Excitation of the membrane-bound protein complexes photosystem I (PSI) and II (PSII) by light must be optimized to ensure the highest efficiency of photosynthetic electron transport. Redistribution of excitation energy between both photosystems as an immediate and dynamic response to changing illumination conditions occurs during the process termed 'State transitions', where State 1 is induced by excess PSI light and State 2 by excess PSII light [1]. State 1 to State 2 transition occurs in response to the reduction of the plastoquinone pool, triggering the activation of thylakoid-bound kinases which in turn phosphorylate the mobile light-harvesting complex II (LHCII) antenna [2][3][4][5]. The phosphorylated LHCII is proposed to transfer physically from PSII to PSI to balance energy distribution between, and optimize the rate of electron transfer through, the two photosystems or induce cyclic electron flow around PSI [6][7][8][9]. Conversely, in PSI-favouring light, oxidation of plastoquinone occurs, leading to deactivation of LHCII-specific kinases and dephosphorylation of mobile LHCII by redox-independent phosphatases. As a consequence, LHCII detaches from PSI and functionally couples to PSII (State 2 to State 1 transition). Recent studies of the mutants that were blocked in State 1 revealed that thylakoid protein kinase Stt7 from green alga Chlamydomonas reinhardtii and its higher plant orthologue STN7 are required for phosphorylation of several LHCII polypeptides [4,5], thus providing further evidence that protein phosphorylation is essential for State transitions. The State 1 to State 2 transition in the photosynthetic membranes of plants and green algae involves the functional coupling of phosphorylated lightharvesting complexes of photosystem II (LHCII) to photosystem I (PSI). We present evidence suggesting that in Chlamydomonas reinhardtii this coupling may be aided by a hyper-phosphorylated form of the LHCII-like CP29 protein (Lhcbm4). MS analysis of CP29 showed that Thr6, Thr16 and Thr32, and Ser102 are phosphorylated in State 2, whereas in State 1-exposed cells only phosphorylation of Thr6 and Thr32 could be detected. The LHCI-PSI supercomplex isolated from the alga in State 2 was found to contain strongly associated CP29 in phosphorylated form. Electron microscopy suggests that the binding site for this highly phosphorylated CP29 is close to the PsaH protein. It is therefore postulated that redoxdependent multiple phosphorylation of CP29 in green algae is an integral part of the State transition process in which the structural changes of CP29, induced by reversible phosphorylation, determine the affinity of LHCII for either of the two photosystems.Abbreviations Chl, chlorophyll; DDM, b-dodecyl maltoside; EM, electron microscopy; IMAC, immobilized metal affinity chromatography; LHCII, lightharvesting complex II; PSI, photosystem I; PSII, photosystem II; S1 and S2, State 1 and State 2.

Mapping of in vivoThe thylakoid membranes in chloroplasts of plants and green algae carry out oxygenic photosynthesis. Two multisubunit pigment-containing protein complexes, photosystem II (PSII) 1 and photosystem I (PSI), located in this membrane system work in series to generate an electrochemical potential gradient of protons across the membrane following vectorial electron flow from PSII to PSI via the cytochrome b 6 f complex. PSII uses light to oxidize water, whereas PSI, via a second photoact, uses reducing equivalents derived from PSII to reduce NADP ϩ to NADPH. The electrochemical potential gradient of protons is used to power conversion of ADP to ATP (1). Several thylakoid membrane proteins that make up the PSII complex and its LHCII (light-harvesting chlorophyll a/b-binding proteins of PSII) antennae undergo light-and redox-dependent phosphorylation (2, 3) as discovered more than 2 decades ago (4 -6). Phosphorylation of LHCII in plants and algae controls photosynthetic state transitions, which optimize efficient use of the absorbed light energy by both photosystems. Thus, in State 1 more energy is transferred to PSII, whereas in State 2 a proportion of the excitation energy is redistributed to PSI (7-9). The essential role of protein phosphorylation in state transitions has recently been proven in the studies using mutants of Arabidopsis thaliana plants and the green alga Chlamydomonas reinhardtii deficient in protein kinases STN7 (9) and Stt7 (8), respectively. However, despite the generally assumed similarity in thylakoid protein phosphorylation between plants and algae and a high homology between the plant STN7 and the algal Stt7 protein kinases (8), the extent of photosynthetic state transitions differs between these species. In plant thylakoids, only 15-20% of LHCII participates in the lateral migration between the photosystems, whereas up to 80% of the excitation energy absorbed by the LHCII antenna can be redistributed from PSII to PSI in green alga (8 -10).

