Lipid dependence of diadinoxanthin solubilization and de-epoxidation in artificial membrane systems resembling the lipid composition of the natural thylakoid membrane

In the present study, the solubility and enzymatic de-epoxidation of diadinoxanthin (Ddx) was investigated in three different artificial membrane systems: (1) Unilamellar liposomes composed of different concentrations of the bilayer forming lipid phosphatidylcholine (PC) and the inverted hexagonal phase (H(II) phase) forming lipid monogalactosyldiacylglycerol (MGDG), (2) liposomes composed of PC and the H(II) phase forming lipid phosphatidylethanolamine (PE), and (3) an artificial membrane system composed of digalactosyldiacylglycerol (DGDG) and MGDG, which resembles the lipid composition of the natural thylakoid membrane. Our results show that Ddx de-epoxidation strongly depends on the concentration of the inverted hexagonal phase forming lipids MGDG or PE in the liposomes composed of PC or DGDG, thus indicating that the presence of inverted hexagonal structures is essential for Ddx de-epoxidation. The difference observed for the solubilization of Ddx in H(II) phase forming lipids compared with bilayer forming lipids indicates that Ddx is not equally distributed in the liposomes composed of different concentrations of bilayer versus non-bilayer lipids. In artificial membranes with a high percentage of bilayer lipids, a large part of Ddx is located in the membrane bilayer. In membranes composed of equal proportions of bilayer and H(II) phase forming lipids, the majority of the Ddx molecules is located in the inverted hexagonal structures. The significance of the pigment distribution and the three-dimensional structure of the H(II) phase for the de-epoxidation reaction is discussed, and a possible scenario for the lipid dependence of Ddx (and violaxanthin) de-epoxidation in the native thylakoid membrane is proposed.     

Lipid dependence of diadinoxanthin solubilization and de-epoxidation in artificial membrane systems resembling the lipid composition of the natural thylakoid membrane

In the present study, the solubility and enzymatic de-epoxidation of diadinoxanthin (Ddx) was investigated in three different artificial membrane systems: (1) Unilamellar liposomes composed of different concentrations of the bilayer forming lipid phosphatidylcholine (PC) and the inverted hexagonal phase (H_II phase) forming lipid monogalactosyldiacylglycerol (MGDG), (2) liposomes composed of PC and the H_II phase forming lipid phosphatidylethanolamine (PE), and (3) an artificial membrane system composed of digalactosyldiacylglycerol (DGDG) and MGDG, which resembles the lipid composition of the natural thylakoid membrane. Our results show that Ddx de-epoxidation strongly depends on the concentration of the inverted hexagonal phase forming lipids MGDG or PE in the liposomes composed of PC or DGDG, thus indicating that the presence of inverted hexagonal structures is essential for Ddx de-epoxidation. The difference observed for the solubilization of Ddx in H_II phase forming lipids compared with bilayer forming lipids indicates that Ddx is not equally distributed in the liposomes composed of different concentrations of bilayer versus non-bilayer lipids. In artificial membranes with a high percentage of bilayer lipids, a large part of Ddx is located in the membrane bilayer. In membranes composed of equal proportions of bilayer and H_II phase forming lipids, the majority of the Ddx molecules is located in the inverted hexagonal structures. The significance of the pigment distribution and the three-dimensional structure of the H_II phase for the de-epoxidation reaction is discussed, and a possible scenario for the lipid dependence of Ddx (and violaxanthin) de-epoxidation in the native thylakoid membrane is proposed.     

Thylakoid membrane landscape in the sixties: a tribute to Andrew Benson

Prior to the 1960s, the model for the molecular structure of cell membranes consisted of a lipid bilayer held in place by a thin film of electrostatically-associated protein stretched over the bilayer surface: (the Danielli–Davson–Robertson “unit membrane” model). Andrew Benson, an expert in the lipids of chloroplast thylakoid membranes, questioned the relevance of the unit membrane model for biological membranes, especially for thylakoid membranes, instead of emphasizing evidence in favour of hydrophobic interactions of membrane lipids within complementary hydrophobic regions of membrane-spanning proteins. With Elliot Weier, Benson postulated a remarkable subunit lipoprotein monolayer model for thylakoids. Following the advent of freeze fracture microscopy and the fluid lipid-protein mosaic model by Singer and Nicolson, the subunits, membrane-spanning integral proteins, span a dynamic lipid bilayer. Now that high resolution X-ray structures of photosystems I and II are being revealed, the seminal contribution of Andrew Benson can be appreciated.     

