Molecular Mechanism of Protein Transport into Chloroplasts

细胞生物学中一个重要的悬而未决的问题是了解细胞质合成的蛋白质是如何靶向于真核细胞的各个隔室的。在植物细胞中，叶绿体为研究这一问题提供了一个极好的系统。大多数叶绿体蛋白在细胞核中编码，在细胞质中合成为更高分子量的前体，并在翻译后通过两个包膜输入。这项研究计划的长期目标是了解氯化塑料蛋白进口的分子细节。关于这一过程的细节，目前的工作将检验两个具体的假设。首先，Hsp93是Hsp100家族中的一种间质分子伴侣，它为前体蛋白在两个被膜上的运输提供了动力。我们将使用两种策略来评估这一假设。第一个将是使用纯化的伴侣蛋白和纯化的前体蛋白建立体外分析，以确定是否可以在体外测量任何预测在蛋白质转位过程中发生的事件。主要焦点将放在Hsp93和前体蛋白之间的相互作用上，但也将研究伙伴蛋白的可能参与。最后，将测量Hsp93的ATPase活性，以试图了解能量在支持可能驱动蛋白质输入的伴侣循环中的作用。第二种策略是进行反向遗传学，以删除或改变Hsp93，并试图确定这些突变在体内和体外的影响。需要评估的第二个假设是，GTP循环在进口过程的早期运作，可能有助于识别前体蛋白所需的特异性。由于目前体外分析的局限性，短期内将采用反向遗传学的策略，以产生缺乏GTP结合蛋白的植物，GTP结合蛋白是外膜受体复合体的一部分，或者这些蛋白发生变化。这些研究的目标将是评估是否存在这样的GTP循环，并确定其对蛋白质输入过程的重要性。理解蛋白质输入到叶绿体中的重要性至少有两个原因。首先，它增加了我们对一个基本的细胞过程，即细胞内蛋白质运输的理解。在过去的二十年里，关于真核细胞内蛋白质运输的重大发现已经取得。由于叶绿体蛋白输入装置的组成与线粒体和内质网运输系统的组成没有序列相似性，因此假设叶绿体系统是独立进化的。如果正确，那么澄清蛋白质运输到叶绿体的细节，并将它们与其他细胞内运输系统进行比较，应该会揭示在收敛进化过程中独立选择的蛋白质运输系统的重要特征。其次，了解蛋白质进入叶绿体对于利用基因工程改变叶绿体中发生的许多代谢过程具有实际意义。植物新陈代谢的成功修饰不仅需要将新的遗传信息引入细胞核并在适当的时间表达，而且基因产物还需要针对其在细胞内的适当位置。因此，了解以叶绿体为靶标的蛋白质具有相当大的现实意义。

A significant unsolved problem in cell biology is to understand how cytoplasmically synthesized proteins are targeted to the various compartments of eukaryotic cells. In plant cells, chloroplasts offer an excellent system for studying this problem. Most chloroplastic proteins are encoded in the nucleus, synthesized in the cytoplasm as higher molecular weight precursors, and imported posttranslationally across the two envelope membranes. The long-term goal of this research program is to understand the molecular details of chloroplastic protein import. Two specific hypotheses will be tested in the current work regarding the details of this process. The first is that Hsp93, a stromal molecular chaperone in the Hsp100 family of proteins, provides the driving force for the transport of precursor proteins across the two envelope membranes. Two strategies will be used to evaluate this hypothesis. The first will be to establish in vitro assays using purified chaperone proteins and purified precursor proteins to determine whether any of the events that are predicted to occur during protein translocation can be measured in vitro. The major focus will be on interactions between Hsp93 and precursor proteins, however the possible involvement of partner proteins will also be investigated. Finally, the ATPase activity of Hsp93 will be measured in an attempt to understand the role of energy in supporting the chaperone cycle that presumably drives protein import. The second strategy will be to perform reverse genetics to either delete or make changes in Hsp93 and attempt to determine the effects of these mutations both in vivo and in vitro. The second hypothesis to be evaluated is that a GTP cycle operates early in the import process, possibly contributing to the specificity needed during the recognition of precursor proteins. Because of the limitations of current in vitro assays, the strategy to be used for the near term will be that of reverse genetics to generate plants either lacking the GTP-binding proteins that are part of the receptor complex in the outer envelope membrane or with alterations in these proteins. The goal of these studies will be to evaluate whether such a GTP cycle exists and to determine its importance for the protein import process.Understanding protein import into chloroplasts is important for at least two reasons. First, it adds to our understanding of a basic cellular process, i.e. intracellular protein trafficking. Significant discoveries regarding protein trafficking within eukaryotic cells have been made in the last two decades. Because the components of the chloroplastic protein import apparatus have no sequence similarity to components from the mitochondrial and ER transport systems, it is hypothesized that the chloroplast system evolved independently. If correct, then clarifying the details of protein transport into chloroplasts and comparing them with other intracellular transport systems should reveal important features of protein trafficking systems that have been selected independently during convergent evolution. Second, understanding protein import into chloroplasts has practical significance for efforts to employ genetic engineering to alter the many metabolic processes that occur in chloroplasts. Successful modifications of plant metabolism requires not only that new genetic information be introduced into the nucleus and expressed at the proper time, but also that gene products be targeted to their proper locations within cells. Thus, understanding protein targeting to plastids has considerable practical significance.