Analysis of Functional Regions of a Chloroplast Transit Peptide

以前的工作集中在分析蛋白质转运到 利用核酮糖-1，5-二磷酸的小亚基的叶绿体 羧化酶，pSSU作为模型。 基因缺失突变的影响 pSSU转运肽（TP）是蛋白质转运所必需的 研究了 基于这些结果，现在将产生突变体 TP的羧基末端和中心区域发生变化。 定点诱变将用于改变少量的 以前被认为对蛋白质转运至关重要的特定残基 在这些地区。 由于氨基末端的功能残基 区域不太明确，该区域的突变体将通过 随机化学诱变 将分析突变体的膜 绑定、处理步骤和运输的ATP要求。 前蛋白，位于基质或类囊体中 膜，将被隔离。 它们将被用来锁定和隔离 不同的膜受体类别。 合成肽代表 TP的功能区将用于膜的分离， 受体成分，这将被表征。 远景目标 是生成一个运输过程的机械模型。 真核细胞的复杂性使得靶向 蛋白质到分隔代谢途径的特定隔室。 各种主要的亚细胞（和细胞外） 隔间在特定方面有所不同。 即使在不同类型的 细胞，排序可能不同。 例如，当绿色植物 两种不同的双膜蛋白， 封闭的细胞内室，线粒体和叶绿体， 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。 氨基酸序列中编码的信息 特定蛋白质的“翻译”成特定的易位， 合适的隔室是细胞生物学中的基本问题， 这一点在本提案中直接提到。

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.