Coupling between lipid shape and membrane curvature

Using molecular dynamics simulations, we examine the behavior of lipids whose preferred curvature can be systematically varied. This curvature is imposed by controlling the headgroup size of a coarse-grained lipid model recently developed by us. To validate this approach, we examine self-assembly of each individual lipid type and observe the complete range of expected bilayer and micelle phases. We then examine binary systems consisting of lipids with positive and negative preferred curvature and find a definite sorting effect. Lipids with positive preferred curvature are found in greater proportions in outer monolayers with the opposite observed for lipids with negative preferred curvature. We also observe a similar, but slightly stronger effect for lipids in a developing spherical bud formed by adhesion to a colloid (e.g., a viral capsid). Importantly, the magnitude of this effect in both cases was large only for regions with strong mean curvature (radii of curvature <10 nm). Our results suggest that lipid shape must act in concert with other physico-chemical effects such as phase transitions or interactions with proteins to produce strong sorting in cellular pathways.     

Lipid interacting regions in phosphate stress glycosyltransferase atDGD2 from Arabidopsis thaliana

Membrane lipid glycosyltransferases (GTs) in plants are enzymes that regulate the levels of the non-bilayer prone monogalactosyldiacylglycerol (GalDAG) and the bilayer-forming digalactosyldiacylglycerol (GalGalDAG). The relative amounts of these lipids affect membrane properties such as curvature and lateral stress. During phosphate shortage, phosphate is rescued by replacing phospholipids with GalGalDAG. The glycolsyltransferase enzyme in Arabidopsis thaliana responsible for this, atDGD2, senses the bilayer properties and interacts with the membrane in a monotopic manner. To understand the parameters that govern this interaction, we have identified several possible lipid-interacting sites in the protein and studied these by biophysical techniques. We have developed a multivariate discrimination algorithm that correctly predicts the regions in the protein that interact with lipids, and the interactions were confirmed by a variety of biophysical techniques. We show by bioinformatic methods and circular dichroism (CD), fluorescence, and NMR spectroscopic techniques that two regions are prone to interact with lipids in a surface-charge dependent way. Both of these regions contain Trp residues, but here charge appears to be the dominating feature governing the interaction. The sequence corresponding to residues 227-245 in the protein is seen to be able to adapt its structure according to the surface-charge density of a bilayer. All results indicate that this region interacts specifically with lipid molecules and that a second region in the protein, corresponding to residues 130-148, also interacts with the bilayer. On the basis of this, and sequence charge features in the immediate environment of S227-245, a response model for the interaction of atDGD2 with the membrane bilayer interface is proposed.     

Functional roles of the major chloroplast lipids in the violaxanthin cycle

Monogalactosyldiacylglyceride (MGDG) and digalactosyldiacylglyceride (DGDG) are the major membrane lipids of chloroplasts. The question of the specialized functions of these unique lipids has received limited attention. One function is to support violaxanthin de-epoxidase (VDE) activity, an enzyme of the violaxanthin cycle. To understand better the properties of this system, the effects of galactolipids and phosphatidylcholines on VDE activity were examined by two independent methods. The results show that the micelle-forming lipid (MGDG) and bilayer forming lipids (DGDG and phosphatidylcholines) support VDE activity differently. MGDG supported rapid and complete de-epoxidation starting at a threshold lipid concentration (10 μM) coincident with complete solubilization of violaxanthin. In contrast, DGDG supported slow but nevertheless complete to nearly complete de-epoxidation at a lower lipid concentration (6.7 μM) that did not completely solubilize violaxanthin. Phosphotidylcholines showed similar effects as DGDG except that de-epoxidation was incomplete. Since VDE requires solubilized violaxanthin, aggregated violaxanthin in DGDG at low concentration must become solubilized as de-epoxidation proceeds. High lipid concentrations had lower activity possibly due to formation of multilayered structures (liposomes) that restrict accessibility of violaxanthin to VDE. MGDG micelles do not present such restrictions. The results indicate VDE operates throughout the lipid phase of the single bilayer thylakoid membrane and is not limited to putative MGDG micelle domains. Additionally, the results also explain the differential partitioning of violaxanthin between the envelope and thylakoid as due to the relative solubilities of violaxanthin and zeaxanthin in MGDG, DGDG and phospholipids. The violaxanthin cycle is hypothesized to be a linked system of the thylakoid and envelope for signal transduction of light stress.     

Permeability and electrical properties of planar lipid membranes from thylakoid lipids.

