Relationship between a new Membrane Protein Kinase and Thylakoid Protein Translocation

一种新的膜蛋白激酶与类囊体蛋白易位的关系

• 批准号: 9218931
• 负责人: Bruce Kohorn
• 金额: $ 23.11万
• 依托单位: Duke University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-07-01 至 1997-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9218931&HistoricalAwards=false
• 关键词: Relationship; between; new; Membrane; Protein

A number of molecules have recently been described that effect the correct transport and assembly of cytoplasmically synthesized proteins to cellular membranes. Few studies, however, have described factors involved in the post-translational insertion of proteins into the chloroplast thylakoid membrane. To identify proteins that bind or modify other proteins during the process of membrane translocation, we developed a new yeast selection scheme that employs the yeast transcriptional activator, GAL4. This selection facilitates the isolation of cDNAs that encode binding proteins, proteases, or post-translational modifiers specific for known target peptide sequences. With this selection scheme we isolated an Arabidopsis cDNA encoding a 596 amino acid polypeptide (termed PRO25) that can genetically interact with the amino terminus of a light harvesting chlorophyll binding protein (LHCP), a cytoplasmically synthesized protein that is post-translationally inserted into the thylakoid membrane. The Arabidopsis CDNA encodes a protein with at least two functional domains, one with similarity to the family of serine/threonine kinases and another that contains an epidermal growth factor (EGF) repeat, the latter of which has not been previously reported in the plant kingdom. Nucleic acid hybridization and antibody staining indicate that this gene and its product are present in other genera of higher plants. The mRNA is expressed in green leaves but not in other plant tissues or in etiolated plants. Preliminary results with polyclonal antisera indicate that the encoded protein may be present on both the plasma membrane and thylakoid membrane of leaf cells, suggesting an intriguing interaction between the two compartments. This proposal addresses the following questions: 1, where in the cell is PRO25 localized?; 2, is the kinase or amino-terminal domain important for interaction with LHCP?; and 3, what is the function of PRO25? The first question will be addressed using immunoelectron microscopy and Western blotting. These studies will include developmental profiles of expression, and the use of control antisera to known proteins of defined location. It must also be determined whether the antisera are specific to one protein or to a family whose members are in multiple locations. The second will be addressed using the yeast functional assay and expression in E. coli to dissect the two protein domains. The third will be addressed by expression and analysis of dominant mutations of PRO25 in transformed Arabidopsis, which should provide a phenotype that will provide a clue to its biological role. %%% This project is the result of a totally unexpected finding. As such, it is highly speculative, and although it is likely that the research will lead to something very interesting and important, it is not yet possible to predict what that "something" will be. The investigator was studying the problem of how cytoplasmically- encoded plant proteins get imported and assembled into chloroplasts, and developed a clever genetic scheme which was intended to fish out genes and gene products which interacted with a specific imported chloroplast protein, the Light Harvesting Chlorophyll a/b Binding Protein. It was expected that this scheme would identify LHCP-specific proteases responsible for LHCP maturation in the chloroplast. Instead, the gene that was fished out by the selection scheme had no resemblance to a protease, but rather had two domains, one of which appears to be a serine/threonine kinase (i.e., an enzyme that adds phosphate residues to serine and threonine residues of proteins) and the other of which appears to be homologous to the epidermal growth factor of animals (which had never been described in plants before). Expression of this protein appears to be light-dependent, suggesting a functional link to photosynthesis. Homologous proteins have been found in other higher plants, suggesting a highly conserved function. Protein phosphorylation is a major mechanism for cellular regulation of protein functions, and the LHCP is known to get phosphorylated in the chloroplast. The question is, what is the function of this curious, novel, plant protein? Depending on the answers to the questions addressed in this research project, there is the potential to learn some very unexpected and important things about how higher plants convert sunlight into foodstuff.

最近有一些分子被描述为 细胞质合成的蛋白质的正确运输和组装 蛋白质到细胞膜。 然而，很少有研究 描述了参与翻译后插入的因素， 蛋白质进入叶绿体类囊体膜。 以识别 在合成过程中结合或修饰其他蛋白质的蛋白质 膜易位，我们开发了一种新的酵母选择方案， 它使用了酵母转录激活因子GAL 4。 这 选择促进编码结合的cDNA的分离 特异性的蛋白质、蛋白酶或翻译后修饰物， 已知的靶肽序列。 通过这个选择方案，我们 分离了一个拟南芥cDNA，编码596个氨基酸的多肽 （称为PRO25），可以与氨基酸相互作用， 捕光叶绿素结合蛋白（LHCP）的末端， 一种细胞质合成的蛋白质， 插入到类囊体膜中。 拟南芥cDNA编码 至少有两个功能域的蛋白质，其中一个具有相似性 属于丝氨酸/苏氨酸激酶家族， 表皮生长因子（EGF）重复序列，后者具有 以前在植物界没有报道过。 核酸 杂交和抗体染色表明，该基因及其 产物存在于其他高等植物属中。 将mrna 在绿色叶中表达，但在其它植物组织中不表达， 黄化的植物 多克隆抗血清的初步结果 这表明编码的蛋白质可能存在于血浆 膜和类囊体膜，表明 两个隔间之间的有趣互动。 这项建议 解决了以下问题：1，PRO25在细胞中的位置 本地化？2，激酶或氨基末端结构域对 与LHCP的相互作用？PRO25的功能是什么？ 的 第一个问题将使用免疫电子显微镜来解决 和Western印迹。 这些研究将包括发展 表达谱，以及使用已知的 确定位置的蛋白质。 还必须确定是否 所述抗血清特异于一种蛋白质或家族，所述家族 成员分布在多个地点。 第二个问题将在 利用酵母功能测定和在E.杆菌以 分解两个蛋白质结构域。 第三个问题将由 PRO25基因显性突变的表达和分析 转化的拟南芥，这应该提供一个表型， 为它的生物学作用提供了线索 %%% 这个项目是一个完全出乎意料的发现的结果。 作为 这是高度投机性的，虽然很可能， 研究将导致一些非常有趣和重要的事情，它 现在还不可能预测那个“东西”会是什么。 的 研究人员正在研究细胞质如何- 编码的植物蛋白被输入并组装成 叶绿体，并开发了一个聪明的遗传方案， 目的是找出基因和基因产物， 一种特定的输入叶绿体蛋白，光收集 叶绿素a/B结合蛋白。 据估计，这一计划 将确定LHCP特异性蛋白酶负责LHCP 在叶绿体中成熟。 相反， 通过选择方案，它与蛋白酶没有相似之处，但 而有两个领域，其中一个似乎是一个 丝氨酸/苏氨酸激酶（即，一种能将磷酸盐 蛋白质的丝氨酸和苏氨酸残基）和 另一种似乎与表皮生长同源 动物因子（从未在植物中描述过 之前）。 这种蛋白质的表达似乎是光依赖性的， 这表明了与光合作用的功能性联系。 同源 在其他高等植物中也发现了这种蛋白质， 高度保守的功能。 蛋白质磷酸化是 蛋白质功能的细胞调节机制，以及 已知LHCP在叶绿体中被磷酸化。 的 问题是，这种奇怪的、新奇的植物的功能是什么？ 蛋白质？ 取决于对 这个研究项目，有可能学到一些非常 关于高等植物如何转化 阳光进入食物。