IMPORT OF POLYPEPTIDES INTO CHLOROPLASTS

核酮糖-1，5-二磷酸羧基酶的小亚基 捕光叶绿素a/b的组成(AB)多肽 以胞质核糖体为前体合成蛋白质复合体 多肽。这些前体(PRESS和preAB)的特征是 N-末端延伸称为转运肽。我们最近做了 证明了新闻转运肽可以调节细胞周期 异源多肽的翻译后导入叶绿体。这 提案涉及到定义这一观察结果有多普遍 以及转运肽中调节个体的序列 导入步骤。对于这些研究，我们将使用体外表达系统 以产生感兴趣的标记多肽。多肽WE 建议研究包含多种融合多肽的区别 我们将在其中产生的特征和突变多肽 PRESS或preAB转运肽中的缺失。我们会 检查单个多肽进入分离叶绿体的情况 对于那些有缺陷的，我们将确定缺陷是否 在绑定、移位或处理过程中发生。通过这些程序，我们 期望表征转运肽中的介导性结构域 有针对性的进口流程。我们将在这些域中定义序列 通过寡核苷酸引导的突变。拟议的研究将 有助于我们理解多肽的进口过程 转化为细胞器，并将有助于未来对表型的操纵 植物的特征。

The small subunit (SS) of ribulose-1,5-bisphosphate carboxylase and the constituent (AB) polypeptides of the light-harvesting chlorophyll a/b protein complex are synthesized on cytoplasmic ribosomes as precursor polypeptides. These precursors (preSS and preAB) are characterized by an N-terminal extension known as the transit peptide. We have recently demonstrated that the transit peptide for preSS can mediate the posttranslational import into chloroplasts of a foreign polypeptide. This proposal is concerned with defining both how general this observation is and the sequences within the transit peptide that mediate the individual steps of import. For these studies we will use in vitro expression systems to generate the labeled polypeptides of interest. The polypeptides we propose to study include a variety of fusion polypeptides of distinguishing characteristics and mutant polypeptides in which we will have generated deletions within the transit peptide of either preSS or preAB. We will examine the import into isolated chloroplasts for individual polypeptides and for those that are defective we will determine whether the defect occurs with bindings, translocation or processing. By these procedures we expect to characterize domains within the transit peptides that mediate destinct import processes. We will define sequences within these domains by oligonucleotide-directed mutagenesis. The proposed studies will contribute to our understanding of the process of import of polypeptides into organelles and will aid the future manipulation of phenotypic characteristics of plants.