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.
