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

小行星9419287 “信号肽”靶向序列介导蛋白质跨几种进化上相关的膜（从真核生物的内质网到细菌的细胞膜）易位。该建议涉及叶绿体类囊体中发现的新型蛋白质转运机制的表征，该转运机制的独特之处在于仅由pH梯度驱动。由于靶向类囊体腔的蛋白质是在叶绿体外的胞质中合成的，因此有两个膜系统，即外部双膜系统和类囊体膜本身。 靶向通过外部双膜系统的蛋白质的信号从蛋白质上被切割下来，显示出信号肽的第二部分，该第二部分然后与类囊体蛋白转运机制或转位酶相互作用。 该提案的目的是纯化易位酶，鉴定与易位酶相关的孔复合物，评估可以使用其他易位酶的蛋白质对该孔的使用，而不仅仅是由pH梯度驱动，反之亦然，确定类囊体膜系统上的机器的位置，并探索这种易位酶可能存在于E.杆菌 当代基于信号肽的转运系统已经从原型机制进化而来。 然而，每个系统都有独特的靶向、初始插入和链易位方法，这些方法通过特定的组件和易位要求来反映。 在这种情况下，预计一个类囊体系统，其奇异的pH梯度的依赖性，已经演变成一个更简单，可能更有效的机制易位。 这项研究的结果应该提供洞察pH梯度驱动的易位机制的机制。 这也将为探索类囊体形成早期发育阶段被膜与类囊体之间的关系提供工具。 这项工作将对我们了解蛋白质跨膜转运的机制和进化产生影响。 ***

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. ***