Parallel expression of alternate forms of psbA2 gene provides evidence for the existence of a targeted D1 repair mechanism in Synechocystis sp. PCC 6803 |
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Authors: | Aparna Nagarajan Robert L. Burnap |
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Affiliation: | 1. Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK 74078, USA;2. Department of Biology, Washington University in St. Louis, St. Louis, MO 63108, USA |
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Abstract: | The D1 protein of Photosystem II (PSII) is recognized as the main target of photoinhibitory damage and exhibits a high turnover rate due to its degradation and replacement during the PSII repair cycle. Damaged D1 is replaced by newly synthesized D1 and, although reasonable, there is no direct evidence for selective replacement of damaged D1. Instead, it remains possible that increased turnover of D1 subunits occurs in a non-selective manner due for example, to a general up-regulation of proteolytic activity triggered during damaging environmental conditions, such as high light. To determine if D1 degradation is targeted to damaged D1 or generalized to all D1, we developed a genetic system involving simultaneous dual expression of wild type and mutant versions of D1 protein. Dual D1 strains (nS345P:eWT and nD170A:eWT) expressed a wild type (WT) D1 from ectopic and a damage prone mutant (D1-S345P, D1-D170A) from native locus on the chromosome. Characterization of strains showed that all dual D1 strains restore WT like phenotype with high PSII activity. Higher PSII activity indicates increased population of PSII reaction centers with WT D1. Analysis of steady state levels of D1 in nS345P:eWT by immunoblot showed an accumulation of WT D1 only. But, in vivo pulse labeling confirmed the synthesis of both S345P (exists as iD1) and WT D1 in the dual strain. Expression of nS345P:eWT in FtsH2 knockout background showed accumulation of both iD1 and D1 proteins. This demonstrates that dual D1 strains express both forms of D1, yet only damage prone PSII complexes are selected for repair providing evidence that the D1 degradation process is targeted towards damaged PSII complexes. Since the N-terminus has been previously shown to be important for the degradation of damaged D1, the possibility that the highly conserved cysteine 18 residue situated in the N-terminal domain of D1 is involved in the targeted repair process was tested by examining site directed mutants of this and the other cysteines of the D1 protein. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy. |
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Keywords: | Chl, chlorophyll D1, reaction center protein encoded by the psbA gene DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea EmR, erythromycin resistance cassette Hepes, 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid HBG-11, BG-11 growth medium buffered with Hepes&ndash NaOH pH 8 KmR, kanamycin resistance cassette MES, 2-(n-morpholino) ethanesulfonic acid Mn4CaO5, metal cluster functioning in H2O oxidation PAGE, polyacrylamide gel electrophoresis PSII, Photosystem II P680, primary electron donor in PSII PVDF, poly vinylidene difluoride QA, PSII primary electron acceptor strongly influencing fluorescence yield QB, the secondary quinone acceptor of PSII Yz, redox active tyrosine of the D1 protein acting as a secondary electron donor of the reaction center |
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