Correlation of structure and function of chloroplast membranes at the supramolecular level

Freeze-fracture electron microscopy has revealed that different size classes of intramembrane particles of chloroplast membranes are nonrandomly distributed between appressed grana and nonappressed stroma membrane regions. It is now generally assumed that thylakoid membranes contain five major functional complexes, each of which can give rise to an intramembrane particle of a defined size. These are the photosystem II complex, the photosystem I complex, the cytochrome f/b6 complex, the chlorophyll a/b light-harvesting complex, and the CF0 -CF1 ATP synthetase complex. By mapping the distribution of the different categories of intramembrane particles, information on the lateral organization of functional membrane units of thylakoid membranes can be determined. In this review, we present a brief summary of the evidence supporting the correlation of specific categories of intramembrane particles with known biochemical entities. In addition, we discuss studies showing that ions and phosphorylation of the membrane adhesion factor, the chlorophyll a/b light-harvesting complex, can affect the lateral organization of chloroplast membrane components and thereby regulate membrane function.     

Lateral organization of biological membranes

The lateral organization of biological membranes is of great importance in many biological processes, both for the formation of specific structures such as super-complexes and for function as observed in signal transduction systems. Over the last years, AFM studies, particularly of bacterial photosynthetic membranes, have revealed that certain proteins are able to segregate into functional domains with a specific organization. Furthermore, the extended non-random nature of the organization has been suggested to be important for the energy and redox transport properties of these specialized membranes. In the work reported here, using a coarse-grained Monte Carlo approach, we have investigated the nature of interaction potentials able to drive the formation and segregation of specialized membrane domains from the rest of the membrane and furthermore how the internal organization of the segregated domains can be modulated by the interaction potentials. These simulations show that long-range interactions are necessary to allow formation of membrane domains of realistic structure. We suggest that such possibly non-specific interactions may be of great importance in the lateral organization of biological membranes in general and in photosynthetic systems in particular. Finally, we consider the possible molecular origins of such interactions and suggest a fundamental role for lipid-mediated interactions in driving the formation of specialized photosynthetic membrane domains. We call these lipid-mediated interactions a ‘lipophobic effect.’     

Ultrastructural organization of chloroplast membranes in mutants of Pisum sativum L. with impaired activity in the photosystems

The ultrastructural organization and the photosynthesis reactions of chloroplast membranes were studied in three lethal mutants of Pisum sativum, Chl-1, Chl-19 and Chl-5, all lacking the capacity to evolve oxygen. The rates of 2,6-dichloroindophenol reduction, delayed fluorescence and electron-spin-resonance signal 1 indicate that Chl-1 and Chl-19 have an impaired activity in photosystem II (PS II), while in Chl-5 the electron transport is blocked between PS I and the reactions of CO₂ fixation. Ultrathin sectioning demonstrates the presence of giant grana in the chloroplasts of Chl-1 and Chl-19, while the chloroplast structure of the Chl-5 is very similar to that of the wild-type. The grana of the Chl-19 mutant contain large multilamellar regions of tightly packed membranes. When the chloroplast membranes were studied by freeze-fracture, the exoplasmic and protoplasmic fracture faces (EF and PF, respectively) in both stacked and unstacked membranes were found to show large differences in particle concentrations and relative population area (per m²), and also in particle size distribution, between all mutant chloroplast membranes and the wild-type. A close correlation between increasing k_mt (ratio of particle concentrations on PF/EF) and PS II activity was observed. The differences in particle concentrations on both fracture faces in different regions of the intact chloroplast membranes of the wild-type are the consequence of a rearrangement of existing membrane components by lateral particle movements since quantitative measurements demonstrate almost complete conservation of intramembrane particles in number and size during the stacking of stroma thylakoid membranes. The results indicating particle movements strongly support the concept that the chloroplast membranes have a highly dynamic structure.Abbreviations DPIP 2,6-dichloroindophenol - EF and PF exoplasmic and protoplasmic fracture faces, respectively - PS I and PS II photosystems I and II, respectively     

Application of 31P-NMR saturation transfer techniques to investigate phospholipid motion and organization in model and biological membranes

The potential of ³¹P-NMR saturation transfer experiments for determining motional characteristics (in the millisecond to second time scale) of phospholipids in model and biological membranes is demonstrated. A technique to separate membrane phospholipid ³¹P-NMR signals from those of small water-soluble phosphates in intact cells in liver tissue is also illustrated.     