Electrical measurements were carried out on planar lipid membranes from thylakoid lipids. The specific capacitance of membranes formed from decane-containing monogalactosyldiacylglycerol (MGDG), which accounts for 57% of the total lipid content of thylakoids, showed that it adopted a bilayer structure. Solvent-free bilayers of MGDG were not formed, with very rare exceptions, indicating that decane is required to stabilize the planar conformation. However, this cone-shaped lipid produces bilayer structures in combination with other cylindrical thylakoid lipids even in the absence of organic solvent. We compared the properties of solvent-free and decane-containing bilayers from MGDG, soybean lecithin, and the quaternary mixture of lipids similar to that found in vivo. The conductance of decane-MGDG was 26 times higher than that of decane-lecithin. The flux through the decane-lecithin bilayer was found to be slightly dependent on pH, whereas the decane-MGDG membrane was not. The specific conductance of bilayers formed from the quaternary mixture of lipids was 5 to 10 times larger than lecithin (with alkane or not). Further experiments with bilayers made in the presence of a KCl gradient showed that decane-MGDG, decane-MGDG/DGDG/SQDG/PG, and solvent-free MGDG/DGDG/SQDG/PG were cation-selective. The permeability coefficient for potassium ranged from 4.9 to 8.3 x 10(-11) cm s-1. The permeability coefficient for protons in galactolipids, however, was determined to be about six orders of magnitude higher than the value for potassium ions. The HCl permeation mechanism through the lipid membranes was determined from diffusion potentials measured in HCl gradients. Our results suggest that HCl was not transported as neutral molecules. The data is discussed with regard to the function of galactolipids in the ion transport through thylakoid membranes.     

Increased thermal stability of photosystem II and the macro-organization of thylakoid membranes,induced by co-solutes,associated with changes in the lipid-phase behaviour of thylakoid membranes

Photosynthetica - The principal function of the thylakoid membrane depends on the integrity of the lipid bilayer, yet almost half of the thylakoid lipids are of non-bilayer-forming type, whose...     

Molecular mechanisms of spontaneous curvature and softening in complex lipid bilayer mixtures

Membrane reshaping is an essential biological process. The chemical composition of lipid membranes determines their mechanical properties and thus the energetics of their shape. Hundreds of distinct lipid species make up native bilayers, and this diversity complicates efforts to uncover what compositional factors drive membrane stability in cells. Simplifying assumptions, therefore, are used to generate quantitative predictions of bilayer dynamics based on lipid composition. One assumption commonly used is that “per lipid” mechanical properties are both additive and constant—that they are an intrinsic property of lipids independent of the surrounding composition. Related to this is the assumption that lipid bulkiness, or “shape,” determines its curvature preference, independently of context. In this study, all-atom molecular dynamics simulations on three separate multilipid systems were used to explicitly test these assumptions, applying methodology recently developed to isolate properties of single lipids or nanometer-scale patches of lipids. The curvature preference experienced by populations of lipid conformations were inferred from their redistribution on a dynamically fluctuating bilayer. Representative populations were extracted by both structural similarity and semi-automated hidden Markov model analysis. The curvature preferences of lipid dimers were then determined and compared with an additive model that combines the monomer curvature preference of both the individual lipids. In all three systems, we identified conformational subpopulations of lipid dimers that showed non-additive curvature preference, in each case mediated by a special chemical interaction (e.g., hydrogen bonding). Our study highlights the importance of specific chemical interactions between lipids in multicomponent bilayers and the impact of interactions on bilayer stiffness. We identify two mechanisms of bilayer softening: diffusional softening, driven by the dynamic coupling between lipid distributions and membrane undulations, and conformational softening, driven by the inter-conversion between distinct dimeric conformations.     

Lipid-engineered Escherichia coli membranes reveal critical lipid headgroup size for protein function

