The Identification and Characterization of Cytosolic FactorsInvolved in Targeting of Nuclear-Encoded Precursors to Higher Plant Chloroplasts

9401840布鲁斯，巴里，D。这是为一位植物生物学博士后研究员提供的创业资助。在植物和其他真核生物中，核编码基因的翻译可以分为两条不同的途径：第一条途径，蛋白质在与内质网相关的核糖体上翻译，并被送往内质网/高尔基复合体分泌或进入质膜；第二条途径，蛋白质在细胞质中的游离核糖体上合成。在植物中，由游离细胞质核糖体合成的蛋白质可以留在细胞质中，也可以通过翻译后途径输入到四种不同的细胞器中：核、过氧化物体、线粒体或叶绿体。这种翻译后的导入需要含有靶向序列的蛋白质的前体形式，该序列将其定向到特定的细胞器。有人提出，进口这些前体的一个先决条件是它们必须保持一种合格的进口配置。今天，人们普遍认为，将蛋白质输入线粒体需要它们处于不稳定的构象。蛋白质构象对进入叶绿体的作用还不是很确定。这项建议的目的是确定什么定义叶绿体靶标前体的输入能力，以及什么细胞质因子参与维持这种能力。核酮糖二磷酸羧基酶的小亚基被选为一个模型来解决这个问题。这项研究将集中于与转运肽相互作用的胞质因子的纯化；通过双杂交克隆技术鉴定反式作用的叶绿体进口因子，允许通过运输激活剂的重组在体内鉴定特定的蛋白质-蛋白质相互作用；以及在体外分析提高二磷酸核酮糖羧基酶SMA 11亚单位前体的进口能力的可溶性因子。在对蛋白质进入细胞器问题的博士后研究中，PI取得了很好的进展，并获得了一些结果，表明叶绿体输入和线粒体输入的构象要求可能不同。这个项目的目标是研究蛋白质运输到叶绿体的特殊要求。这是他在Keegstra实验室进行博士后研究时开始的主题。他将把这个项目带到诺克斯维尔的田纳西大学，在那里他将于1994年1月开始终身教职。因此，这笔为NSF植物生物学博士后研究员提供的启动资金将是帮助他建立自己的实验室和科学地位的关键。***

9401840 Bruce, Barry, D. This is a start-up grant for a Post Doctoral Fellow in Plant Biology. In plants and other eukaryotes, the translation of nuclear encoded genes can be divided into two diverging pathways: one, where proteins are translated on ribosomes associated with the endoplasmic reticulum and are destined either for the ER/Golgi complex for secretion, or for the plasma membrane; and a second pathway, where proteins are synthesized on free ribosomes in the cytoplasm. In plants, proteins synthesized on free cytoplasmic ribosomes can either remain in the cytoplasm, or being imported into one of four different organelles: the nucleus, the peroxisome, the mitochondria or the chloroplast via a post translational pathway. This post translational import requires a precursor form of the protein containing a targeting sequence which directs it to a specific organelle. It has been proposed that one prerequisite for the import of these precursors is that they maintain an import competent configuration. It is commonly accepted today that import of proteins into mitochondria requires that they be in a destabilized conformation. The role of protein conformation on import into chloroplasts is much less certain. The goal of this proposal is to determine what defines the import competence of chloroplast targeted precursors, and also what cytosolic factors are involved in maintaining this competence. The small subunit of ribulose bisphosphate carboxylase has been chosen as a model to address this question. The investigation will focus on purification of cytosolic factors that interact with the transit peptide for chloroplastic transport; on identification of trans-acting chloroplast import factors by the two-hybrid cloning technique, that allows specific protein-protein interactions to be identified in vivo through the reconstitution of a transport activator; and assaying in vitro the soluble factors in increasing import competence of the precursor of the sma ll subunit of ribulose bisphosphate carboxylase. %%% The PI made good progress during his post doctoral research on the problems concerning protein import into organelles, and obtained results which suggest that the conformational requirements for chloroplast import and mitochondrial import may be different. The goal of this project is to investigate the specific requirements for protein transport into chloroplasts. This is the topic that the PI started during his post doctoral research in the Keegstra laboratory. He will be taking the project with him to the University of Tennessee at Knoxville, where he starts a tenure track position in January 1994. This start-up grant for NSF Post Doctoral fellows in Plant Biology therefore will be essential to help him establish his own laboratory, and also scientific status. ***