Role of isotropic lipid phase in the fusion of photosystem II membranes.

It has been thoroughly documented, by using 31P-NMR spectroscopy, that plant thylakoid membranes (TMs), in addition to the bilayer (or lamellar, L) phase, contain at least two isotropic (I) lipid phases and an inverted hexagonal (HII) phase. However, our knowledge concerning the structural and functional roles of the non-bilayer phases is still rudimentary. The objective of the present study is to elucidate the origin of I phases which have been hypothesized to arise, in part, from the fusion of TMs (Garab et al. 2022 Progr Lipid Res 101,163). We take advantage of the selectivity of wheat germ lipase (WGL) in eliminating the I phases of TMs (Dlouhý et al. 2022 Cells 11: 2681), and the tendency of the so-called BBY particles, stacked photosystem II (PSII) enriched membrane pairs of 300-500 nm in diameter, to form large laterally fused sheets (Dunahay et al. 1984 BBA 764: 179). Our 31P-NMR spectroscopy data show that BBY membranes contain L and I phases. Similar to TMs, WGL selectively eliminated the I phases, which at the same time exerted no effect on the molecular organization and functional activity of PSII membranes. As revealed by sucrose-density centrifugation, magnetic linear dichroism spectroscopy and scanning electron microscopy, WGL disassembled the large laterally fused sheets. These data provide direct experimental evidence on the involvement of I phase(s) in the fusion of stacked PSII membrane pairs, and strongly suggest the role of non-bilayer lipids in the self-assembly of the TM system.

Chloroplast Acetyltransferase GNAT2 is Involved in the Organization and Dynamics of Thylakoid Structure.

Higher plants acclimate to changes in light conditions by adjusting the thylakoid membrane ultrastructure. Additionally, excitation energy transfer between photosystem II (PSII) and photosystem I (PSI) is balanced in a process known as state transition. These modifications are mediated by reversible phosphorylation of Lhcb1 and Lhcb2 proteins in different pools of light-harvesting complex (LHCII) trimers. Our recent study demonstrated that chloroplast acetyltransferase NUCLEAR SHUTTLE INTERACTING (NSI)/GNAT2 (general control non-repressible 5 (GCN5)-related N-acetyltransferase 2) is also needed for the regulation of light harvesting, evidenced by the inability of the gnat2 mutant to perform state transitions although there are no defects in LHCII phosphorylation. Here, we show that despite contrasting phosphorylation states of LHCII, grana packing in the gnat2 and state transition 7 (stn7) mutants possesses similar features, as the thylakoid structure of the mutants does not respond to the shift from darkness to light, which is in striking contrast to wild type (Wt). Circular dichroism and native polyacrylamide gel electrophoresis analyses further revealed that the thylakoid protein complex organization of gnat2 and stn7 resembles each other, but differ from that of Wt. Also, the location of the phosphorylated Lhcb2 as well as the LHCII antenna within the thylakoid network in gnat2 mutant is different from that of Wt. In gnat2, the LHCII antenna remains largely in grana stacks, where the phosphorylated Lhcb2 is found in all LHCII trimer pools, including those associated with PSII. These results indicate that in addition to phosphorylation-mediated regulation through STN7, the GNAT2 enzyme is involved in the organization and dynamics of thylakoid structure, probably through the regulation of chloroplast protein acetylation.

Heparan sulfate proteoglycan (HSPG) can take part in cell division: inside and outside.

Prior to the cytokinesis, the cell-matrix interactions should be disrupted, and the mitotic cells round up. Prerequisite of mitosis, the centrosomes duplicate, spindle fibers are generated and move away from each other to opposite sides of the cells marking the cell poles. Later, an invagination in the plasma membrane is formed a few minutes after anaphase. This furrow ingression is driven by a contractile actomyosin ring, whose assembly is regulated by RhoA GTPase. At the completion of cytokinesis, the two daughter cells are still connected by a thin intercellular bridge, which is subjected to abscission, as the terminal step of cytokinesis. Here, it is overviewed, how syndecan-4, a transmembrane, heparan sulfate proteoglycan, can contribute to these processes in a phosphorylation-dependent manner.