Escherichia coli membranes have a substantial bilayer curvature stress due to a large fraction of the nonbilayer-prone lipid phosphatidylethanolamine, and a mutant (AD93) lacking this lipid is severely crippled in several membrane-associated processes. Introduction of four lipid glycosyltransferases from Acholeplasma laidlawii and Arabidopsis thaliana, synthesizing large amounts of two nonbilayer-prone, and two bilayer-forming gluco- and galacto-lipids, (i) restored the curvature stress with the two nonbilayer lipids, and (ii) diluted the high negative lipid surface charge in all AD93 bilayers. Surprisingly, the bilayer-forming diglucosyl-diacylglycerol was almost as good in improving AD93 membrane processes as the two nonbilayer-prone glucosyl-diacylglycerol and galactosyl-diacylglycerol lipids, strongly suggesting that lipid surface charge dilution by these neutral lipids is very important for E. coli. Increased acyl chain length and unsaturation, plus cardiolipin (nonbilayer-prone) content, were probably also beneficial in the modified strains. However, despite a correct transmembrane topology for the transporter LacY in the diglucosyl-diacylglycerol clone, active transport failed in the absence of a nonbilayer-prone glycolipid. The corresponding digalactosyl-diacylglycerol bilayer lipid did not restore AD93 membrane processes, despite analogous acyl chain and cardiolipin contents. Chain ordering, probed by bis-pyrene lipids, was substantially lower in the digalactosyl-diacylglycerol strain lipids due to its extended headgroup. Hence, a low surface charge density of anionic lipids is important in E. coli membranes, but is inefficient if the headgroup of the diluting lipid is too large. This strongly indicates that a certain magnitude of the curvature stress is crucial for the bilayer in vivo.     

Membrane curvature stress controls the maximal conversion of violaxanthin to zeaxanthin in the violaxanthin cycle--influence of alpha-tocopherol, cetylethers, linolenic acid, and temperature

Zeaxanthin, an important component in protection against overexcitation in higher plants, is formed from violaxanthin by the enzyme violaxanthin de-epoxidase. We have investigated factors that may control the maximal degree of conversion in the violaxanthin cycle. The conversion of violaxanthin to zeaxanthin in isolated spinach thylakoids was followed at different temperatures and in the presence of lipid packing modifiers. The maximum degree of conversion was found to be 35%, 70% and 80% at 4 degrees C, 25 degrees C and 37 degrees C respectively. In the presence of membrane modifying agents, known to promote non-lamellar structures (H(II)), such as linolenic acid the conversion increased, and the maximal level of violaxanthin de-epoxidation obtained was close to 100%. In contrast, substances promoting lamellar phases (L(alpha)), such as alpha-tocopherol and 8-cetylether (C(16)EO(8)), only 55% and 35% of the violaxanthin was converted at 25 degrees C, respectively. The results are interpreted in light of the lipid composition of the thylakoid membrane, and we propose a model where a negative curvature elastic stress in the thylakoid lipid bilayer is required for violaxanthin de-epoxidase activity. In this model zeaxanthin with its longer hydrophobic stretch is proposed to promote lamellar arrangements of the membrane. As a result, zeaxanthin relieves the curvature elastic stress, which in turn leads to inactivation of violaxanthin de-epoxidase.     

Cytochrome c-lipid interactions studied by resonance Raman and 31P NMR spectroscopy. Correlation between the conformational changes of the protein and the lipid bilayer.

The interaction of cytochrome c with negatively charged lipids has been studied by resonance Raman spectroscopy of the protein heme group and 31P NMR of the phospholipid headgroups. The gel-to-fluid-phase transition of dimyristoylphosphatidylglycerol induces shifts in the conformational and coordination equilibria of the bound cytochrome c, as recorded by the resonance Raman spectra in the fingerprint and marker band regions. Conformational and coordination shifts of the bound cytochrome are also induced on admixture of dioleoylglycerol or dioleoylphosphatidylcholine with dioleoylphosphatidylglycerol. In the case of dioleoylglycerol, significant changes take place even at levels as low as 5 mol %. Binding of cytochrome c induces or increases the content of near isotropically diffusing lipid registered by the 31P NMR spectra of the different lipids studied. Admixture of dioleoylglycerol also increases the bilayer curvature of dioleoylphosphatidylglycerol, inducing an inverted hexagonal phase at 50 mol % concentration; the tendency to spontaneous curvature in the lipid appears to relax the conformational change detected in the protein.     

Solid-state NMR investigation of the depth of insertion of protegrin-1 in lipid bilayers using paramagnetic Mn2+

