The Role of 54CP in Chloroplast Protein Targeting

叶绿体蛋白转运的主要尚未解决的问题之一是蛋白质如何整合到叶绿体膜的机制。由于在光合作用的光反应中起作用的大多数蛋白质都嵌入到类囊体膜中，因此蛋白质嵌入到这种膜中的过程非常重要。研究人员最近发现，在体外，叶绿体蛋白54CP对于主要光收集蛋白（LHCP）整合到类囊体膜中至关重要。本研究的主要目的是通过检测54CP在体内的功能来验证54CP在叶绿体内蛋白靶向中起基础作用的假设。为了实现这一目标，正在创造54CP含量降低或表达54CP突变形式的植物，这些突变形式赋予了显性负表型。这些植物将使研究人员能够在体内研究54CP在蛋白质靶向中的作用。蛋白质靶向缺陷将通过测量叶片叶绿素含量、分析叶绿体膜的多肽组成、测定叶绿体热休克蛋白的表达以及进行敏感的运输试验来评估。叶片将在体内用35S -Met标记，通过免疫沉淀法确定可溶性和膜组分中特异性类囊体蛋白的分布。这些实验将揭示LHCP的靶向是否严格依赖于54CP通路，以及其他叶绿体蛋白是否需要54CP才能靶向。这些实验也可能揭示替代转运途径是否可以弥补54CP通路的缺陷。第二个目标是阐明54CP功能所需的GTP水解循环的细节。在这方面计划了几种类型的实验。首先，纯化成熟的54CP和鸟嘌呤核苷酸结合区域突变的形式，并测量核苷酸结合和水解活性。其次，这些蛋白质将被预先装载到分离的叶绿体中，并用于研究LHCP在细胞器中的运输。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部位。该蛋白的突变形式可能与54CP依赖性靶向途径的其他组分形成稳定的复合物，并促进其分离。所提出的研究将促进我们对类囊体膜生物发生的认识。一种与54CP相关的蛋白质存在于所有真核生物的细胞质中，并在将蛋白质引导到分泌系统中起作用。在原核生物中也发现了类似的蛋白质，但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进化和功能的方式。植物含有叶绿体，这是一种高度区隔的细胞器，有一层被称为类囊体的内部膜，它将阳光的能量转化为储存在我们食物中的化学能。在光合作用的光反应中起作用的大多数蛋白质都嵌入在这层膜中。叶绿体蛋白转运的主要问题之一是蛋白质如何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证据，证明叶绿体蛋白54CP对于主要光收集蛋白（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验证54CP在叶绿体内蛋白靶向中起基础作用的假设。为了实现这一目标，正在创建转基因植物，减少54CP含量或表达干扰内源性54CP功能的蛋白质突变形式。这些植物将使研究人员能够在体内研究54CP在蛋白质靶向中的作用。这些实验将揭示LHCP的靶向是否严格依赖于54CP通路，以及其他叶绿体蛋白是否需要54CP才能靶向。这些实验也可能揭示替代转运途径是否可以弥补54CP通路的缺陷。所提出的研究将促进我们对类囊体膜生物发生的认识。一种与54CP相关的蛋白质存在于所有真核生物的细胞质中，并在将蛋白质引导到分泌系统中起作用。在原核生物中也发现了类似的蛋白质，但其功能尚未明确确定。因此，拟议的研究应该揭示54p适应精确和有效的机制的方式，这种机制已经进化到将蛋白质传递到特定的细胞内目的地。蛋白质分选是一个必不可少的过程；异常会导致细胞死亡。除了蛋白质靶向在维持生命中的基本重要性之外，对该过程的基本理解具有相关的实际应用。例如，现在有可能将外源基因引入许多生物体。通过了解生物体如何控制蛋白质分类，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，这种应用包括生产基因产品，增强对疾病或压力的抵抗力，提高生产力和营养特性，或合成生物产品，如化学工业的药品和原料。* * *

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. ***