PtdIns4P is required for the autophagosomal recruitment of STX17 (syntaxin 17) to promote lysosomal fusion.

The autophagosomal SNARE STX17 (syntaxin 17) promotes lysosomal fusion and degradation, but its autophagosomal recruitment is incompletely understood. Notably, PtdIns4P is generated on autophagosomes and promotes fusion through an unknown mechanism. Here we show that soluble recombinant STX17 is spontaneously recruited to negatively charged liposomes and adding PtdIns4P to liposomes containing neutral lipids is sufficient for its recruitment. Consistently, STX17 colocalizes with PtdIns4P-positive autophagosomes in cells, and specific inhibition of PtdIns4P synthesis on autophagosomes prevents its loading. Molecular dynamics simulations indicate that C-terminal positively charged amino acids establish contact with membrane bilayers containing negatively charged PtdIns4P. Accordingly, Ala substitution of Lys and Arg residues in the C terminus of STX17 abolishes membrane binding and impairs its autophagosomal recruitment. Finally, only wild type but not Ala substituted STX17 expression rescues the autophagosome-lysosome fusion defect of STX17 loss-of-function cells. We thus identify a key step of autophagosome maturation that promotes lysosomal fusion.Abbreviations: Cardiolipin: 1',3'-bis[1-palmitoyl-2-oleoyl-sn-glycero-3-phospho]-glycerol; DMSO: dimethyl sulfoxide; GST: glutathione S-transferase; GUV: giant unilamellar vesicles; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PA: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate; PC/POPC: 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine; PG: 1-palmitoyl-2-linoleoyl-sn-glycero-3-phospho-(1'-rac-glycerol); PI: L-α-phosphatidylinositol; PI4K2A: phosphatidylinositol 4-kinase type 2 alpha; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; POPE/PE: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; PS: 1-stearoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine; PtdIns(3,5)P2: 1,2-dioleoyl-sn-glycero-3-phospho-(1"-myo-inositol-3',5'-bisphosphate); PtdIns3P: 1,2- dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol-3'-phosphate); PtdIns4P: 1,2-dioleoyl-sn-glycero-3-phospho-(1"-myo-inositol-4'-phosphate); SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; STX17: syntaxin 17.

Lipid profiles of autophagic structures isolated from wild type and Atg2 mutant Drosophila.

Autophagy is mediated by membrane-bound organelles and it is an intrinsic catabolic and recycling process of the cell, which is very important for the health of organisms. The biogenesis of autophagic membranes is still incompletely understood. In vitro studies suggest that Atg2 protein transports lipids presumably from the ER to the expanding autophagic structures. Autophagy research has focused heavily on proteins and very little is known about the lipid composition of autophagic membranes. Here we describe a method for immunopurification of autophagic structures from Drosophila melanogaster (an excellent model to study autophagy in a complete organism) for subsequent lipidomic analysis. Western blots of several organelle markers indicate the high purity of the isolated autophagic vesicles, visualized by various microscopy techniques. Mass spectrometry results show that phosphatidylethanolamine (PE) is the dominant lipid class in wild type (control) membranes. We demonstrate that in Atg2 mutants (Atg2-), phosphatidylinositol (PI), negatively charged phosphatidylserine (PS), and phosphatidic acid (PA) with longer fatty acyl chains accumulate on stalled, negatively charged phagophores. Tandem mass spectrometry analysis of lipid species composing the lipid classes reveal the enrichment of unsaturated PE and phosphatidylcholine (PC) in controls versus PI, PS and PA species in Atg2-. Significant differences in the lipid profiles of control and Atg2- flies suggest that the lipid composition of autophagic membranes dynamically changes during their maturation. These lipidomic results also point to the in vivo lipid transport function of the Atg2 protein, pointing to its specific role in the transport of short fatty acyl chain PE species.

Phycobilisome integrity and functionality in lipid unsaturation and xanthophyll mutants in Synechocystis.