Lipid-polymorphism of plant thylakoid membranes. Enhanced non-bilayer lipid phases associated with increased membrane permeability.

Earlier experiments, using 31 P-NMR and time-resolved merocyanine fluorescence spectroscopy, have shown that isolated intact, fully functional plant thylakoid membranes, in addition to the bilayer phase, contain three non-bilayer (or non-lamellar) lipid phases. It has also been shown that the lipid polymorphism of thylakoid membranes can be characterized by remarkable plasticity, i.e. by significant variations in 31 P-NMR signatures. However, changes in the lipid-phase behaviour of thylakoids could not be assigned to changes in the overall membrane organization and the photosynthetic activity, as tested by circular dichroism and 77 K fluorescence emission spectroscopy and the magnitude of the variable fluorescence of photosystem II, which all showed only marginal variations. In this work, we investigated in more detail the temporal stability of the different lipid phases by recording 31 P-NMR spectra on isolated thylakoid membranes that were suspended in sorbitol- or NaCl-based media. We observed, at 5°C during 8 h in the dark, substantial gradual enhancement of the isotropic lipid phases and diminishment of the bilayer phase in the sorbitol-based medium. These changes compared well with the gradually increasing membrane permeability, as testified by the gradual acceleration of the decay of flash-induced electrochromic absorption changes and characteristic changes in the kinetics of fast chlorophyll a-fluorescence transients; all variations were much less pronounced in the NaCl-based medium. These observations suggest that non-bilayer lipids and non-lamellar lipid phases play significant roles in the structural dynamics and functional plasticity of thylakoid membranes.

Excitation energy transfer between Light-harvesting complex II and Photosystem I in reconstituted membranes.

Light-harvesting complex II (LHCII), the major peripheral antenna of Photosystem II in plants, participates in several concerted mechanisms for regulation of the excitation energy and electron fluxes in thylakoid membranes. In part, these include interaction of LHCII with Photosystem I (PSI) enhancing the latter's absorption cross-section - for example in the well-known state 1 - state 2 transitions or as a long-term acclimation to high light. In this work we examined the capability of LHCII to deliver excitations to PSI in reconstituted membranes in vitro. Proteoliposomes with native plant thylakoid membrane lipids and different stoichiometric ratios of LHCII:PSI were reconstituted and studied by steady-state and time-resolved fluorescence spectroscopy. Fluorescence emission from LHCII was strongly decreased in PSI-LHCII membranes due to trapping of excitations by PSI. Kinetic modelling of the time-resolved fluorescence data revealed the existence of separate pools of LHCII distinguished by the time scale of energy transfer. A strongly coupled pool, equivalent to one LHCII trimer per PSI, transferred excitations to PSI with near-unity efficiency on a time scale of less than 10ps but extra LHCIIs also contributed significantly to the effective antenna size of PSI, which could be increased by up to 47% in membranes containing 3 LHCII trimers per PSI. The results demonstrate a remarkable competence of LHCII to increase the absorption cross-section of PSI, given the opportunity that the two types of complexes interact in the membrane.

Role of MGDG and Non-bilayer Lipid Phases in the Structure and Dynamics of Chloroplast Thylakoid Membranes.

In this chapter we focus our attention on the enigmatic structural and functional roles of the major, non-bilayer lipid monogalactosyl-diacylglycerol (MGDG) in the thylakoid membrane. We give an overview on the state of the art on the role of MGDG and non-bilayer lipid phases in the xanthophyll cycles in different organisms. We also discuss data on the roles of MGDG and other lipid molecules found in crystal structures of different photosynthetic protein complexes and in lipid-protein assemblies, as well as in the self-assembly of the multilamellar membrane system. Comparison and critical evaluation of different membrane models--that take into account and capitalize on the special properties of non-bilayer lipids and/or non-bilayer lipid phases, and thus to smaller or larger extents deviate from the 'standard' Singer-Nicolson model--will conclude this review. With this chapter the authors hope to further stimulate the discussion about, what we think, is perhaps the most exciting question of membrane biophysics: the why and wherefore of non-bilayer lipids and lipid phases in, or in association with, bilayer biological membranes.

