ASSEMBLY OF CAB POLYPEPTIDES

CAB多肽的组装

• 批准号: 3467735
• 负责人: NEIL E HOFFMAN
• 金额: $ 10.63万
• 依托单位: CARNEGIE INSTITUTION OF WASHINGTON, D.C.
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-07-01 至 1994-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3467735
• 关键词: acylation; autoradiography; binding proteins; biotechnology; chimeric proteins; chlorophyll; fatty acids; fusion gene; genetic manipulation; membrane proteins; membrane reconstitution /synthesis; molecular cloning; phosphorylation; photosynthesis; posttranslational modifications; protein biosynthesis; scintillation counter; thin layer chromatography; tomato

The aim of this study is to define and to characterize those factors which influence the assembly of chlorophyll a/b binding polypeptides (CAs polypeptides; into the light harvesting complexes associated with photosystem I (LHC I) and photosystem II (LHCII). Assembly of CAB polypeptides into LHC involves a series of complicated processes that include apoprotein insertion into the membrane, attachment to pigments and assembly into either LHC I or LHC II asymmetrically located in the membrane. In addition PS II- CAs polypeptides redistribute themselves within the membrane in response to environmental conditions. Our aim is to dissect these complicated processes into understandable and predictable biochemical reactions. We will develop a system in isolated chloroplasts for defining protein domains involved in the assembly of a given CAB polypeptide into LHC I versus LHC II. Chimaeric genes will be constructed between PS I and PS II cab genes. These constructs will be transcribed and translated in vitro and the resulting radiolabeled fusion proteins will be imported, processed and assembled into LHC in organello. Subsequent fractionation of the chloroplasts into photosystem. I and II will enable us to determine which protein domains mediate photosystem specific assembly. We will further exploit the in organello system to test whether phosphorylation of the precursor affects the distribution of the polypeptides into appressed or nonappressed membranes. This question is of relevance because phosphorylation-mediated redistribution of CAB polypeptides is thought to influence the relative excitation of is I and PS II. To complement our in organello approach, we will attempt to develop a subchloroplast system in which we can reconstitute the assembly pathway. Presently a system exists in which a single CAB polypeptide has been inserted in isolated thylakoid membranes supplemented with a stromal extract. We will extend such studies to include the insertion of a wide range of CAB polypeptides, the involvement of fatty acid acylation in this process, and the requirements of pigments for assembly. We will attempt to reconstitute assembly in subthylakoid systems using membranes enriched in either PS I or PS II and artificial membranes reconstituted with reaction centers devoid of LHC. Such studies will help to characterize the machinery required for the integration of CAB polypeptides into the membrane and their assembly into LHC. The aforementioned approaches should help us understand how to account for the nonrandom distribution of proteins within physically continuous membranes.

本研究的目的是定义并描述这些 影响叶绿素 a/b 结合组装的因素 多肽（CAs多肽；进入光捕获复合物 与光系统​​ I (LHC I) 和光系统 II (LHCII) 相关。 将 CAB 多肽组装到 LHC 中涉及一系列步骤 复杂的过程，包括脱辅基蛋白插入 膜、颜料附着并组装成 LHC I 或 LHC II 不对称地位于膜中。 另外 PS II- CAs 多肽在膜内重新分布 对环境条件的反应。 我们的目的是剖析这些 复杂的过程变得可理解和可预测 生化反应。 我们将开发一个分离叶绿体系统来定义 参与给定 CAB 多肽组装的蛋白质结构域 分为 LHC I 与 LHC II。 将构建嵌合基因 PS I 和 PS II cab 基因之间。 这些构造将是 体外转录和翻译以及由此产生的放射性标记 融合蛋白将被导入、加工并组装到 LHC 中 在器官中。 随后将叶绿体分离成 光系统。 I 和 II 将使我们能够确定哪种蛋白质 域介导光系统特异性组装。 我们将进一步 利用细胞器内系统来测试是否磷酸化 前体影响多肽的分布 贴壁或非贴壁膜。 这个问题很有意义 因为 CAB 多肽的磷酸化介导的重新分布 被认为影响 is I 和 PS II 的相对激发。 为了补充我们的细胞器方法，我们将尝试开发 我们可以在其中重建组件的叶绿体下系统 途径。 目前存在一个系统，其中单个 CAB 多肽已插入分离的类囊体膜中 补充基质提取物。 我们将扩大此类研究 包括插入多种 CAB 多肽， 脂肪酸酰化参与该过程，并且 组装颜料的要求。 我们将尝试 使用膜重建亚类囊体系统中的组装 富含 PS I 或 PS II 以及人造膜 用没有大型强子对撞机的反应中心重建。 此类研究 将有助于表征所需的机械 CAB 多肽整合到膜中及其 组装进大型强子对撞机。 上述方法应该对我们有帮助 了解如何解释非随机分布 物理连续膜内的蛋白质。