The purpose of this proposal is to understand the relationship between membrane architecture and function in photosynthetic membranes. Our approach will be via discrete, yet correlative, techniques using the unicellular, transformable cyanobacterium Anacystis nidulans R2. The emphasis will be on Photosystem II and the oxygen-evolving apparatus. Our strategy will consist of seven major experimental techniques: (1) the isolation and characterization of an oxygen-evolving complex that contains Photosystem II and phycobilisomes; (2) site-specific labeling and cross-linking of proteins to determine their position in the membrane; (3) reconstitution of photosynthetic electron transport from H2O to NADP+ in liposomes using isolated complexes; (4) isolation of photosynthetic mutants and herbicide-resistant mutants. In addition to temperature-sensitive mutants, strains will be isolated that must be maintained by heterotrophic growth; (5) complementation analysis with cloned genes; (6) cloned photosynthesis genes will be used for biochemical and genetic experiments; and (7) fluorescence analysis of chlorophyllprotein complexes in normal and developing cells. This project is a combined genetic and biochemical study of membrane structure and function and is thus of great benefit to membrane biology in general. The specific information that should be obtained from this proposal includes: (1) the protein composition of the major membrane complexes, such as the O2-evolving apparatus and chlorophyll-proteins; (2) the precise location of proteins in the membrane; (3) the effects of mutation on membrane structure and function; (4) the effect of adding cloned photosynthesis genes to wild-type and mutant cells; (5) the identity of genes coding for photosynthetic proteins; (6) the identity of the chlorophyll components that give rise to the 77 K fluorescence emission peaks; and (7) the mechanism of membrane assembly in iron-deficient cells after iron addition and in heterotrophic cells after light induction. The combination of biochemistry and genetics makes A. nidulans a very valuable organism for the study of bioenergetics and membrane structure.
Showing the most recent 10 out of 14 publications
