Processing the Polyprotein Precursor to Euglena LHCPII

96-30817施瓦茨巴赫叶绿体中的大多数蛋白质都是核编码的，在细胞质中合成，并在翻译后输入到叶绿体中。叶绿体蛋白前体含有针对叶绿体和输入叶绿体的拓扑性信息。光系统11捕光叶绿素a/b结合蛋白(PLHCP11)和核酮糖二磷酸羧化酶小亚基(PSSU)的前体是由成熟的多肽通过保守的十肽共价连接而成的多肽。体内脉冲追逐实验表明，pLHCP11和pSSU在叶绿体定位之前作为完整的膜蛋白从内质网(ER)运输到高尔基体，而不是直接输入到叶绿体中。一旦多蛋白进入叶绿体，它们就会被一种内切蛋白水解性多蛋白加工肽酶处理。PLHCP11和pSSU各自含有靶向内质网的信号肽和信号肽的停止转移信号C端。利用犬微粒体和缺失结构物进行的研究表明，停止转移信号将蛋白质锚定在膜上，N端在微体腔内，C端在细胞质中。前人的工作描述了Euglena叶绿体蛋白的输入和加工途径。本研究的目的是了解裸藻叶绿体蛋白这一新的运输途径的分子机制。这将通过开发一个体外系统来实现，该系统重组高尔基体到叶绿体运输事件。该系统应考虑到这一步骤的生化特征以及对这一过程至关重要的成分的鉴定。这包括多蛋白加工多肽酶的分离和鉴定(S)。叶绿体中的大多数蛋白质都是核编码的，在细胞质中合成，并在翻译后进入叶绿体。叶绿体蛋白前体含有针对叶绿体和输入叶绿体的拓扑性信息。本研究的目的是探索一种新的核编码的叶绿体蛋白向真核藻叶绿体运输的途径。叶绿体蛋白的前体首先被定位于内质网，然后被运输到高尔基体，然后被运输到叶绿体。关于叶绿体蛋白前体向叶绿体的这种新的运输途径的分子机制尚不清楚。***

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. ***