Label-free LC-MS/MS identification of phosphatidylglycerol-regulated proteins in Synechocystis sp. PCC6803.

We present a proteomics dataset combining SDS-PAGE prefractionation and data-dependent LC-MS/MS that enables the identification of phosphatidylglycerol-regulated proteins in the pgsA(-) mutant of Synechocystis sp. PCC6803, a cyanobacterium strain that grows with this indispensable phospholipid added exogenously. We searched the acquired raw data against a composite protein sequence database of Synechocystis using MASCOT, and employed Progenesis LC-MS software for label-free quantification based on extracted peptide intensities to detect changes in protein abundances upon phospholipid withdrawal. Protein identifications were validated using rigorous criteria, and our analysis of the dataset revealed 80 phosphatidylglycerol-regulated proteins involved in various cellular processes including photosynthesis, respiration, metabolism, transport, transcription, and translation. The data have been deposited to the ProteomeXchange with identifier PXD000363 (http://proteomecentral.proteomexchange.org/dataset/PXD000363).

Two functional sites of phosphatidylglycerol for regulation of reaction of plastoquinone Q(B) in photosystem II.

Functional roles of an anionic lipid phosphatidylglycerol (PG) were studied in pgsA-gene-inactivated and cdsA-gene-inactivated/phycobilisome-less mutant cells of a cyanobacterium Synechocystis sp. PCC 6803, which can grow only in PG-supplemented media. 1) A few days of PG depletion suppressed oxygen evolution of mutant cells supported by p-benzoquinone (BQ). The suppression was recovered slowly in a week after PG re-addition. Measurements of fluorescence yield indicated the enhanced sensitivity of Q(B) to the inactivation by BQ. It is assumed that the loss of low-affinity PG (PG(L)) enhances the affinity for BQ that inactivates Q(B). 2) Oxygen evolution without BQ, supported by the endogenous electron acceptors, was slowly suppressed due to the direct inactivation of Q(B) during 10 days of PG depletion, and was recovered rapidly within 10h upon the PG re-addition. It is concluded that the loss of high-affinity PG (PG(H)) displaces Q(B) directly. 3) Electron microscopy images of PG-depleted cells showed the specific suppression of division of mutant cells, which had developed thylakoid membranes attaching phycobilisomes (PBS). 4) Although the PG-depletion for 14 days decreased the chlorophyll/PBS ratio to about 1/4, flourescence spectra/lifetimes were not modified indicating the flexible energy transfer from PBS to different numbers of PSII. Longer PG-depletion enhanced allophycocyanin fluorescence at 683nm with a long 1.2ns lifetime indicating the suppression of energy transfer from PBS to PSII. 5) Action sites of PG(H), PG(L) and other PG molecules on PSII structure are discussed.

Reconsidering Dogmas about the Growth of Bacterial Populations.

The growth of bacterial populations has been described as a dynamic process of continuous reproduction and cell death. However, this is far from the reality. In a well fed, growing bacterial population, the stationary phase inevitably occurs, and it is not due to accumulated toxins or cell death. A population spends the most time in the stationary phase, where the phenotype of the cells alters from the proliferating ones, and only the colony forming unit (CFU) decreases after a while, not the total cell concentration. A bacterial population can be considered as a virtual tissue as a result of a specific differentiation process, in which the exponential-phase cells develop to stationary-phase cells and eventually reach the unculturable form. The richness of the nutrient had no effect on growth rate or on stationary cell density. The generation time seems not to be a constant value, but it depended on the concentration of the starter cultures. Inoculations with serial dilutions of stationary populations reveal a so-called minimal stationary cell concentration (MSCC) point, up to which the cell concentrations remain constant upon dilutions; that seems to be universal among unicellular organisms.

