Relationship between a new Membrane Protein Kinase and Thylakoid Protein Translocation

最近已经描述了一些分子，它们影响细胞质合成的蛋白质到细胞膜的正确运输和组装。然而，很少有研究描述蛋白质翻译后插入叶绿体类囊体膜的因素。为了识别在膜转位过程中结合或修饰其他蛋白质的蛋白质，我们开发了一种新的酵母选择方案，该方案使用了酵母转录激活因子GAL4。这种选择有助于分离编码结合蛋白、蛋白水解酶或针对已知靶肽序列的翻译后修饰物的cDNA。通过这个选择方案，我们分离到了一个编码596个氨基酸的多肽(称为PRO25)的拟南芥cDNA，它可以与捕光叶绿素结合蛋白(LHCP)的氨基末端发生遗传作用，LHCP是一种细胞质合成的蛋白质，翻译后插入类囊体膜。拟南芥CDNA编码的蛋白质至少有两个功能结构域，一个与丝氨酸/苏氨酸激酶家族相似，另一个含有表皮生长因子(EGF)重复序列，后者在植物界尚未见报道。核酸杂交和抗体染色表明，该基因及其产物存在于高等植物的其他属中。该基因在绿叶中表达，但在其他植物组织或黄化植物中不表达。多克隆抗血清的初步结果表明，编码的蛋白质可能存在于叶细胞的质膜和类囊体膜上，这表明这两个隔室之间存在有趣的相互作用。这个建议解决了以下问题：1，PRO25在细胞中定位在哪里？2，与LHCP相互作用的激酶或氨基末端结构域重要吗？3，PRO25的功能是什么？第一个问题将使用免疫电子显微镜和Western blotting来解决。这些研究将包括表达的发育概况，以及针对已知蛋白质的已知位置的对照抗血清的使用。还必须确定抗血清是针对一种蛋白质还是针对成员位于多个位置的家族。第二个将使用酵母功能分析并在大肠杆菌中表达，以剖析这两个蛋白结构域。第三个问题将通过在转化的拟南芥中表达和分析PRO25的显性突变来解决，这将提供一个将为其生物学作用提供线索的表型。%这个项目是一个完全出乎意料的发现的结果。因此，它具有很高的投机性，尽管这项研究很可能会带来一些非常有趣和重要的东西，但还不可能预测那个“东西”会是什么。这位研究人员正在研究细胞质编码的植物蛋白如何输入并组装到叶绿体中的问题，并开发了一种聪明的遗传方案，旨在筛选出与特定的进口叶绿体蛋白--捕光叶绿素a/b结合蛋白--相互作用的基因和基因产物。预计该方案将鉴定负责叶绿体中LHCP成熟的LHCP特异性酶。相反，通过选择方案捕获的基因与蛋白酶没有相似之处，而是有两个结构域，其中一个似乎是丝氨酸/苏氨酸激酶(即，一种将磷酸残基添加到蛋白质的丝氨酸和苏氨酸残基上的酶)，另一种似乎与动物的表皮生长因子同源(以前从未在植物中描述过)。这种蛋白的表达似乎依赖于光，这表明它与光合作用有功能联系。在其他高等植物中也发现了同源蛋白，表明其具有高度保守的功能。蛋白质磷酸化是细胞调节蛋白质功能的主要机制，而LHCP已知在叶绿体中被磷酸化。问题是，这种奇特、新奇的植物蛋白质有什么功能？根据这项研究项目中提出的问题的答案，有可能了解到一些关于高等植物如何将阳光转化为食物的非常意想不到和重要的事情。

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.