Assembly of a Multimeric Membrane Protein Complex

许多酶和结构蛋白是由许多相同或不同的亚基组成的多聚体复合体。对于那些位于亚细胞细胞器内的复合体，通常直到细胞质合成的亚基转移到细胞器中才能进行组装。叶绿体的光合放氧酶复合体(OEC)就是由3个核编码多肽和5个叶绿体编码亚基组成的复合体。这个项目的重点是通过将从克隆基因体外合成的三个核编码亚基呈现给分离的完整叶绿体来组装OEC。调查将分两个顺序进行。首先，将优化允许三个外在亚基与PS II有效结合的实验条件，并对新组装的OEC的状态进行评估。不同的条件将检验它们产生新组装的复合体的能力，这些复合体最接近于体内形成的那些。在第二阶段，关于OEC组装的一些机制细节将被阐明，例如叶绿体中存在未组装的亚基池，对叶绿体编码的PS II反应中心蛋白从头合成的要求，以及未组装的亚基在管腔中的寿命。在可能的情况下，这些结果将被用来评估蛋白质复合体组装的一般模型及其调控。活细胞包含复杂的机械来完成它的许多功能。这些复杂的组合是由组成细胞不同隔间的成分(蛋白质)组成的。这些细胞的制造、运输和组装过程控制着细胞的新陈代谢。这个项目研究的是光合作用的复合体，但所阐明的一般原理适用于许多细胞过程。

Many enzymes and structural proteins are multimeric complexes made up of a number of identical or different subunits. For those complexes located within sub-cellular organelles, assembly often does not proceed until the cytoplasmically synthesized subunits are translocated into the organelle. The photosynthetic oxygen-evolving enzyme complex (OEC) of chloroplasts is one such complex which is minimally composed of three nuclear-encoded polypeptides in association with five chloroplast-encoded subunits. This project focuses on the assembly of the OEC in organello by presenting the three nuclear-encoded subunits, synthesized in vitro from cloned genes, to isolated intact chloroplasts. The investigation will proceed in two sequential phases. In the first, the experimental conditions allowing the efficient association of the three extrinsic subunits with PS II will be optimized, and the state of the newly assembled OEC will be evaluated. Different conditions will be examined for their ability to produce newly assembled complexes which most closely resemble those formed in vivo. In the second phase, some of the mechanistic details concerning OEC assembly will be elucidated, such as the presence of pools of unassembled subunits within the chloroplasts, the requirement for de novo synthesis of chloroplast-encoded PS II reaction center proteins, and the lifetimes of unassembled subunits in the lumen. Where possible, these results will be used to evaluate general models of protein complex assembly, and the regulation and control thereof. The living cell contains complex machinery for many of its functions. These complex assemblages are made up of components (proteins) which are made in different compartments of the cell. The process by which these are made, transported and assembled serves to control the cell's metabolism. This project studies a complex of photosynthesis but the general principles being elucidated apply to many cell processes.