organelles. %%% Plants contain chloroplasts, a highly compartmentalized organelle having an internal membrane called the thylakoid which converts the energy of sunlight into the chemical energy stored in our foodstuffs. Most of the proteins that function in the light reactions of photosynthesis are embedded in this membrane. One of the major unresolved problems of chloroplast protein transport is the mechanism by which proteins are integrated into the thylakoid membrane. The investigators have recently obtained evidence from a cell free system that a chloroplast protein, 54CP, is essential for the integration of the major light harvesting protein (LHCP) into thylakoid membranes. The investigators now propose to test the hypothesis that 54CP plays a fundamental role in intrachloroplast protein targeting by examining the function of the protein in vivo. To accomplish this objective, transgenic plants are being created that are reduced in 54CP content or express mutant forms of the protein that will interfere with endogenous 54CP function. Such plants will allow the investigators to study the role of 54CP in protein targeting in vivo. These experiments should reveal whether the targeting of LHCP is strictly dependent on a 54CP pathway and whether other chloroplast proteins require 54CP for targeting. These experiments might also reveal whether alternate transport pathways can compensate for defects in the 54CP pathway. The proposed studies should advance our knowledge of the biogenesis of thylakoid membranes. A protein related to 54CP is found in the cytoplasm of all eukaryotes and plays a role in directing proteins to the secretory system. Similar proteins have also been found in prokaryotes though a function has not been clearly established.. Thus the proposed studies should reveal insight into the ways in which 54p fits into the accurate and efficient mechanisms which have evolved for delivering proteins to specific intracellular destinations. Protein sorting is an essential process; abnormalities lead to cell death. In addition to the fundamental importance of protein targeting in sustaining life, a basic understanding of the process has relevant practical applications. For example, it is now possible to introduce foreign genes into many organisms. By understanding how organisms control protein sorting, it will be possible to control the precise localization of the proteins expressed from these foreign genes. In plants, such applications include the production of gene products that confer increased resistance to disease or stress, lead to improvements in productivity and nutritional characteristics, or enable the synthesis of bioproducts such as pharmaceuticals and raw materials f or the chemical industry. ***

MCB-9507745 Hoffman 叶绿体蛋白质运输尚未解决的主要问题之一是蛋白质整合到叶绿体膜中的机制。由于大多数在光合作用的光反应中发挥作用的蛋白质都嵌入类囊体膜中，因此蛋白质插入该膜的过程非常重要。研究人员最近发现，叶绿体蛋白 54CP 对于主要光捕获蛋白 (LHCP) 在体外整合到类囊体膜中至关重要。本提案的主要目的是通过检查体内蛋白质的功能来检验 54CP 在叶绿体内蛋白质靶向中发挥基本作用的假设。为了实现这一目标，人们正在培育减少 54CP 含量或表达 54CP 突变体形式的植物，从而赋予显性失活表型。 此类植物将使研究人员能够研究 54CP 在体内蛋白质靶向中的作用。 将通过测量叶片叶绿素含量、分析叶绿体膜的多肽组成、确定叶绿体热休克蛋白的表达以及进行灵敏的转运测定来评估蛋白质靶向的缺陷。叶子将在体内用 35S-Met 标记，并且可溶性和膜级分中特定类囊体蛋白的分布将通过免疫沉淀测定。这些实验应该揭示LHCP的靶向是否严格依赖于54CP途径以及其他叶绿体蛋白是否需要54CP来靶向。 这些实验还可能揭示替代转运途径是否可以弥补 54CP 途径的缺陷。第二个目标是阐明 54CP 功能所需的 GTP 水解循环的细节。在这方面计划进行几种类型的实验。首先，纯化成熟的54CP和鸟嘌呤核苷酸结合结构域突变的形式，并测量核苷酸结合和水解活性。 其次，这些蛋白质将被预加载到分离的叶绿体中，并用于研究细胞器中 LHCP 的运输。如果蛋白质抑制转运，将进行进一步的生化研究以确定作用位点。该蛋白的突变形式可能与 54CP 依赖性靶向途径的其他成分形成稳定的复合物并促进其分离。 拟议的研究应该增进我们对类囊体膜生物发生的了解。与 54CP 相关的蛋白质存在于所有真核生物的细胞质中，并在将蛋白质引导至分泌系统中发挥作用。在原核生物中也发现了类似的蛋白质，尽管其功能尚未明确确定。因此，拟议的研究应该揭示 54p 在不同生物体和细胞器中的进化和功能方式。 %%% 植物含有叶绿体，这是一种高度分隔的细胞器，具有称为类囊体的内膜，可将阳光的能量转化为储存在我们食品中的化学能。 大多数在光合作用的光反应中起作用的蛋白质都嵌入在该膜中。叶绿体蛋白质运输尚未解决的主要问题之一是蛋白质整合到类囊体膜中的机制。研究人员最近从无细胞系统中获得了证据，表明叶绿体蛋白 54CP 对于将主要光捕获蛋白 (LHCP) 整合到类囊体膜中至关重要。研究人员现在提议通过检查体内蛋白质的功能来检验 54CP 在叶绿体内蛋白质靶向中发挥基本作用的假设。 为了实现这一目标，人们正在培育转基因植物，这些植物的 54CP 含量会降低，或者表达会干扰内源 54CP 功能的蛋白质突变形式。 此类植物将使研究人员能够研究 54CP 在体内蛋白质靶向中的作用。 这些实验应该揭示LHCP的靶向是否严格依赖于54CP途径以及其他叶绿体蛋白是否需要54CP来靶向。 这些实验还可能揭示替代转运途径是否可以弥补 54CP 途径的缺陷。 拟议的研究应该增进我们对类囊体膜生物发生的了解。与 54CP 相关的蛋白质存在于所有真核生物的细胞质中，并在将蛋白质引导至分泌系统中发挥作用。 在原核生物中也发现了类似的蛋白质，尽管其功能尚未明确确定。因此，拟议的研究应该揭示 54p 如何适应准确而有效的机制，这些机制已进化为将蛋白质递送到特定的细胞内目的地。 蛋白质分选是一个重要的过程； 异常会导致细胞死亡。除了蛋白质靶向对于维持生命的根本重要性之外，对该过程的基本了解也具有相关的实际应用。 例如，现在可以将外源基因引入许多生物体中。通过了解生物体如何控制蛋白质分选，将有可能控制这些外源基因表达的蛋白质的精确定位。 在植物中，此类应用包括生产基因产物，这些产物可以增强对疾病或应激的抵抗力，从而提高生产力和营养特性，或者能够合成生物产品，例如药物和化学工业的原材料。 ***