Analysis of Functional Regions of a Chloroplast Transit Peptide

以前的工作主要集中在分析蛋白质转运到叶绿体中，使用核酮糖-1,5-二磷酸羧化酶（pSSU）的小亚基作为模型。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，突变体将在TP的羧基末端和中心区域产生变化。位点定向诱变将用于改变先前确定的对这些区域的蛋白质运输至关重要的少数特定残基。由于氨基末端区域的功能残基不太明确，该区域的突变体将通过随机化学诱变产生。将分析突变体的膜结合、处理步骤和运输所需的ATP。将分离出位于基质或类囊体膜中的前蛋白。它们将用于靶向和分离不同的膜受体类。合成肽代表TP的功能区域将用于分离膜受体组分，并对其进行表征。长期目标是生成运输过程的机械模型。真核细胞的复杂性使得有必要将蛋白质靶向到分离代谢途径的特定区室。通往各种主要的亚细胞（和细胞外）区室的途径以特定的方式不同。即使在不同类型的细胞中，分类也可能不同。例如，绿色植物对两种截然不同的双膜封闭细胞内区室（线粒体和叶绿体）的蛋白质进行分类，而酵母细胞（没有叶绿体）将使用“叶绿体输入信号”将蛋白质定位到线粒体中。特定蛋白质氨基酸序列中编码的信息被“翻译”成特定易位到适当区室的机制是细胞生物学中的一个基本问题，本提案直接解决了这一问题。

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.