A novel chloroplast super-complex consisting of the ATP synthase and photosystem I reaction center.
ABSTRACT: Several 'super-complexes' of individual hetero-oligomeric membrane protein complexes, whose function is to facilitate intra-membrane electron and proton transfer and harvesting of light energy, have been previously characterized in the mitochondrial cristae and chloroplast thylakoid membranes. We report the presence of an intra-membrane super-complex dominated by the ATP-synthase, photosystem I (PSI) reaction-center complex and the ferredoxin-NADP+ Reductase (FNR) in the thylakoid membrane. The presence of the super-complex has been documented by mass spectrometry, clear-native PAGE and Western Blot analyses. This is the first documented presence of ATP synthase in a super-complex with the PSI reaction-center located in the non-appressed stromal domain of the thylakoid membrane.
Project description:<h4>Background</h4>The thylakoid system in plant chloroplasts is organized into two distinct domains: grana arranged in stacks of appressed membranes and non-appressed membranes consisting of stroma thylakoids and margins of granal stacks. It is argued that the reason for the development of appressed membranes in plants is that their photosynthetic apparatus need to cope with and survive ever-changing environmental conditions. It is not known however, why different plant species have different arrangements of grana within their chloroplasts. It is important to elucidate whether a different arrangement and distribution of appressed and non-appressed thylakoids in chloroplasts are linked with different qualitative and/or quantitative organization of chlorophyll-protein (CP) complexes in the thylakoid membranes and whether this arrangement influences the photosynthetic efficiency.<h4>Results</h4>Our results from TEM and in situ CLSM strongly indicate the existence of different arrangements of pea and bean thylakoid membranes. In pea, larger appressed thylakoids are regularly arranged within chloroplasts as uniformly distributed red fluorescent bodies, while irregular appressed thylakoid membranes within bean chloroplasts correspond to smaller and less distinguished fluorescent areas in CLSM images. 3D models of pea chloroplasts show a distinct spatial separation of stacked thylakoids from stromal spaces whereas spatial division of stroma and thylakoid areas in bean chloroplasts are more complex. Structural differences influenced the PSII photochemistry, however without significant changes in photosynthetic efficiency. Qualitative and quantitative analysis of chlorophyll-protein complexes as well as spectroscopic investigations indicated a similar proportion between PSI and PSII core complexes in pea and bean thylakoids, but higher abundance of LHCII antenna in pea ones. Furthermore, distinct differences in size and arrangements of LHCII-PSII and LHCI-PSI supercomplexes between species are suggested.<h4>Conclusions</h4>Based on proteomic and spectroscopic investigations we postulate that the differences in the chloroplast structure between the analyzed species are a consequence of quantitative proportions between the individual CP complexes and its arrangement inside membranes. Such a structure of membranes induced the formation of large stacked domains in pea, or smaller heterogeneous regions in bean thylakoids. Presented 3D models of chloroplasts showed that stacked areas are noticeably irregular with variable thickness, merging with each other and not always parallel to each other.
Project description:Nuclear-encoded light-harvesting chlorophyll- and carotenoid-binding proteins (LHCPs) are imported into the chloroplast and transported across the stroma to thylakoid membrane assembly sites by the chloroplast signal recognition particle (CpSRP) pathway. The LHCP translocation defect (LTD) protein is essential for the delivery of imported LHCPs to the CpSRP pathway in Arabidopsis. However, the function of the LTD protein in Chlamydomonas reinhardtii has not been investigated. Here, we generated a C. reinhardtii ltd (Crltd) knockout mutant by using CRISPR-Cas9, a new target-specific knockout technology. The Crltd1 mutant showed a low chlorophyll content per cell with an unusual increase in appressed thylakoid membranes and enlarged cytosolic vacuoles. Profiling of thylakoid membrane proteins in the Crltd1 mutant showed a more severe reduction in the levels of photosystem I (PSI) core proteins and absence of functional LHCI compared with those of photosystem II, resulting in a much smaller PSI pool size and diminished chlorophyll antenna size. The lack of CrLTD did not prevent photoautotrophic growth of the cells. These results are substantially different from those for Arabidopsis ltd null mutant, indicating LTD function in LHCP delivery and PSI assembly may not be as stringent in C. reinhardtii as it is in higher plants.
