This proposal is focused on the study of the membrane protein complex Photosystem II (PSII). PSII is an intriguing light-driven molecular machine. Localized in the thylakoid membranes, this pigment-protein complex mediates electron transfer from water to quinones. Even though light is a substrate for PSH, it creates damage that results in the degradation of this protein complex. To survive, photosynthetic organisms have developed elaborate repair systems. The long range goal of this project is to understand the biochemical mechanisms that protect PII from such light-induced damages. Our experimental organism is Synechocystis 6803, a unicellular, naturally transformable cyanobacterium. This proposal contains plans for detailed genetic and biochemical studies of the functional roles of D1 and cytochrome b559, two core polypeptide components of PSII, as well as CtpA, a recently identified carboxyl- terminal processing protease, in the damage-repair cycle of the PSII complex exposed to light.
Our aims are : (1) To elucidate the function of the carboxyl-terminal extension of the D1 protein. Following its synthesis and integration into the PSII complex, the precursor form of D1 is processed, after which PSII becomes catalytically active. We will investigate how the presence of this extension peptide in D1 influences the biogenesis and function of PSII. (2) To dissect the mechanism of CtpA, a novel processing protease that cleaves the C-terminal extension in D1. (3) To use light-tolerant mutants to identify key determinants in the protection of PSII from photodamage. We will generate random mutations in the psbEF gene cluster encoding cyt b559, to isolate Synechocystis mutants that can grow under high light intensities. Characterization of these mutant strains will aid in our understanding of the roles of these two proteins in alleviating light stress. (4) To elucidate the function of cyt b559 in PSII. Our experiments will be aimed at understanding the mechanism of photoprotection mediated by this protein. We will also use second site suppressor analysis to determine the overall function of cyt b559 in PSII. In view of the rapid turnover of individual polypeptide components of PSII constantly facing potential damage by visible light, the current challenge is to understand the mechanisms that carefully control the formation, stability and function of this pigment-protein complex. We will use tools of genetics, biochemistry and spectroscopy to pursue this challenge. These studies will also provide valuable insights about proteolytic degradation and subsequent repair of membrane bound proteins, key processes related to a number of human diseases as well as aging.