Long- and short-term acclimation of the photosynthetic apparatus to salinity in Chlamydomonas reinhardtii. The role of Stt7 protein kinase.

Salt stress triggers an Stt7-mediated LHCII-phosphorylation signaling mechanism similar to light-induced state transitions. However, phosphorylated LHCII, after detaching from PSII, does not attach to PSI but self-aggregates instead. Salt is a major stress factor in the growth of algae and plants. Here, our study mainly focuses on the organization of the photosynthetic apparatus to the long-term responses of Chlamydomonas reinhardtii to elevated NaCl concentrations. We analyzed the physiological effects of salt treatment at a cellular, membrane, and protein level by microscopy, protein profile analyses, transcripts, circular dichroism spectroscopy, chlorophyll fluorescence transients, and steady-state and time-resolved fluorescence spectroscopy. We have ascertained that cells that were grown in high-salinity medium form palmelloids sphere-shaped colonies, where daughter cells with curtailed flagella are enclosed within the mother cell walls. Palmelloid formation depends on the presence of a cell wall, as it was not observed in a cell-wall-less mutant CC-503. Using the stt7 mutant cells, we show Stt7 kinase-dependent phosphorylation of light-harvesting complex II (LHCII) in both short- and long-term treatments of various NaCl concentrations-demonstrating NaCl-induced state transitions that are similar to light-induced state transitions. The grana thylakoids were less appressed (with higher repeat distances), and cells grown in 150 mM NaCl showed disordered structures that formed diffuse boundaries with the flanking stroma lamellae. PSII core proteins were more prone to damage than PSI. At high salt concentrations (100-150 mM), LHCII aggregates accumulated in the thylakoid membranes. Low-temperature and time-resolved fluorescence spectroscopy indicated that the stt7 mutant was more sensitive to salt stress, suggesting that LHCII phosphorylation has a role in the acclimation and protection of the photosynthetic apparatus.

Controlling cellular P-TEFb activity by the HIV-1 transcriptional transactivator Tat.

The human immunodeficiency virus 1 (HIV-1) transcriptional transactivator (Tat) is essential for synthesis of full-length transcripts from the integrated viral genome by RNA polymerase II (Pol II). Tat recruits the host positive transcription elongation factor b (P-TEFb) to the HIV-1 promoter through binding to the transactivator RNA (TAR) at the 5'-end of the nascent HIV transcript. P-TEFb is a general Pol II transcription factor; its cellular activity is controlled by the 7SK small nuclear RNA (snRNA) and the HEXIM1 protein, which sequester P-TEFb into transcriptionally inactive 7SK/HEXIM/P-TEFb snRNP. Besides targeting P-TEFb to HIV transcription, Tat also increases the nuclear level of active P-TEFb through promoting its dissociation from the 7SK/HEXIM/P-TEFb RNP by an unclear mechanism. In this study, by using in vitro and in vivo RNA-protein binding assays, we demonstrate that HIV-1 Tat binds with high specificity and efficiency to an evolutionarily highly conserved stem-bulge-stem motif of the 5'-hairpin of human 7SK snRNA. The newly discovered Tat-binding motif of 7SK is structurally and functionally indistinguishable from the extensively characterized Tat-binding site of HIV TAR and importantly, it is imbedded in the HEXIM-binding elements of 7SK snRNA. We show that Tat efficiently replaces HEXIM1 on the 7SK snRNA in vivo and therefore, it promotes the disassembly of the 7SK/HEXIM/P-TEFb negative transcriptional regulatory snRNP to augment the nuclear level of active P-TEFb. This is the first demonstration that HIV-1 specifically targets an important cellular regulatory RNA, most probably to promote viral transcription and replication. Demonstration that the human 7SK snRNA carries a TAR RNA-like Tat-binding element that is essential for the normal transcriptional regulatory function of 7SK questions the viability of HIV therapeutic approaches based on small drugs blocking the Tat-binding site of HIV TAR.