The major light-harvesting system in cyanobacteria, the phycobilisome, is an essential component of the photosynthetic apparatus that regulates the utilization of the natural light source-the Sun. Earlier works revealed that the thylakoid membrane composition and its physical properties might have an important role in antennas docking. Polyunsaturated lipids and xanthophylls are among the most significant modulators of the physical properties of thylakoid membranes. In the nature, the action of these molecules is orchestrated in response to environmental stimuli among which the growth temperature is the most influential. In order to further clarify the significance of thylakoid membrane physical properties for the phycobilisomes assembly (i.e. structural integrity) and their ability to efficiently direct the excitation energy towards the photosynthetic complexes, in this work, we utilize cyanobacterial Synechocystis sp. PCC 6803 mutants deficient in polyunsaturated lipids (AD mutant) and xanthophylls (RO mutant), as well as a strain depleted of both xanthophylls and polyunsaturated lipids (ROAD multiple mutant). For the first time, we discuss the effect of those mutations on the phycobilisomes assembly, integrity and functionality at optimal (30 °C) and moderate low (25 °C) and high (35 °C) temperatures. Our results show that xanthophyll depletion exerts a much stronger effect on both phycobilisome's integrity and the response of cells to growth at suboptimal temperatures than lipid unsaturation level. The strongest effects were observed for the combined ROAD mutant, which exhibited thermally destabilized phycobilisomes and a population of energetically uncoupled phycocyanin units.

Trimeric organization of photosystem I is required to maintain the balanced photosynthetic electron flow in cyanobacterium Synechocystis sp. PCC 6803.

In Synechocystis sp. PCC 6803 and some other cyanobacteria photosystem I reaction centres exist predominantly as trimers, with minor contribution of monomeric form, when cultivated at standard optimized conditions. In contrast, in plant chloroplasts photosystem I complex is exclusively monomeric. The functional significance of trimeric organization of cyanobacterial photosystem I remains not fully understood. In this study, we compared the photosynthetic characteristics of PSI in wild type and psaL knockout mutant. The results show that relative to photosystem I trimer in wild-type cells, photosystem I monomer in psaL- mutant has a smaller P700+ pool size under low and moderate light, slower P700 oxidation upon dark-to-light transition, and slower P700+ reduction upon light-to-dark transition. The mutant also shows strongly diminished photosystem I donor side limitations [quantum yield Y(ND)] at low, moderate and high light, but enhanced photosystem I acceptor side limitations [quantum yield Y(NA)], especially at low light (22 µmol photons m-2 s-1). In line with these functional characteristics are the determined differences in the relative expression genes encoding of selected electron transporters. The psaL- mutant showed significant (ca fivefold) upregulation of the photosystem I donor cytochrome c6, and downregulation of photosystem I acceptors (ferredoxin, flavodoxin) and proteins of alternative electron flows originating in photosystem I acceptor side. Taken together, our results suggest that photosystem I trimerization in wild-type Synechocystis cells plays a role in the protection of photosystem I from photoinhibition via maintaining enhanced donor side electron transport limitations and minimal acceptor side electron transport limitations at various light intensities.

Photosystem I oligomerization affects lipid composition in Synechocystis sp. PCC 6803.

In cyanobacteria, increasing growth temperature decreases lipid unsaturation and the ratio of monomer/trimer photosystem I (PSI) complexes. In the present study we applied Fourier-transform infrared (FTIR) spectroscopy and lipidomic analysis to study the effects of PSI monomer/oligomer ratio on the physical properties and lipid composition of thylakoids. To enhance the presence of monomeric PSI, a Synechocystis sp. PCC6803/ΔpsaL mutant strain (PsaL) was used which, unlike both trimeric and monomeric PSI-containing wild type (WT) cells, contain only the monomeric form. The protein-to-lipid ratio remained unchanged in the mutant but, due to an increase in the lipid disorder in its thylakoids, the gel to liquid-crystalline phase transition temperature (Tm) is lower than in the WT. In thylakoid membranes of the mutant, digalactosyldiacylglycerol (DGDG), the most abundant bilayer-forming lipid is accumulated, whereas those in the WT contain more monogalactosyldiacylglycerol (MGDG), the only non-bilayer-forming lipid in cyanobacteria. In PsaL cells, the unsaturation level of sulphoquinovosyldiacylglycerol (SQDG), a regulatory anionic lipid, has increased. It seems that merely a change in the oligomerization level of a membrane protein complex (PSI), and thus the altered protein-lipid interface, can affect the lipid composition and, in addition, the whole dynamics of the membrane. Singular value decomposition (SVD) analysis has shown that in PsaL thylakoidal protein-lipid interactions are less stable than in the WT, and proteins start losing their native secondary structure at much milder lipid packing perturbations. Conclusions drawn from this system should be generally applicable for protein-lipid interactions in biological membranes.

Understanding the importance of autophagy in human diseases using Drosophila.

