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

9401840布鲁斯，巴里，D. 这是植物生物学博士后研究员的启动补助金。 在植物和其他真核生物中，核编码基因的翻译可以分为两个不同的途径：一个是蛋白质在与内质网相关的核糖体上翻译，并被指定为ER/高尔基体复合体用于分泌，或用于质膜;第二个途径是蛋白质在细胞质中的游离核糖体上合成。 在植物中，在游离细胞质核糖体上合成的蛋白质可以留在细胞质中，或者通过翻译后途径输入到四种不同的细胞器之一：细胞核，过氧化物酶体，线粒体或叶绿体。这种翻译后输入需要蛋白质的前体形式，其含有将其引导至特定细胞器的靶向序列。 据建议，进口这些前体的一个先决条件是它们保持有进口能力的结构。 今天普遍接受的是，将蛋白质输入线粒体需要它们处于不稳定的构象。 蛋白质构象在输入叶绿体中的作用还不太确定。 这个建议的目标是确定什么定义的叶绿体靶向前体的进口能力，也有哪些胞质因子参与维持这种能力。 核酮糖二磷酸羧化酶的小亚基已被选为解决这个问题的模型。 研究将集中于与叶绿体转运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输入因子，该技术允许通过重组转运激活剂在体内鉴定特定的蛋白质-蛋白质相互作用;以及体外测定可溶性因子在增加核酮糖二磷酸羧化酶的small亚基的前体的输入能力中的作用。 该PI在其博士后研究中对有关蛋白质输入细胞器的问题取得了良好的进展，并获得的结果表明叶绿体输入和线粒体输入的构象要求可能不同。 本项目的目标是研究蛋白质运输到叶绿体的具体要求。 这是PI在Keegstra实验室进行博士后研究期间开始的主题。 他将把这个项目带到田纳西大学诺克斯维尔分校，1994年1月在那里开始终身教职。 因此，NSF植物生物学博士后研究员的启动补助金对于帮助他建立自己的实验室和科学地位至关重要。 ***

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. ***