The depth of insertion of an antimicrobial peptide, protegrin-1 (PG-1), in lipid bilayers is investigated using solid-state NMR. Paramagnetic Mn(2+) ions bind to the surface of lipid bilayers and induce distance-dependent dipolar relaxation of nuclear spins. By comparing the signal dephasing of the peptide with that of the lipids, whose segmental depths of insertion are known, we determined the depths of several residues of PG-1 in 1,2 dilauryl-sn-glycero-3-phosphotidylcholine (DLPC) bilayers. We found that residues G2 at the N-terminus and F12 at the beta-turn of the peptide reside near the membrane surface, whereas L5 and V16 are embedded in the acyl chain region. The depths increase in the order of G2 < F12 < L5 < V16. These intensity-dephasing results are confirmed by direct measurement of the paramagnetically enhanced (13)C transverse relaxation rates. The relative depths indicate that PG-1 is tilted from the bilayer normal, which is consistent with independent solid-state NMR measurements of PG-1 orientation in the same lipids (Yamaguchi et al., 2001). They also indicate that PG-1 is fully immersed in the lipid bilayer. However, a quantitative mismatch between the bilayer thickness and PG-1 length suggests a local thinning of the DLPC bilayer by 8-10 A. The depth sensitivity of this Mn(2+) dephasing technique is tunable with the Mn(2+) concentration to focus on different regions of the lipid bilayer.     

Cholesterol and phosphatidylethanolamine lipids exert opposite effects on membrane modulations caused by the M2 amphipathic helix

Membrane curvature remodeling induced by amphipathic helices (AHs) is essential in many biological processes. Here we studied a model amphipathic peptide, M2AH, derived from influenza A M2. We are interested in how M2AH may promote membrane curvature by altering membrane physical properties. We used atomic force microscopy (AFM) to examine changes in membrane topographic and mechanical properties. We used electron paramagnetic resonance (EPR) spectroscopy to explore changes in lipid chain mobility and chain orientational order. We found that M2AH perturbed lipid bilayers by generating nanoscale pits. The structural data are consistent with lateral expansion of lipid chain packing, resulting in a mechanically weaker bilayer. Our EPR spectroscopy showed that M2AH reduced lipid chain mobility and had a minimal effect on lipid chain orientational order. The EPR data are consistent with the surface-bound state of M2AH that acts as a chain mobility inhibitor. By comparing results from different lipid bilayers, we found that cholesterol enhanced the activity of M2AH in inducing bilayer pits and altering lipid chain mobility. The results were explained by considering specific M2AH-cholesterol recognition and/or cholesterol-induced expansion of interlipid distance. Both AFM and EPR experiments revealed a modest effect of anionic lipids. This highlights that membrane interaction of M2AH is mainly driven by hydrophobic forces. Lastly, we found that phosphatidylethanolamine (PE) lipids inhibited the activity of M2AH. We explained our data by considering interlipid hydrogen-bonding that can stabilize bilayer organization. Our results of lipid-dependent membrane modulations are likely relevant to M2AH-induced membrane restructuring.     

Modulation of PI-specific phospholipase C by membrane curvature and molecular order

Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus has been assayed on large and small unilamellar vesicles consisting of PI, either pure or in mixtures with other lipids. Vesicle diameter (in the 50-300 nm range) influences PI-PLC activity, enzyme rates increasing with decreasing curvature radii. With sonicated unilamellar vesicles of pure PI, two apparent K(s) values are observed, one in the 0-2 mM concentration range and the other in the 2-12 mM concentration range. The latter ( approximately 4.2 mM) corresponds to previously published values, while the low-concentration K(s) is on the same order of magnitude as the single apparent K(m) value found with large unilamellar liposomes ( approximately 0.30 mM). PI-PLC appears to be very sensitive to bilayer composition. Certain nonsubstrate lipids, e.g., galactosylceramide or cholesterol, inhibit PI-PLC in a dose-dependent way, at least up to 33 mol % in the bilayers, under conditions with a constant PI concentration. Simultaneous measurements of enzyme activity, interfacial enzyme binding, and fluorescence of different probes, on a variety of bilayer compositions, reveal that both the level of enzyme binding and activity decrease with increasing lipid order, as measured by the fluorescence polarization of the hydrophobic probe diphenylhexatriene. In contrast, no correlation is found for enzyme activity with fluorescence changes of probes, e.g., laurdan, that report on phenomena occurring mainly at the lipid-water interface. Sphingomyelin has a dual effect. Up to 40 mol %, it increases PI-PLC activity, with little effect on bilayer molecular order. At higher proportions, the increased lipid chain order causes a decrease in enzyme activity. The same effects are observed for distearoylphosphatidylcholine when added to PI bilayers. These results support the "two-stage model" for binding of PI-PLC to lipid bilayers, and underline the significance of the enzyme partial penetration into the membrane hydrophobic matrix for its catalytic activity.     

