A Novel Signal Peptide-Based Protein Translocation System in the Thylakoids of Chloroplasts

9419287 Cline“信号肽”靶向序列介导蛋白质在几种进化相关的膜上的转运，从真核生物的内质网到细菌的细胞膜。这项建议涉及到在叶绿体类囊体中发现的新的蛋白质运输机制的特征，这一机制的独特之处在于转位仅由pH梯度驱动。由于针对类囊体腔的蛋白质是在胞质中的叶绿体外合成的，所以有两个膜系统被大量传递，即外部双膜系统和类囊体膜本身。通过外层双膜系统以蛋白质为靶标的信号被从蛋白质中切割出来，揭示出信号肽的第二部分，然后信号肽与类囊体蛋白运输机制或转位酶相互作用。该提案的目的是纯化转位酶，鉴定与转位酶相关的孔复合体，评估蛋白质对该孔的利用，这些蛋白质可以使用不完全由pH梯度驱动的其他转位酶，反之亦然，确定机械在类囊体膜系统上的位置，并探索此类转位酶可能在大肠杆菌中发生的可能性。当代基于信号肽的转运系统是从一个原型机制进化而来的。然而，每个系统都有独特的靶向、初始插入和链转位方法，这些方法反映在特定的成分和转位要求中。在这种情况下，可以预期，一个类囊体系凭借其对pH梯度的单一依赖，已经进化出一种更简单、可能更有效的转位机制。这项研究的结果应该为深入了解pH梯度驱动的易位机制提供依据。它还将提供工具来探索囊体和类囊体膜之间的关系，在类囊体体形成的早期发育阶段。这项拟议的工作将对我们对蛋白质跨膜转运的机制和进化的认识产生影响。***

9419287 Cline "Signal peptide" targeting sequences mediate protein translocation across several evolutionarily related membranes, ranging from the endoplasmic reticulum of eukaryotes to the cell membrane of bacteria. This proposal is concerned with the characterization of the novel protein transport machinery found in chloroplast thylakoids which is unique in that translocation is driven only by a pH gradient. Because proteins targeted to the thylakoid lumen are synthesized outside the chloroplast in the cytosol, there are two membrane systems which much be passed, the outer double membrane system and the the thylakoid membrane itself. The signal which targets the proteins past the outer double membrane system is cleaved from the protein, revealing the second part of the signal peptide which then interacts with the thylakoid protein transport machinery, or translocase. The objectives of the proposal are to purify the translocase, identify the pore complex associated with the translocase, assess the use of this pore by proteins which can use other translocases not driven solely by a pH gradient and vice versa, determine the location of the machinery on the thylakoid membrane system, and explore the possibility that such translocases might occur in E. coli. %%% Contemporary signal peptide-based transport systems have evolved from an archetypal mechanism. Yet each system has unique methods for targeting, initial insertion, and chain translocation, which are reflected by specific components and translocation requirements. In this context, it is expected that one thylakoid system, with its singular dependence on a pH gradient, has evolved a simpler, possibly more efficient mechanism for translocation. Results of this study should provide insight into the mechanism of pH gradient-driven translocation mechanisms. It will also provide tools to explore the relationship between envelope and thylakoids during the early developmental stages of thylakoid forma tion. The work proposed will have impact on our knowledge of the mechnanism and evolution of protein translocation across membranes in general. ***