Reversible membrane reorganizations during photosynthesis in vivo: revealed by small-angle neutron scattering.

In the present study, we determined characteristic repeat distances of the photosynthetic membranes in living cyanobacterial and eukaryotic algal cells, and in intact thylakoid membranes isolated from higher plants with time-resolved small-angle neutron scattering. This non-invasive technique reveals light-induced reversible reorganizations in the seconds-to-minutes time scale, which appear to be associated with functional changes in vivo.

Structural Entities Associated with Different Lipid Phases of Plant Thylakoid Membranes-Selective Susceptibilities to Different Lipases and Proteases.

It is well established that plant thylakoid membranes (TMs), in addition to a bilayer, contain two isotropic lipid phases and an inverted hexagonal (HII) phase. To elucidate the origin of non-bilayer lipid phases, we recorded the 31P-NMR spectra of isolated spinach plastoglobuli and TMs and tested their susceptibilities to lipases and proteases; the structural and functional characteristics of TMs were monitored using biophysical techniques and CN-PAGE. Phospholipase-A1 gradually destroyed all 31P-NMR-detectable lipid phases of isolated TMs, but the weak signal of isolated plastoglobuli was not affected. Parallel with the destabilization of their lamellar phase, TMs lost their impermeability; other effects, mainly on Photosystem-II, lagged behind the destruction of the original phases. Wheat-germ lipase selectively eliminated the isotropic phases but exerted little or no effect on the structural and functional parameters of TMs-indicating that the isotropic phases are located outside the protein-rich regions and might be involved in membrane fusion. Trypsin and Proteinase K selectively suppressed the HII phase-suggesting that a large fraction of TM lipids encapsulate stroma-side proteins or polypeptides. We conclude that-in line with the Dynamic Exchange Model-the non-bilayer lipid phases of TMs are found in subdomains separated from but interconnected with the bilayer accommodating the main components of the photosynthetic machinery.

Remodeling of phosphatidylglycerol in Synechocystis PCC6803.

The phosphatidylglycerol deficient DeltapgsA mutant of Synechocystis PCC6803 provided a unique experimental system for investigating in vivo retailoring of exogenously added dioleoylphosphatidylglycerol in phosphatidylglycerol-depleted cells. Gas chromatographic analysis of fatty acid composition suggested that diacyl-phosphatidylglycerols were synthesized from the artificial synthetic precursor. The formation of new, retailored lipid species was confirmed by negative-ion electrospray ionization-Fourier-transform ion cyclotron resonance and ion trap tandem mass spectrometry. Various isomeric diacyl-phosphatidylglycerols were identified indicating transesterification of the exogenously added dioleoylphosphatidyl-glycerol at the sn-1 or sn-2 positions. Polyunsaturated fatty acids were incorporated selectively into the sn-1 position. Our experiments with Synechocystis PCC6803/DeltapgsA mutant cells demonstrated lipid remodeling in a prokaryotic photosynthetic bacterium. Our data suggest that the remodeling of diacylphosphatidylglycerol likely involves reactions catalyzed by phospholipase A(1) and A(2) or acyl-hydrolase, lysophosphatidylglycerol acyltransferase and acyl-lipid desaturases.

Identification of thylakoid membrane thermal transitions in Synechocystis sp. PCC6803 photosynthetic mutants.

