The Role of Non-Bilayer Forming Lipids in Chloroplast Protein Transport

9604535布鲁斯 技术 这一提议解决了关于蛋白质跨膜转运机制的基本问题。 虽然正在研究的蛋白质运输系统是靶向和易位的核编码的蛋白质进入高等植物叶绿体，潜在的机制是潜在的相关的所有蛋白质运输系统。 主要的焦点是调查的非双层形成脂质，单半乳糖基-二酰基甘油（MGDG），在叶绿体蛋白质运输的作用。 我们建议，叶绿体前体已经发展出特定的结构域在其转运肽，特别是与MGDG相互作用，改变双层组织。 转运肽-脂质相互作用可能是短暂的，时间和空间，但可能代表叶绿体蛋白质运输的重要的第一步，也许在所有的运输过程。 我们的初步数据表明，前体的Rubisco（prSSU）的小亚基包含一个脂质相互作用的结构域，主要位于转运肽（SS-TP）的C-末端。 使用其组成可以控制的脂质体，我们已经通过电子显微镜和荧光测量表明，纯化的prSSU和全长SS-TP破坏双层的能力完全取决于非双层形成脂质MGDG的存在。 本提案中概述的实验是我们对SS-TP/膜相互作用的体外分析的直接延伸。第一个目标是完善负责脂质相互作用/膜不稳定的prSSU序列的映射。 将合成一套完整的重叠肽，其完全跨越SS-tp并延伸到SSU的成熟结构域。以这种方式，将精确地确定对于脂质相互作用而言既必要又足够的prSSU序列，并且将解决成熟结构域中序列的潜在参与。 第二个目标是检查MGDG的化学性质，占其在转运肽介导的膜不稳定的作用。 通过利用含有替代的非双层形成脂质如磷脂酰乙醇胺和心磷脂的脂质体，将有可能区分MGDG的非双层形成性质与其他特征，如qugar头基的氢键合活性。 第三个目的是通过利用一种或多种脂肪酸去饱和酶fad缺陷的拟南芥突变体来改变MGDG在体内的非双层形成趋势。 这些fad突变体对于我们的目的是理想的，因为早期的病毒研究清楚地表明，MGDG仅在其脂肪酸高度不饱和时才倾向于形成非双层结构。 通过利用各种单、双和三重fad突变体，我们可以产生含有MGDG变体的叶绿体，所述MGDG变体被预测在它们形成非双层结构的偏好上变化。 这些突变体将提供最完整的系统，以直接评估非双层结构在叶绿体蛋白质转运中的作用。 非技术性生物膜由脂质双层组成，其中包埋有蛋白质。为了组装细胞内结构，蛋白质必须穿过膜移位。许多研究都致力于确定膜的蛋白质组分，这些组分在蛋白质穿过膜时促进蛋白质的运动和加工。布鲁斯博士将从不同的角度来看待这一过程，他将进行研究，以检查什么脂质成分是必不可少的，以促进叶绿体蛋白质的运输。叶绿体是进行这些研究的有用模型系统，因为叶绿体具有在其他细胞内细胞器中未发现的独特脂质。布鲁斯博士将利用脂质合成的突变体，以提供实验模型来探测蛋白质易位到叶绿体中的过程。这项工作很重要，因为它提供了细胞内细胞器组装的基本过程的基础。 ***

9604535 Bruce Technical This proposal addresses fundamental questions regarding the mechanism of protein translocation across membranes. Although the protein transport system being investigated is the targeting and translocation of a nuclear encoded protein into higher plant chlormplasts, the underlying mechanisms are potentially relevant to all protein transport systems. The primary focus is to investigate the role of the non-bilayer forming lipid, monogalactosyl-diacyglycerol (MGDG), in chloroplast protein transport. We propose that chloroplast precursors have evolved specific domains in their transit peptide that interact specifically with MGDG to alter the bilayer organization. Transit peptide-lipid interactions may be transient, temporally and spatially, yet may represent an essential first step in chloroplast protein transport, and perhaps in all transport processes. Our preliminary data indicates that the precursor to the small subunit of Rubisco (prSSU) contains a lipid interacting domain that is located primarily at the C-terminus of the transit peptide (SS-tp). Using liposomes whose composition can be controlled, we have shown by electron microscopy and fluorescence measurements that the ability of both purified prSSU and the full-length SS-tp to disrupt bilayers is totally dependent on the presence of the non-bilayer forming lipid, MGDG. The experiments outlined in this proposal are a direct extension of our in vitro analysis of the SS-tp/membrane interaction. The first objective is to refine the mapping of prSSU sequences that are responsible for lipid interaction/membrane destabilization. A complete set overlapping peptides will be synthesized that span SS-tp entirely and extend into the mature domain of SSU. In this way, the sequences of prSSU that are both necessary and sufficient for lipid interaction will be determined precisely and the potential involvement of sequences in the mature domain will be addressed. The second objective is to examine the chemical proper ties of MGDG that account for its role in transit peptide-mediated membrane destabilization. By utilizing liposomes containing alternative non-bilayer forming lipids such as phosphatidylethanolamine and cardiolipin, it will be possible to distinguish between the non-bilayer forming property of MGDG vs. other traits, such as hydrogen bonding activity of the qugar head group. The third objective is to alter the non-bilayer forming tendency of MGDG in vivo by utilizing Arabidopsis mutants that are deficient in one or more of their fatty acid desaturases fad. These fad mutants are ideal for our purpose since earlier in viro research clearly indicates that MGDG prefers to form non-bilayer structures only when its fatty acids are highly unsaturated. By utilizing various single, double, and triple fad mutants, we can produce chloroplasts that contain MGDG variants which are predicted to vary in their preferences to form non-bilayer structures. These mutants will ppovide the most intact system to directly evaluate the role of non-bilayer structures in chloroplast protein translocation. Non-technical Biological membranes consist of a bilayer of lipids in which are embedded proteins. In order to assemble intracellular structures it is necessary for proteins to be translocated through the membrane. Much research has been directed at determining the protein components of membranes that facilitate the movement and processing of proteins as they transit through a membrane. Dr. Bruce will look at this process from a different perspective and he will conduct research to examine what lipid components are essential to facilitate trafficking of chloroplast proteins. The chloroplast is a useful model system to conduct these studies because chloroplasts possess unique lipids not found in other intracellular organelles. Dr. Bruce will use mutants of lipid synthesis in order to provide experimental models to probe the process of protein translocation into the chloroplast. This work is important because it provides a basis of the fundamental process of the assembly of intracellular organelles. ***