Analysis of Functional Regions of a Chloroplast Transit Peptide

以前的工作集中在利用核酮糖-1，5-二磷酸羧化酶的小亚基pSSU作为模型来分析蛋白质进入叶绿体的运输。研究了蛋白质输入所必需的pSSU转运肽(TP)缺失突变的影响。基于这些结果，现在将随着TP的羧基末端和中心区的变化而产生突变。定点突变将被用来改变以前被认为对这些区域的蛋白质运输至关重要的少量特定残基。由于氨基末端区域的功能残基定义较少，该区域的突变将通过随机化学突变产生。将分析突变体的膜结合、加工步骤和运输的ATP要求。将分离位于基质或类囊体膜中的前蛋白。它们将被用来靶向和分离不同的膜受体类别。代表TP功能区的合成肽将用于膜受体组分的分离，并对其进行表征。长期目标是建立一个运输过程的机械化模型。真核细胞的复杂性使得有必要将蛋白质定位于分离代谢途径的特定隔间。通往各种主要亚细胞(和细胞外)隔室的路线在特定方面有所不同。即使在不同类型的单元格之间，排序也可能不同。例如，虽然绿色植物将蛋白质分拣到两个截然不同的双膜封闭的细胞内隔间--线粒体和叶绿体，但确切地说，酵母细胞(缺乏叶绿体)将使用“叶绿体输入信号”将蛋白质靶向线粒体。编码在特定蛋白质的氨基酸序列中的信息被“翻译”成特定的移位到适当的隔间的机制(S)是细胞生物学中的一个基本问题，在本提案中直接涉及到这一问题。

Previous work focussed on analysis of protein transport into chloroplasts using the small subunit of ribulose-1,5-bisphosphate carboxylase, pSSU, as the model. The effects of deletion mutations in the pSSU transit peptide (TP) which is essential for protein import were studied. Based on those results, mutants will now be generated with changes in the carboxyterminal and central regions of the TP. Site-directed mutagenesis will be used to alter a small number of specific residues previously identified as crucial to protein transport in those regions. Since functional residues in the aminoterminal region are less well defined, mutants in this region will be created by random chemical mutagenesis. Mutants will be analyzed for membrane binding, processing steps, and ATP requirements for transport. Preproteins, which are either located in the stroma or in the thylakoid membrane, will be isolated. They will be used to target and isolate different membrane receptor classes. Synthetic peptides representing functional regions of TP will be used for the isolation of membrane receptor components, which will be characterized. The long-term goal is to generate a mechanistic model of the transport process. The complexity of eucaryotic cells makes it necessary to target proteins to specific compartments which separate metabolic pathways. The routes to various major subcellular (and extracellular) compartments differ in specific ways. Even among different types of cells, the sorting may differ. For example, while green plants sort proteins destined for the two distinctly different double-membrane- enclosed intracellular compartments, mitochondria and for chloroplasts, precisely distinctly, yeast cells (which lack chloroplasts) will use the "chloroplast import signal" to target proteins into mitochondria. The mechanism(s) whereby information encoded in the amino acid sequence of specific proteins is "translated" into specific translocation into appropriate compartments is a fundamental problem in cell biology, which is addressed directly in this proposal.