Photosynthetic energy conversion under extreme conditions--II: the significance of lipids under light limited growth in Antarctic sea ice diatoms

Low photosynthetic active radiation is a strong determinant in the development and growth of sea ice algae. The algae appear to have universal mechanisms to overcome light limitation. One important process, which is induced under light limitation, is the desaturation of chloroplast membrane lipids. In order to discover whether this process is universally valid in sea ice diatoms, we investigated three species coexisting in chemostats illuminated with 15 and 2 micromol photons m(-2) s(-1) at -1 degrees C. Growth under 2 micromol photons m(-2) s(-1) caused a 50% increase in monogalactosyldiacylglycerols (MGDG) thylakoid membrane related 20:5 n-3 fatty acids. This fatty acid supports the fluidity of the thylakoid membrane and therefore the velocity of electron flow, which is indicated by increasing rate constants for the electron transport between Q(A) (first stable electron acceptor) and bound Q(B) (second stable electron acceptor) (11.16 +/- 1.34 to 23.24 +/- 1.35 relative units). Two micromol photons m(-2) s(-1) furthermore resulted in higher amounts of non-lipid bilayer forming MGDG in relation to other bilayer forming lipids, especially digalactosydiacylglycerol (DGDG). The ratio of MGDG:DGDG increased from 3.4 +/- 0.3 to 5.7 +/- 0.3. The existence of bilayer thylakoid membranes with high proportions of non. bilayer forming lipids is only possible when sufficient thylakoid pigment-protein complexes are present. If more thylakoid pigment-protein complexes are present in membranes, as found under extreme light limitation, less bilayer forming lipids such as DGDG are required to stabilize the bilayer structure. Differences in protein contents between both light intensities were not found. Consequently pigment contents which nearly doubled under 2 micromol photons m(-2) s(-1) must be responsible in balancing the potential stability loss resulting from an increase in MGDG:DGDG ratio.     

The lipid dependence of diadinoxanthin de-epoxidation presents new evidence for a macrodomain organization of the diatom thylakoid membrane

The present study shows that thylakoid membranes of the diatom Cyclotella meneghiniana contain much higher amounts of negatively charged lipids than higher plant or green algal thylakoids. Based on these findings, we examined the influence of SQDG on the de-epoxidation reaction of the diadinoxanthin cycle and compared it with results from the second negatively charged thylakoid lipid PG. SQDG and PG exhibited a lower capacity for the solubilization of the hydrophobic xanthophyll cycle pigment diadinoxanthin than the main membrane lipid MGDG. Although complete pigment solubilization took place at higher concentrations of the negatively charged lipids, SQDG and PG strongly suppressed the de-epoxidation of diadinoxanthin in artificial membrane systems. In in vitro assays employing the isolated diadinoxanthin cycle enzyme diadinoxanthin de-epoxidase, no or only a very weak de-epoxidation reaction was observed in the presence of SQDG or PG, respectively. In binary mixtures of the inverted hexagonal phase forming lipid MGDG with the negatively charged bilayer lipids, comparable suppression took place. This is in contrast to binary mixtures of MGDG with the neutral bilayer lipids DGDG and PC, where rapid and efficient de-epoxidation was observed. In complex lipid mixtures resembling the lipid composition of the native diatom thylakoid membrane, we again found strong suppression of diadinoxanthin de-epoxidation due to the presence of SQDG or PG. We conclude that, in the native thylakoids of diatoms, a strict separation of the MGDG and SQDG domains must occur; otherwise, the rapid diadinoxanthin de-epoxidation observed in intact cells upon illumination would not be possible.     

Phase behavior of phosphatidylglycerol in spinach thylakoid membranes as revealed by 31P-NMR

Non-bilayer lipids account for about half of the total lipid content in chloroplast thylakoid membranes. This lends high propensity of the thylakoid lipid mixture to participate in different phases which might be functionally required. It is for instance known that the chloroplast enzyme violaxanthin de-epoxidase (VDE) requires a non-bilayer phase for proper functioning in vitro but direct evidence for the presence of non-bilayer lipid structures in thylakoid membranes under physiological conditions is still missing. In this work, we used phosphatidylglycerol (PG) as an intrinsic bulk lipid label for 31P-NMR studies to monitor lipid phases of thylakoid membranes. We show that in intact thylakoid membranes the characteristic lamellar signal is observed only below 20 degrees C. But at the same time an isotropic phase is present, which becomes even dominant between 14 and 28 degrees C despite the presence of fully functional large membrane sheets that are capable of generating and maintaining a transmembrane electric field. Tris-washed membranes show a similar behavior but the lamellar phase is present up to higher temperatures. Thus, our data show that the location of the phospholipids is not restricted to the bilayer phase and that the lamellar phase co-exists with a non-bilayer isotropic phase.