Autophagy is a lysosome-dependent intracellular degradation pathway that has been implicated in the pathogenesis of various human diseases, either positively or negatively impacting disease outcomes depending on the specific context. The majority of medical conditions including cancer, neurodegenerative diseases, infections and immune system disorders and inflammatory bowel disease could probably benefit from therapeutic modulation of the autophagy machinery. Drosophila represents an excellent model animal to study disease mechanisms thanks to its sophisticated genetic toolkit, and the conservation of human disease genes and autophagic processes. Here, we provide an overview of the various autophagy pathways observed both in flies and human cells (macroautophagy, microautophagy and chaperone-mediated autophagy), and discuss Drosophila models of the above-mentioned diseases where fly research has already helped to understand how defects in autophagy genes and pathways contribute to the relevant pathomechanisms.

Structural integrity of Synechocystis sp. PCC 6803 phycobilisomes evaluated by means of differential scanning calorimetry.

Phycobilisomes (PBSs) are supramolecular pigment-protein complexes that serve as light-harvesting antennae in cyanobacteria. They are built up by phycobiliproteins assembled into allophycocyanin core cylinders (ensuring the physical interaction with the photosystems) and phycocyanin rods (protruding from the cores and having light-harvesting function), the whole PBSs structure being maintained by linker proteins. PBSs play major role in light-harvesting optimization in cyanobacteria; therefore, the characterization of their structural integrity in intact cells is of great importance. The present study utilizes differential scanning calorimetry and spectroscopy techniques to explore for the first time, the thermodynamic stability of PBSs in intact Synechocystis sp. PCC 6803 cells and to probe its alteration as a result of mutations or under different growth conditions. As a first step, we characterize the thermodynamic behavior of intact and dismantled PBSs isolated from wild-type cells (having fully assembled PBSs) and from CK mutant cells (that lack phycocyanin rods and contain only allophycocyanin cores), and identified the thermal transitions of phycocyanin and allophycocyanin units in vitro. Next, we demonstrate that in intact cells PBSs exhibit sharp, high amplitude thermal transition at about 63 °C that strongly depends on the structural integrity of the PBSs supercomplex. Our findings implicate that calorimetry could offer a valuable approach for the assessment of the influence of variety of factors affecting the stability and structural organization of phycobilisomes in intact cyanobacterial cells.

Zeaxanthin and echinenone modify the structure of photosystem I trimer in Synechocystis sp. PCC 6803.

The function of xanthophylls in the organisation and structure of the photosynthetic complexes is not completely clarified yet. Recently, we observed a reduced level of the photosystem oligomers upon xanthophyll deficiency, although xanthophylls are not considered to be part of the photosynthetic complexes of cyanobacteria. The present study aimed at further investigating the relationship between xanthophylls and photosytem I (PSI) complex in the cyanobacterium Synechocystis sp. PCC 6803. Interestingly, we recorded the presence of echinenone and zeaxanthin in the isolated PSI trimers. These two xanthophyll species are among the most abundant xanthophylls in this cyanobacterial species. Various xanthophyll biosynthesis mutants were used to investigate the specific role of these xanthophylls. Our spectroscopic results revealed specific structural changes manifested in altered pigment-pigment or pigment-protein interactions within PSI complex in the absence of zeaxanthin and echinenone. These structural modifications of the complexes seem to destabilize the PSI trimeric complexes and eventually result in an increased propensity for monomerization. Our results clearly demonstrate that xanthophylls are important for the fine-tuning of the PSI trimer structure. These xanthophylls could be part of the complex or be embedded in the membrane in the vicinity of PSI.

Lipid and carotenoid cooperation-driven adaptation to light and temperature stress in Synechocystis sp. PCC6803.

Polyunsaturated lipids are important components of photosynthetic membranes. Xanthophylls are the main photoprotective agents, can assist in protection against light stress, and are crucial in the recovery from photoinhibition. We generated the xanthophyll- and polyunsaturated lipid-deficient ROAD mutant of Synechocystis sp. PCC6803 (Synechocystis) in order to study the little-known cooperative effects of lipids and carotenoids (Cars). Electron microscopic investigations confirmed that in the absence of xanthophylls the S-layer of the cellular envelope is missing. In wild-type (WT) cells, as well as the xanthophyll-less (RO), polyunsaturated lipid-less (AD), and the newly constructed ROAD mutants the lipid and Car compositions were determined by MS and HPLC, respectively. We found that, relative to the WT, the lipid composition of the mutants was remodeled and the Car content changed accordingly. In the mutants the ratio of non-bilayer-forming (NBL) to bilayer-forming (BL) lipids was found considerably lower. Xanthophyll to ß-carotene ratio increased in the AD mutant. In vitro and in vivo methods demonstrated that saturated, monounsaturated lipids and xanthophylls may stabilize the trimerization of Photosystem I (PSI). Fluorescence induction and oxygen-evolving activity measurements revealed increased light sensitivity of RO cells compared to those of the WT. ROAD showed a robust increase in light susceptibility and reduced recovery capability, especially at moderate low (ML) and moderate high (MH) temperatures, indicating a cooperative effect of xanthophylls and polyunsaturated lipids. We suggest that both lipid unsaturation and xanthophylls are required for providing the proper structure and functioning of the membrane environment that protects against light and temperature stress.