We used differential scanning calorimetry (DSC) as a technique capable of identifying photosynthetic complexes on the basis of their calorimetric transitions. Annotation of thermal transitions was carried out with thylakoid membranes isolated from various photosynthetic mutants of Synechocystis sp. PCC6803. The thylakoid membranes exhibited seven major DSC bands between 40 and 85°C. The heat sorption curves were analyzed both by mathematical deconvolution of the overall endotherms and by a subsequent annealing procedure. The successive annealing procedure proved to be more reliable technique than mathematical deconvolution in assigning thermal transitions. The main DSC band, around 47°C, resulting from the high enthalpy change that corresponds to non-interacting complex of PSII, was assigned using the PSI-less/apcE(-) mutant cells. Another band around 68-70°C relates to the denaturation of PSII surrounded by other proteins of the photosynthetic complexes in wild type and PSI-less/apcE(-) cells. A further major transition found at 82-84°C corresponds to the PSI core complex of wild type and PSII-deficient BE cells. Other transition bands between 50-67 and 65-75°C are believed to relate to ATP synthase and cytochrome b(6)f, respectively. These thermal transitions were obtained with thylakoids isolated from PSI(-)/PSII(-) mutant cells. Some minor bands determined at 59 and 83-84°C correspond to an unknown complex and NADH dehydrogenase, respectively. These annotations were done by PSI-less/apcE(-) and PSI(-)/PSII(-) mutants.

Salt Stress Induces Paramylon Accumulation and Fine-Tuning of the Macro-Organization of Thylakoid Membranes in Euglena gracilis Cells.

The effects of salt stress condition on the growth, morphology, photosynthetic performance, and paramylon content were examined in the mixotrophic, unicellular, flagellate Euglena gracilis. We found that salt stress negatively influenced cell growth, accompanied by a decrease in chlorophyll (Chl) content. Circular dichroism (CD) spectroscopy revealed the changes in the macro-organization of pigment-protein complexes due to salt treatment, while the small-angle neutron scattering (SANS) investigations suggested a reduction in the thylakoid stacking, an effect confirmed by the transmission electron microscopy (TEM). At the same time, the analysis of the thylakoid membrane complexes using native-polyacrylamide gel electrophoresis (PAGE) revealed no significant change in the composition of supercomplexes of the photosynthetic apparatus. Salt stress did not substantially affect the photosynthetic activity, as reflected by the fact that Chl fluorescence yield, electron transport rate (ETR), and energy transfer between the photosystems did not change considerably in the salt-grown cells. We have observed notable increases in the carotenoid-to-Chl ratio and the accumulation of paramylon in the salt-treated cells. We propose that the accumulation of storage polysaccharides and changes in the pigment composition and thylakoid membrane organization help the adaptation of E. gracilis cells to salt stress and contribute to the maintenance of cellular processes under stress conditions.

Involvement of carotenoids in the synthesis and assembly of protein subunits of photosynthetic reaction centers of Synechocystis sp. PCC 6803.

The crtB gene of Synechocystis sp. PCC 6803, encoding phytoene synthase, was inactivated in the Delta crtH mutant to generate a carotenoidless Delta crtH/B double mutant. Delta crtH mutant cells were used because they had better transformability than wild-type cells, most probably due to their adaptation to partial carotenoid deficiency. Cells of the Delta crtH/B mutant were light sensitive and could grow only under light-activated heterotrophic growth conditions in the presence of glucose. Carotenoid deficiency did not significantly affect the cellular content of phycobiliproteins while the chlorophyll content of the mutant cells decreased. The mutant cells exhibited no oxygen-evolving activity, suggesting the absence of photochemically active PSII complexes. This was confirmed by 2D electrophoresis of photosynthetic membrane complexes. Analyses identified only a small amount of a non-functional PSII core complex lacking CP43, while the monomeric and dimeric PSII core complexes were absent. On the other hand, carotenoid deficiency did not prevent formation of the cytochrome b(6)f complex and PSI, which predominantly accumulated in the monomeric form. Radioactive labeling revealed very limited synthesis of inner PSII antennae, CP47 and especially CP43. Thus, carotenoids are indispensable constituents of the photosynthetic apparatus, being essential not only for antioxidative protection but also for the efficient synthesis and accumulation of photosynthetic proteins and especially that of PSII antenna subunits.