Unavoidable side reactions of photosynthetic energy conversion can damage the water-splitting photosystem II (PSII) holocomplex embedded in the thylakoid membrane system inside chloroplasts. Plant survival is crucially dependent on an efficient molecular repair of damaged PSII realized by a multistep repair cycle. The PSII repair cycle requires a brisk lateral protein traffic between stacked grana thylakoids and unstacked stroma lamellae that is challenged by the tight stacking and low protein mobility in grana. We demonstrated that high light stress induced two main structural changes that work synergistically to improve the accessibility between damaged PSII in grana and its repair machinery in stroma lamellae: lateral shrinkage of grana diameter and increased protein mobility in grana thylakoids. It follows that high light stress triggers an architectural switch of the thylakoid network that is advantageous for swift protein repair. Studies of the thylakoid kinase mutant stn8 and the double mutant stn7/8 demonstrate the central role of protein phosphorylation for the structural alterations. These findings are based on the elaboration of mathematical tools for analyzing confocal laser-scanning microscopic images to study changes in the sophisticated thylakoid architecture in intact protoplasts.confocal microscopy | macromolecular crowding | photosynthesis P hotosynthetic transformation of sunlight into metabolic energy equivalents is a dangerous venture, because toxic side products of the primary photochemical processes can lead to uncontrolled damage. Harmful photosynthetic side reactions cannot be avoided completely and become a serious problem under stress (e.g., high light stress). The main target of photoinhibition (PI) is the D1 subunit of the water-splitting photosystem II (PSII) (1, 2). The D1 subunit is buried in the massive (1,400 kDa) PSII holocomplex that is organized as a dimer and binds between two and four trimeric light-harvesting complex IIs (LHCIIs) (3, 4). Estimates predict that without repair of damaged PSII, the efficiency for photosynthetic energy conversion would drop below 5% (5). Consequently, plants would not survive in a highly dynamic and competitive natural environment. Plants address this challenge through the evolutionary invention of one of the fastest and most efficient molecular repair mechanisms in nature, the PSII repair cycle (5-7).The PSII repair cycle consist of a series of events, including phosphorylation/ dephosphorylation of PSII subunits, holocomplex disassembly/reassembly, and D1 degradation/de novo synthesis (8-10). All of these reactions are harbored in or at the thylakoid membrane system inside the chloroplast. The complex folding of the thylakoid membrane leads to a structural differentiation into stacked grana regions interconnected by unstacked stroma lamellae (11)(12)(13). This structural heterogeneity is accompanied by, and partly driven by, differential protein distributions. PSII and LHCII are concentrated in grana stacks, photosystem I (PSI) and the ATPas...

Photosynthetic organisms are able to adapt to changes in light conditions by balancing the light excitation energy between the light-harvesting systems of photosystem (PS) II and photosystem I to optimize the photosynthetic yield. A key component in this process, called state transitions, is the chloroplast protein kinase Stt7/STN7, which senses the redox state of the plastoquinone pool. Upon preferential excitation of photosystem II, this kinase is activated through the cytochrome b 6 f complex and required for the phosphorylation of the light-harvesting system of photosystem II, a portion of which migrates to photosystem I (state 2). Preferential excitation of photosystem I leads to the inactivation of the kinase and to dephosphorylation of light-harvesting complex (LHC) II and its return to photosystem II (state 1). Here we compared the thylakoid phosphoproteome of the wild-type strain and the stt7 mutant of Chlamydomonas under state 1 and state 2 conditions. This analysis revealed that under state 2 conditions several Stt7-dependent phosphorylations of specific Thr residues occur in Lhcbm1/Lhcbm10, Lhcbm4/Lhcbm6/Lhcbm8/Lhcbm9, Lhcbm3, Lhcbm5, and CP29 located at the interface between PSII and its light-harvesting system. Among the two phosphorylation sites detected specifically in CP29 under state 2, one is Stt7-dependent. This phosphorylation may play a crucial role in the dissociation of CP29 from PSII and/or in its association to PSI where it serves as a docking site for LHCII in state 2. Moreover, Stt7 was required for the phosphorylation of the thylakoid protein kinase Stl1 under state 2 conditions, suggesting the existence of a thylakoid protein kinase cascade. Stt7 itself is phosphorylated at Ser 533 in state 2, but analysis of mutants with a S533A/D change indicated that this phosphorylation is not required forstatetransitions.Moreover,wealsoidentifiedphosphorylation sites that are redox (state 2)-dependent but independent of Stt7 and additional phosphorylation sites that are redox-independent. Molecular & Cellular Proteomics 9:1281-1295, 2010.The primary photochemical reactions of photosynthesis are catalyzed by the pigment-protein complexes photosystem II (PSII) 1 and PSI (PSI), which are linked in series through the plastoquinone pool, the cytochrome b 6 f complex, and plastocyanin in the thylakoid membranes. Upon light absorption by the antenna systems of PSII and PSI, charge separations occur across the membrane that lead to the oxidation of water by PSII and electron flow to PSI and ultimately to the reduction of NADP ϩ . Because the antenna systems of PSII and PSI have different pigment composition, they are differentially sensitized upon changes in light quality and quantity. However, photosynthetic organisms have the ability to adapt to changes in light. They balance energy input and consumption in the short term through dissipation of excess absorbed light energy into heat through non-photochemical quenching and regulate absorption of excitation energy between PSII and PSI through state tra...

Quorum sensing (QS) signaling allows bacteria to control gene expression once a critical population density is achieved. The Gram-negative human pathogen Pseudomonas aeruginosa uses N-acylhomoserine lactones (AHL) as QS signals, which coordinate the production of virulence factors and biofilms. These bacterial signals can also modulate human cell behavior. Little is known about the mechanisms of the action of AHL on their eukaryotic targets. Here, we found that N-3-oxo-dodecanoyl-L-homoserine lactone 3O-C12-HSL modulates human intestinal epithelial Caco-2 cell migration in a dose- and time-dependent manner. Using new 3O-C12-HSL biotin and fluorescently-tagged probes for LC-MS/MS and confocal imaging, respectively, we demonstrated for the first time that 3O-C12-HSL interacts and co-localizes with the IQ-motif-containing GTPase-activating protein IQGAP1 in Caco-2 cells. The interaction between IQGAP1 and 3O-C12-HSL was further confirmed by pull-down assay using a GST-tagged protein with subsequent Western blot of IQGAP1 and by identifying 3O-C12-HSL with a sensor bioassay. Moreover, 3O-C12-HSL induced changes in the phosphorylation status of Rac1 and Cdc42 and the localization of IQGAP1 as evidenced by confocal and STED microscopy and Western blots. Our findings suggest that the IQGAP1 is a novel partner for P.aeruginosa 3O-C12-HSL and likely the integrator of Rac1 and Cdc42- dependent altered cell migration. We propose that the targeting of IQGAP1 by 3O-C12-HSL can trigger essential changes in the cytoskeleton network and be an essential component in bacterial – human cell communication.