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Genetic and Biochemical Studies of Photosystem II Assembly

Abstract

Photosystem II (PSII) is the protein-pigment complex in oxygenic photosynthesis that uses light energy to catalyze the oxidation of water. How the subunits and cofactors that make up this enzyme are properly assembled into a functional photosystem remains unknown. I sought to find and characterize previously unknown proteins involved in this process by screening a population of insertional mutants of the unicellular green alga Chlamydomonas reinhardtii. One mutant, which I named the second photosystem assembly component (2pac), contained a deletion of a gene encoding a membrane-bound rubredoxin (RBD1) and, as a result, displayed a PSII-specific phenotype. I then characterized plant and cyanobacterial mutants lacking the RBD1 ortholog and found that they too displayed a PSII-specific phenotype.

To uncover the precise role of this rubredoxin in the assembly of PSII, I further characterized the 2pac mutant and found that PSII subunits were translated in the mutant but unstable. I found that the low level of PSII subunits that did accumulate in 2pac were assembled into PSII monomers but not dimers. By analyzing 2D-PAGE gels, I observed a comigration of RBD1 and the PSII subunit CP43, supporting a possible interaction between the two proteins. We attempted to further characterize interacting partners of RBD1 by coexpression of RBD1 and genes encoding PSII subunits in yeast and found an interaction between RBD1 and the PsbI protein. Taken together, the data are consistent with a model in which RBD1 aids in the fusion of a CP43-containing precomplex with the nascent PSII reaction center during formation of the PSII monomer.

Because rubredoxins are known to be redox-active proteins in non-photosynthetic organisms, we hypothesized that RBD1 might be required for a redox modification of PSII during its biogenesis. We tested this by combining the 2pac mutation with a mutation in the gene encoding the chloroplast protease FtsH1 (which is known to degrade damaged or misassembled PSII) in order to accumulate more PSII in the 2pac mutant background for study. We found that PSII did indeed accumulate to a higher level in this double mutant strain (2pac ftsh1-1) than in the 2pac mutant. We also found the PSII in this strain to be much more light-sensitive than a wild-type PSII, supporting our hypothesis. We were unable to resolve any differences in cofactor or subunit composition, but we were able to rule out several candidate subunits and a cofactor.

In order to find other genes like RBD1 that are responsible for PSII function, I devised a phylogenomic approach to search the publicly available genomes of oxygenic photoautotrophs for genes that had a specific phylogenetic signature. I hypothesized that genes required for PSII function would be present in the genomes of all oxygenic photoautotrophs except for the cyanobacterium UCYN-A, which lacks genes for PSII due to its symbiotic relationship with a diatom. The preliminary results indicated that some genes found through this approach might indeed be associated with PSII.

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