Project description:Thylakoid membranes scaffold an assortment of large protein complexes that work together to harness the energy of light. It has been a longstanding challenge to visualize how the intricate thylakoid network organizes these protein complexes to finely tune the photosynthetic reactions. Previously, we used in situ cryo-electron tomography to reveal the native architecture of thylakoid membranes (Engel et al., 2015). Here, we leverage technical advances to resolve the individual protein complexes within these membranes. Combined with a new method to visualize membrane surface topology, we map the molecular landscapes of thylakoid membranes inside green algae cells. Our tomograms provide insights into the molecular forces that drive thylakoid stacking and reveal that photosystems I and II are strictly segregated at the borders between appressed and non-appressed membrane domains. This new approach to charting thylakoid topology lays the foundation for dissecting photosynthetic regulation at the level of single protein complexes within the cell.
Project description:In wild type plants, decreasing CO2 lowers the activity of the chloroplast ATP synthase, slowing proton efflux from the thylakoid lumen resulting in buildup of thylakoid proton motive force (pmf). The resulting acidification of the lumen regulates both light harvesting, via the qE mechanism, and photosynthetic electron transfer through the cytochrome b6f complex. Here, we show that the cfq mutant of Arabidopsis, harboring single point mutation in its ?-subunit of the chloroplast ATP synthase, increases the specific activity of the ATP synthase and disables its down-regulation under low CO2. The increased thylakoid proton conductivity (gH+) in cfq results in decreased pmf and lumen acidification, preventing full activation of qE and more rapid electron transfer through the b6f complex, particularly under low CO2 and fluctuating light. These conditions favor the accumulation of electrons on the acceptor side of PSI, and result in severe loss of PSI activity. Comparing the current results with previous work on the pgr5 mutant suggests a general mechanism where increased PSI photodamage in both mutants is caused by loss of pmf, rather than inhibition of CEF per se. Overall, our results support a critical role for ATP synthase regulation in maintaining photosynthetic control of electron transfer to prevent photodamage.
Project description:The thylakoid membrane inside chloroplasts hosts the light-dependent reactions of photosynthesis. Its embedded protein complexes are responsible for light harvesting, excitation energy transfer, charge separation, and transport. In higher plants, when the illumination conditions vary, the membrane adapts its composition and nanoscale morphology, which is characterized by appressed and non-appressed regions known as grana and stroma lamellae, respectively. Here we investigate the nanophotonic regime of light propagation in chloroplasts of higher plants and identify novel mechanisms in the optical response of the thylakoid membrane. Our results indicate that the relative contributions of light scattering and absorption to the overall optical response of grana strongly depend on the concentration of the light-harvesting complexes. For the pigment concentrations typically found in chloroplasts, the two mechanisms have comparable strengths, and their relative value can be tuned by variations in the protein composition or in the granal diameter. Furthermore, we find that collective modes in ensembles of grana significantly increase light absorption at selected wavelengths, even in the presence of moderate biological disorder. Small variations in the granal separation or a large disorder can dismantle this collective response. We propose that chloroplasts use this mechanism as a strategy against dangerously high illumination conditions, triggering a transition to low-absorbing states. We conclude that the morphological separation of the thylakoid membrane in higher plants supports strong nanophotonic effects, which may be used by chloroplasts to regulate light absorption. This adaptive self-organization capability is of interest as a model for novel bioinspired optical materials for artificial photosynthesis, imaging, and sensing.
Project description:Assembly of Photosystem (PS) II in plants has turned out to be a highly complex process which, at least in part, occurs in a sequential order and requires many more auxiliary proteins than subunits present in the complex. Owing to the high evolutionary conservation of the subunit composition and the three-dimensional structure of the PSII complex, most plant factors involved in the biogenesis of PSII originated from cyanobacteria and only rarely evolved de novo. Furthermore, in chloroplasts the initial assembly steps occur in the non-appressed stroma lamellae, whereas the final assembly including the attachment of the major LHCII antenna proteins takes place in the grana regions. The stroma lamellae are also the place where part of PSII repair occurs, which very likely also involves assembly factors. In cyanobacteria initial PSII assembly also occurs in the thylakoid membrane, in so-called thylakoid centers, which are in contact with the plasma membrane. Here, we provide an update on the structures, localisations, topologies, functions, expression and interactions of the low molecular mass PSII subunits PsbY, PsbW and the auxiliary factors HCF136, PsbN, TerC and ALB3, assisting in PSII complex assembly and protein insertion into the thylakoid membrane.