Carotenoids Assist in Cyanobacterial Photosystem II Assembly and Function.

Carotenoids (carotenes and xanthophylls) are ubiquitous constituents of living organisms. They are protective agents against oxidative stresses and serve as modulators of membrane microviscosity. As antioxidants they can protect photosynthetic organisms from free radicals like reactive oxygen species that originate from water splitting, the first step of photosynthesis. We summarize the structural and functional roles of carotenoids in connection with cyanobacterial Photosystem II. Although carotenoids are hydrophobic molecules, their complexes with proteins also allow cytoplasmic localization. In cyanobacterial cells such complexes are called orange carotenoid proteins, and they protect Photosystem II and Photosystem I by preventing their overexcitation through phycobilisomes (PBS). Recently it has been observed that carotenoids are not only required for the proper functioning, but also for the structural stability of PBSs.

Elevated growth temperature can enhance photosystem I trimer formation and affects xanthophyll biosynthesis in Cyanobacterium Synechocystis sp. PCC6803 cells.

In the thylakoid membranes of the mesophilic cyanobacterium Synechocystis PCC6803, PSI reaction centers (RCs) are organized as monomers and trimers. PsaL, a 16 kDa hydrophobic protein, a subunit of the PSI RC, was previously identified as crucial for the formation of PSI trimers. In this work, the physiological effects accompanied by PSI oligomerization were studied using a PsaL-deficient mutant (ΔpsaL), not able to form PSI trimers, grown at various temperatures. We demonstrate that in wild-type Synechocystis, the monomer to trimer ratio depends on the growth temperature. The inactivation of the psaL gene in Synechocystis grown phototropically at 30°C induces profound morphological changes, including the accumulation of glycogen granules localized in the cytoplasm, resulting in the separation of particular thylakoid layers. The carotenoid composition in ΔpsaL shows that PSI monomerization leads to an increased accumulation of myxoxantophyll, zeaxanthin and echinenone irrespective of the temperature conditions. These xanthophylls are formed at the expense of ß-carotene. The measured H2O→CO2 oxygen evolution rates in the ΔpsaL mutant are higher than those observed in the wild type, irrespective of the growth temperature. Moreover, circular dichroism spectroscopy in the visible range reveals that a peak attributable to long-wavelength-absorbing carotenoids is apparently enhanced in the trimer-accumulating wild-type cells. These results suggest that specific carotenoids are accompanied by the accumulation of PSI oligomers and play a role in the formation of PSI oligomer structure.

Effect of partial or complete elimination of light-harvesting complexes on the surface electric properties and the functions of cyanobacterial photosynthetic membranes.

Influence of the modification of the cyanobacterial light-harvesting complex [i.e. phycobilisomes (PBS)] on the surface electric properties and the functions of photosynthetic membranes was investigated. We used four PBS mutant strains of Synechocystis sp. PCC6803 as follows: PAL (PBS-less), CK (phycocyanin-less), BE (PSII-PBS-less) and PSI-less/apcE(-) (PSI-less with detached PBS). Modifications of the PBS content lead to changes in the cell morphology and surface electric properties of the thylakoid membranes as well as in their functions, such as photosynthetic oxygen-evolving activity, P700 kinetics and energy transfer between the pigment-protein complexes. Data reveal that the complete elimination of PBS in the PAL mutant causes a slight decrease in the electric dipole moments of the thylakoid membranes, whereas significant perturbations of the surface charges were registered in the membranes without assembled PBS-PSII macrocomplex (BE mutant) or PSI complex (PSI-less mutant). These observations correlate with the detected alterations in the membrane structural organization. Using a polarographic oxygen rate electrode, we showed that the ratio of the fast to the slow oxygen-evolving PSII centers depends on the partial or complete elimination of light-harvesting complexes, as the slow operating PSII centers dominate in the PBS-less mutant and in the mutant with detached PBS.

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.

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.

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.

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.

