Processing the Polyprotein Precursor to Euglena LHCPII

96-30817 Schwartzbach 叶绿体中的大多数蛋白质是核编码的，在细胞质中合成，并在分裂后输入叶绿体。叶绿体蛋白质的前体含有定位和输入叶绿体的拓扑信息。裸藻光系统11叶绿素a/B结合蛋白（pLHCP 11）和核酮糖二磷酸羧化酶小亚基（pSSU）的前体是由保守的十肽共价连接的成熟多肽组成的多聚蛋白。在体内脉冲追踪实验表明，pLHCP 11和pSSU作为整体膜蛋白从内质网（ER）运输到高尔基体之前，叶绿体本地化，而不是被直接输入到叶绿体。一旦多聚蛋白进入叶绿体，它们就被一种内切蛋白水解的多聚蛋白加工肽酶加工。PLHCP 11和pSSU各自含有用于靶向ER的信号肽和该信号肽的终止转移信号C末端。使用犬微粒体和缺失构建体的研究表明，停止转移信号将蛋白质锚定在膜中，N-末端在微粒体腔中，C-末端在细胞质中。前人的工作描述了眼虫叶绿体蛋白的输入和加工途径。本研究的目的是了解这一新的运输途径眼虫叶绿体蛋白的分子机制。这将通过开发一种体外系统来实现，该系统重建高尔基体到叶绿体运输事件。该系统应允许该步骤的生化表征以及该过程所必需的组分的鉴定。这包括多蛋白加工肽酶的分离和表征。 %%% 叶绿体中的大多数蛋白质是核编码的，在细胞质中合成，并在叶绿体中输入。叶绿体蛋白质的前体含有定位和输入叶绿体的拓扑信息。本研究的目的是探讨一种新的运输途径，核编码的叶绿体蛋白质的眼虫叶绿体。叶绿体蛋白质的前体首先靶向内质网，运输到高尔基体，然后运输到叶绿体。没有什么是已知的这种新的运输途径的叶绿体蛋白质的前体的叶绿体的分子机制。 ***

96-30817 Schwartzbach Most of the proteins in chloroplasts are nuclear-encoded, synthesized in the cytoplasm, and imported post-translationally into the chloroplast. Precursors of chloroplast proteins contain topogenic information for targeting to and import into chloroplasts. Precursors of Euglena light harvesting chlorophyll a/b binding protein of photosystem 11 (pLHCP11) and of the small subunit of ribulose bisphosphate carboxylase (pSSU) are polyproteins that are composed of mature polypeptides covalently linked by a conserved decapeptide. In vivo pulse chase experiments demonstrated that pLHCP11 and pSSU are transported as integral membrane proteins from the endoplasmic reticulum (ER) to the Golgi apparatus prior to chloroplast localization rather than being imported directly into the chloroplast. Once the polyproteins are in the chloroplast they are processed by an endoproteolytic polyprotein processing peptidase. PLHCP11 and pSSU each contain a signal peptide for targeting to the ER and a stop-transfer signal C-terminal of the signal peptide. Studies using canine microsomes and deletion constructs demonstrated that the stop-transfer signal anchors the protein in the membrane with the N-terminus in the microsomal lumen and the C-terminus in the cytoplasm. Previous work described the import and processing pathway of Euglena chloroplast proteins. The objective of this research is to understand the molecular mechanism of this novel trafficking pathway for Euglena chloroplast proteins. This will be achieved by developing an in vitro system that reconstitutes the Golgi apparatus to chloroplast transport event. This system should allow for the biochemical characterization of this step as well as for the identification of components that are essential for this process. This includes the isolation and characterization of the polyprotein processing peptidase(s). %%% Most of the proteins in chloroplasts are nuclear-encoded, synthesized in the cytoplasm, and imported post-translationally int o the chloroplast. Precursors of chloroplast proteins contain topogenic information for targeting to and import into chloroplasts. The objective of this research is to investigate a novel trafficking pathway for nuclear-encoded chloroplast proteins to Euglena chloroplasts. The precursors of chloroplast proteins are first targeted to the endoplasmic reticulum, transported to the Golgi apparatus and are then transported to the chloroplast. Nothing is known about the molecular mechanism of this novel trafficking pathway for precursors of chloroplast proteins to the chloroplast. ***