ITRAQ analysis of Triton X-100 extractable thylakoid proteins contributing to thermotolerance of PSII in Synechocystis PCC6803
Ontology highlight
ABSTRACT:
INSTRUMENT(S): instrument model, QSTAR Pulsar
ORGANISM(S): Synechocystis Sp. Pcc 6803
SUBMITTER: 
Bill Simon
LAB HEAD: Bill Simon
PROVIDER: PRD000815 | Pride | 2010-02-19
REPOSITORIES: Pride
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Project description:In cyanobacteria and chloroplasts, exposure to HL damages the photosynthetic apparatus, especially the D1 subunit of Photosystem II. To avoid chronic photoinhibition, a PSII repair cycle operates to replace damaged PSII subunits with newly synthesised versions. To determine the sub-cellular location of this process, we examined the localisation of FtsH metalloproteases, some of which are directly involved in degrading damaged D1. We generated transformants of the cyanobacterium Synechocystis sp. PCC6803 expressing GFP-tagged versions of its four FtsH proteases. The ftsH2-gfp strain was functional for PSII repair under our conditions. Confocal microscopy shows that FtsH1 is mainly in the cytoplasmic membrane, while the remaining FtsH proteins are in patches either in the thylakoid or at the interface between the thylakoid and cytoplasmic membranes. HL exposure which increases the activity of the Photosystem II repair cycle led to no detectable changes in FtsH distribution, with the FtsH2 protease involved in D1 degradation retaining its patchy distribution in the thylakoid membrane. We discuss the possibility that the FtsH2-GFP patches represent Photosystem II 'repair zones' within the thylakoid membranes, and the possible advantages of such functionally specialised membrane zones. Anti-GFP affinity pull-downs provide the first indication of the composition of the putative repair zones.
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Project description:Remodeling of thylakoid membranes in response to illumination is an important process for the regulation of photosynthesis. We investigated the thylakoid network from Arabidopsis thaliana using atomic force microscopy to capture dynamic changes in height, elasticity, and viscosity of isolated thylakoid membranes caused by changes in illumination. We also correlated the mechanical response of the thylakoid network with membrane ultrastructure using electron microscopy. We find that the elasticity of the thylakoid membranes increases immediately upon PSII-specific illumination, followed by a delayed height change. Direct visualization by electron microscopy confirms that there is a significant change in the packing repeat distance of the membrane stacks in response to illumination. Although experiments with Gramicidin show that the change in elasticity depends primarily on the transmembrane pH gradient, the height change requires both the pH gradient and STN7-kinase-dependent phosphorylation of LHCII. Our studies indicate that lumen expansion in response to illumination is not simply a result of the influx of water, and we propose a dynamic model in which protein interactions within the lumen drive these changes.
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Project description:The photosynthetic machinery of the cyanobacterium Synechocystis sp. PCC 6803 resides in flattened membrane sheets called thylakoids, situated in the peripheral part of the cellular cytoplasm. Under photosynthetic conditions these thylakoid membranes undergo various dynamical processes that could be coupled to their energetic functions. Using Neutron Spin Echo Spectroscopy (NSE), we have investigated the undulation dynamics of Synechocystis sp. PCC 6803 thylakoids under normal photosynthetic conditions and under chemical treatment with DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea), an herbicide that disrupts photosynthetic electron transfer. Our measurements show that DCMU treatment has a similar effect as dark conditions, with differences in the undulation modes of the untreated cells compared to the chemically inhibited cells. We found that the disrupted membranes are 1.5-fold more rigid than the native membranes during the dark cycle, while in light they relax approximately 1.7-fold faster than native and they are 1.87-fold more flexible. The strength of the herbicide disruption effect is characterized further by the damping frequency of the relaxation mode and the decay rate of the local shape fluctuations. In the dark, local thicknesses and shape fluctuations relax twice as fast in native membranes, at 17% smaller mode amplitude, while in light the decay rate of local fluctuations is 1.2-fold faster in inhibited membranes than in native membranes, at 56% higher amplitude. The disrupted electron transfer chain and the decreased proton motive force within the lumenal space partially explain the variations observed in the mechanical properties of the Synechocystis membranes, and further support the hypothesis that the photosynthetic process is tied to thylakoid rigidity in this type of cyanobacterial cell.
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Project description:Plant chloroplasts originated from an endosymbiotic event by which an ancestor of contemporary cyanobacteria was engulfed by an early eukaryotic cell and then transformed into an organelle. Oxygenic photosynthesis is the specific feature of cyanobacteria and chloroplasts, and the photosynthetic machinery resides in an internal membrane system, the thylakoids. The origin and genesis of thylakoid membranes, which are essential for oxygenic photosynthesis, are still an enigma. Vipp1 (vesicle-inducing protein in plastids 1) is a protein located in both the inner envelope and the thylakoids of Pisum sativum and Arabidopsis thaliana. In Arabidopsis disruption of the VIPP1 gene severely affects the plant's ability to form properly structured thylakoids and as a consequence to carry out photosynthesis. In contrast, Vipp1 in Synechocystis appears to be located exclusively in the plasma membrane. Yet, as in higher plants, disruption of the VIPP1 gene locus leads to the complete loss of thylakoid formation. So far VIPP1 genes are found only in organisms carrying out oxygenic photosynthesis. They share sequence homology with a subunit encoded by the bacterial phage shock operon (PspA) but differ from PspA by a C-terminal extension of about 30 amino acids. In two cyanobacteria, Synechocystis and Anabaena, both a VIPP1 and a pspA gene are present, and phylogenetic analysis indicates that VIPP1 originated from a gene duplication of the latter and thereafter acquired its new function. It also appears that the C-terminal extension that discriminates VIPP1 proteins from PspA is important for its function in thylakoid formation.
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