Lipids, proteins, and their interplay in the dynamics of temperature-stressed membranes of a cyanobacterium, Synechocystis PCC 6803.

Proper responses to low- and high-temperature stresses are essential for the survival of many organisms. It has been established that at low-temperature stress the sufficient microviscosity of the lipids is decisive in this respect. In many organisms, adapting the level of lipid unsaturation to the low growth temperature regulates this feature. At high-temperature stresses, however, there are no unequivocal results concerning the role of the lipids. In these temperature ranges, the lipids are all disordered and fluid and their physical parameters change slowly with increasing temperatures, while biological organisms give characteristic stress responses in rather narrow temperature ranges. Therefore, one may speculate that other membrane parameters/components, which change sharply in the range of the high-temperature stress, may give a signal to initiate the general response of the cells. For such a role, proteins are the trivial candidates. To reveal the role of the lipids and the proteins in these processes, we used a genetically engineered system, based on a cyanobacterium, Synechocystis PCC 6803. In the wild-type cells of this bacterium, by altering the growth temperature, the polyunsaturated lipid content of the cell membranes can be varied considerably (as required by the homeoviscous adaptation principle). In the case of desA(-)/desD(-) mutant cells, which can contain only monounsaturated fatty acyl chains in their lipids, homeoviscous adaptation of the lipids is not possible. Since desA(-)/desD(-) mutation affects only the lipids, additional perturbations (e.g., altered protein content) should minimally disturb the comparison of the lipid behaviors in wild-type and mutant cells. Infrared spectra of thylakoid and cytoplasmic membranes isolated from wild-type and mutant cells were recorded in 3 degrees C steps between 8 and 92 degrees C. By analyzing the rates of protein structural changes, hydrogen-deuterium exchange, in-membrane lipid disorder, and water-membrane interfacial order/hydration as functions of the temperature, we conclude that (i) the gel-to-liquid crystalline phase transition of the lipids correlates with the growth temperature in the wild-type cells but not in the desA(-)/desD(-) mutants, (ii) over the physiological temperature range, both protein and lipid dynamics are regulating/regulated, providing remarkably constant dynamics for both the thylakoid and cytoplasmic membrane, (iii) in the high-temperature stress region, protein structure and dynamics are changing sharply without any correlation with growth temperature and/or mutation, i.e., membrane protein stability does not seem to depend on the lipid composition of the membrane (this finding points to the possible primacy of proteins as initiators/targets of heat-shock alarms), and (iv) there are substantial differences between the dynamics of the proteins of the thylakoid and cytoplasmic membranes, reflecting their different protein complexes and lipid-to-protein ratios.

Phosphatidylglycerol depletion induces an increase in myxoxanthophyll biosynthetic activity in Synechocystis PCC6803 cells.

Phosphatidylglycerol (PG) depletion suppressed the oxygen-evolving activity of Synechocystis PCC6803 pgsA mutant cells. Shortage of PG led to decreased photosynthetic activity, which, similar to the effect of high light exposure, is likely to generate the production of reactive oxygen species (ROS) or free radicals. Protection of the PG-depleted cells against light-induced damage increased the echinenone and myxoxanthophyll content of the cells. The increased carotenoid content was localized in a soluble fraction of the cells as well as in isolated thylakoid and cytoplasmic membranes. The soluble carotenoid fraction contained carotene derivatives, which may bind to proteins. These carotene-protein complexes are similar to orange carotenoid protein that is involved in yielding protection against free radicals and ROS. An increase in the content of myxoxanthophyll and echinenone upon PG depletion suggests that PG depletion regulates the biosynthetic pathway of specific carotenoids.

Lipid-assisted protein-protein interactions that support photosynthetic and other cellular activities.

Glycoglycerolipids are dominant lipids of photosynthetic organisms, i.e. higher plants and cyanobacteria. X-ray crystallographic localization of glycerolipids revealed that they are present at functionally and structurally important sites of both the PS I and PS II reaction centres. Phosphatidylglycerol (PG) is an indispensible member of glycerolipids, including the formation of functionally active oligomers of the reaction centres both PS I and PS II. Lipids assist in the assembly of protein subunits of the photosynthetic machinery by pasting the individual protein components together. PG is needed to glue CP43 to the reaction centre core. PG and digalactosyldiacylglycerol (DGDG) interact in photosynthetic processes: PG alone controls electron transport at the acceptor site of PS II, and together with DGDG is involved in electron transport at the donor site of PS II. PG is crucial for the formation of division rings and is implicated in the fission of cyanobacteria.