Phosphatidylglycerol depletion affects photosystem II activity in Synechococcus sp. PCC 7942 cells.

The role of phosphatidylglycerol (PG) in photosynthetic membranes of cyanobacteria was analyzed in a Synechococcus sp. PCC 7942 mutant produced by inactivating its cdsA gene presumably encoding cytidine 5'-diphosphate-diacylglycerol synthase, a key enzyme in PG synthesis. In a medium supplemented with PG the Synechococcus sp. PCC 7942/DeltacdsA cells grew photoautotrophically. Depletion of PG in the medium resulted (a) in an arrest of cell growth and division, (b) in a suppression of O(2) evolving activity, and (c) in a modification of Chl fluorescence induction curves. Two-dimensional PAGE showed that in the absence of PG (a) the amount of the PSI monomers increased at the expense of the PSI trimers and (b) PSII dimers were decomposed into monomers. [(35)S]methionine labeling confirmed that PG depletion did not block the de novo synthesis of PSII proteins but slowed down the assembly of the newly synthesized D1 protein into PSII core complexes. Retailoring of PG was observed during PG depletion: the exogenously added artificial dioleoyl PG was transformed into photosynthetically more essential PG derivatives. Concomitantly with a decrease in PG content, SQDG content increased, but it could not restore photosynthetic activity.

Modulation of non-bilayer lipid phases and the structure and functions of thylakoid membranes: effects on the water-soluble enzyme violaxanthin de-epoxidase.

The role of non-bilayer lipids and non-lamellar lipid phases in biological membranes is an enigmatic problem of membrane biology. Non-bilayer lipids are present in large amounts in all membranes; in energy-converting membranes they constitute about half of their total lipid content-yet their functional state is a bilayer. In vitro experiments revealed that the functioning of the water-soluble violaxanthin de-epoxidase (VDE) enzyme of plant thylakoids requires the presence of a non-bilayer lipid phase. 31P-NMR spectroscopy has provided evidence on lipid polymorphism in functional thylakoid membranes. Here we reveal reversible pH- and temperature-dependent changes of the lipid-phase behaviour, particularly the flexibility of isotropic non-lamellar phases, of isolated spinach thylakoids. These reorganizations are accompanied by changes in the permeability and thermodynamic parameters of the membranes and appear to control the activity of VDE and the photoprotective mechanism of non-photochemical quenching of chlorophyll-a fluorescence. The data demonstrate, for the first time in native membranes, the modulation of the activity of a water-soluble enzyme by a non-bilayer lipid phase.

Role of phosphatidylglycerol in the function and assembly of Photosystem II reaction center, studied in a cdsA-inactivated PAL mutant strain of Synechocystis sp. PCC6803 that lacks phycobilisomes.

To analyze the role of phosphatidylglycerol (PG) in photosynthetic membranes of cyanobacteria we used two mutants of Synechocystis sp. PCC6803: the PAL mutant which has no phycobilisomes and shows a high PSII/PSI ratio, and a mutant derived from it by inactivating its cdsA gene encoding cytidine 5'-diphosphate diacylglycerol synthase, a key enzyme in PG synthesis. In a medium supplemented with PG the PAL/DeltacdsA mutant cells grew photoautotrophically. Depletion of PG in the medium resulted (a) in an arrest of cell growth and division, (b) in a slowdown of electron transfer from the acceptor Q(A) to Q(B) in PSII and (c) in a modification of chlorophyll fluorescence curve. The depletion of PG affected neither the redox levels of Q(A) nor the S(2) state of the oxygen-evolving manganese complex, as indicated by thermoluminescence studies. Two-dimensional PAGE showed that in the absence of PG (a) the PSII dimer was decomposed into monomers, and (b) the CP43 protein was detached from a major part of the PSII core complex. [(35)S]-methionine labeling confirmed that PG depletion did not block de novo synthesis of the PSII proteins. We conclude that PG is required for the binding of CP43 within the PSII core complex.