Project description:While the composition and function of the major thylakoid membrane complexes are well understood, comparatively little is known about their biogenesis. The goal of this work was to shed more light on the role of auxiliary factors in the biogenesis of photosystem II (PSII). Here we have identified the homolog of LOW PSII ACCUMULATION 2 (LPA2) in Chlamydomonas. A Chlamydomonas reinhardtii lpa2 mutant grew slower in low light, was hypersensitive to high light, and exhibited aberrant structures in thylakoid membrane stacks. Chlorophyll fluorescence (Fv/Fm) was reduced by 38%. Synthesis and stability of newly made PSII core subunits D1, D2, CP43, and CP47 were not impaired. However, complexome profiling revealed that in the mutant CP43 was reduced to ~23% and D1, D2, and CP47 to ~30% of wild type levels. Levels of PSI and the cytochrome b6f complex were unchanged, while levels of the ATP synthase were increased by ~29%. PSII supercomplexes, dimers, and monomers were reduced to ~7%, ~26%, and ~60% of wild type levels, while RC47 was increased ~6-fold and LHCII by ~27%. We propose that LPA2 catalyses a step during PSII assembly without which PSII monomers and further assemblies become unstable and prone to degradation. The LHCI antenna was more disconnected from PSI in the lpa2 mutant, presumably as an adaptive response to reduce excitation of PSI. From the co-migration profiles of 1734 membrane-associated proteins, we identified three novel putative PSII associated proteins with potential roles in regulating PSII complex dynamics, assembly, and chlorophyll breakdown.
Project description:The structural dynamics and flexibility of cell membranes play fundamental roles in the functions of the cells, i.e., signaling, energy transduction, and physiological adaptation. The cyanobacterial thylakoid membrane represents a model membrane that can conduct both oxygenic photosynthesis and respiration simultaneously. In this study, we conducted direct visualization of the global organization and mobility of photosynthetic complexes in thylakoid membranes from a model cyanobacterium, Synechococcus elongatus PCC 7942, using high-resolution atomic force, confocal, and total internal reflection fluorescence microscopy. We visualized the native arrangement and dense packing of photosystem I (PSI), photosystem II (PSII), and cytochrome (Cyt) b6f within thylakoid membranes at the molecular level. Furthermore, we functionally tagged PSI, PSII, Cyt b6f, and ATP synthase individually with fluorescent proteins, and revealed the heterogeneous distribution of these four photosynthetic complexes and determined their dynamic features within the crowding membrane environment using live-cell fluorescence imaging. We characterized red light-induced clustering localization and adjustable diffusion of photosynthetic complexes in thylakoid membranes, representative of the reorganization of photosynthetic apparatus in response to environmental changes. Understanding the organization and dynamics of photosynthetic membranes is essential for rational design and construction of artificial photosynthetic systems to underpin bioenergy development. Knowledge of cyanobacterial thylakoid membranes could also be extended to other cell membranes, such as chloroplast and mitochondrial membranes.
Project description:The chloroplast NAD(P)H dehydrogenase (NDH) complex is involved in photosystem I (PSI) cyclic and chlororespiratory electron transport in higher plants. Although biochemical and genetic evidence for its subunit composition has accumulated, it is not enough to explain the complexes putative activity of NAD(P)H-dependent plastoquinone reduction. We analyzed the NDH complex by using blue native PAGE and found that it interacts with PSI to form a novel supercomplex. Mutants lacking NdhL and NdhM accumulated a pigment-protein complex with a slightly lower molecular mass than that of the NDH-PSI supercomplex; this may be an intermediate supercomplex including PSI. This intermediate is unstable in mutants lacking NdhB, NdhD, or NdhF, implying that it includes some NDH subunits. Analysis of thylakoid membrane complexes using sucrose density gradient centrifugation supported the presence of the NDH-PSI supercomplex in vivo. Although the NDH complex exists as a monomer in etioplasts, it interacts with PSI to form a supercomplex within 48 h during chloroplast development.
Project description:We describe the identification of the first immunophilin associated with the photosynthetic membrane of chloroplasts. This complex 40 kDa immunophilin, designated TLP40 (thylakoid lumen PPIase), located in the lumen of the thylakoids, was found to play a dual role in photosynthesis involving both biogenesis and intraorganelle signalling. It originates in a single-copy nuclear gene, is made as a precursor of 49.2 kDa with a bipartite lumenal targeting transit peptide, and is characterized by a structure including a cyclophilin-like C-terminal segment of 20 kDa, a predicted N-terminal leucine zipper and a potential phosphatase-binding domain. It can exist in different oligomeric conformations and attach to the inner membrane surface. It is confined predominantly to the non-appressed thylakoid regions, the site of protein integration into the photosynthetic membrane. The isolated protein possesses peptidyl-prolyl cis-trans isomerase protein folding activity characteristic of immunophilins, but is not inhibited by cyclosporin A. TLP40 also exerts an effect on dephosphorylation of several key proteins of photosystem II, probably as a constituent of a transmembrane signal transduction chain. This first evidence for a direct role of immunophilins in a photoautotrophic process suggests that light-mediated protein phosphorylation in photosynthetic membranes and the role of the thylakoid lumen are substantially more complex than anticipated.