Cholesterol domains in biological membranes

Membrane cholesterol is distributed asymmetrically both within the cell or within cellular membranes. Elaboration of intracellular cholesterol trafficking, targeting and intramembrane distribution has been spurred by both molecular and structural approaches. The expression of recombinant sterol carrier proteins in L-cell fibroblasts has been especially useful in demonstrating for the first time that such proteins actually elicit intracellular and intra-plasma membrane redistribution of sterol. Additional advances in the use of native fluorescent sterols allowed resolution of transbilayer and lateral cholesterol domains in plasma membranes from cultured fibroblasts, brain synaptosomes and erythrocytes. In all three cell surface membranes, cholesterol is enriched in the inner, cytofacial leaflet. Up to three different cholesterol domains have been identified in the lateral plane of the plasma membrane: a fast exchanging domain comprising less than 10% of cholesterol, a slowly exchanging domain comprising about 30% of cholesterol, and a very slowly or non-exchangeable sterol domain comprising 50–60.

Of plasma membrane cholesterol. Factors modulating plasma membrane cholesterol domains include polyunsaturated fatty acids, expression of intracellular sterol carrier proteins, drugs such as ethanol, and several membrane pathologies (systemic lupus erythematosus, sickle cell anaemia and aging). Disturbances in plasma membrane cholesterol domains after transbilayer fluidity gradients in plasma membranes. Such changes are associated with decreased Ca²⁺ -ATPase and Na ⁺, K⁺ -ATPase activity. Thus, the size, dynamics and distribution of cholesterol domains within membranes not only regulate cholesterol efflux/influx but also modulate plasma membrane protein functions and receptor-effector coupled systems.     

Model for cooperativity of biological membranes

We present a mathematical model for the complex cooperativity observed in biological membranes. In our model, it is assumed that the proteins bound on the membrane are noncooperative and possess a Bohr proton. It is further assumed that the net charge of the unliganded state of the protein is different from that of the liganded state owing to the structural change upon binding the ligand. With this model, we show how an all-or-none response, a graded response, and a noncooperative response arise in the binding curve of such biological membranes. In addition, we show how an effector, which can alter the pK_a involved in the binding site, induces a complex cooperativity.     

Detection of protoporphyrin IX in envelope membranes of pea chloroplasts

Envelope membranes were prepared from mature pea chloroplasts. The tetrapyrrole contents of envelope membranes were analysed. The envelope membranes of pea chloroplasts contained substantial amounts of protoporphyrin IX and trace amounts of Mg-protoporphyrin IX and its monoester in addition to protochlorophyllide. The protoporphyrin IX content of envelope membranes was 89.25 pmol (mg protein)(-1). Its content in pea envelope membrane was higher than that of protochlorophyllide. The proportion of monovinyl and divinyl forms of protochlorophyllide present in pea chloroplast envelope membrane was 3:7. The significance of the presence of protoporphyrin IX in the envelope membrane is discussed in relation to plastidic Chl biosynthesis.     

Domains in biological membranes

Our current view on biological membranes has gradually evolved from the influential fluid mosaic model of the early 1970s to a distinctively more complex picture. Biological membranes are now assumed to encompass multiple membrane domains and a plethora of protein-lipid and protein-protein interactions that compartmentalize and temporarily order what has originally been envisioned to be mostly random. In this minireview, we will first highlight some structural principles that govern membrane domain formation and permit a classification of membrane domains. We will then focus on the still controversial issue of lipid-based membrane domains, or lipid rafts, and discuss recent advances in detecting these enigmatic structures in living cells. Finally we will evaluate biochemical approaches to characterize lipid rafts and discuss their contribution to the emerging topic of lipid raft diversity     

Power transmission along biological membranes

Hypothesis on long-distance power transmission along extended energy-transducing membranes (Skulachev, 1969, 1971, 1980), has been experimentally proven in four different systems, namely, (i) trichomes of filamentous cyanobacterium Phormidium uncinatum; (ii) filamentous mitochondria and mitochondrial network in fibroblasts; (iii) clusters of roundish heart muscle mitochondria interconnected with mitochondrial junctions; (iv) mixed animal cell cultures interconnected with gap junctions. In all cases, energy was shown to be transmitted in the form of a transmembrane electric potential difference. The transmission occurred for distances as long as several tens of micrometers. Since the (a) delta-muH-bearing cytoplasmic membrane of cyanobacteria and the inner mitochondrial membrane and (b) delta-muNa-bearing outer animal cell membrane were found to be competent in such an effect, one may assume that the power transmission is a fundamental function of extended membrane systems. This mechanism can be used at the intracellular level (mitochondrial) as well as at the supracellular level (cytoplasmic and outer cell membranes). Studies on the possible involvement of membranes in lateral transport of oxygen, ions, fatty acids and membrane proteins seem to hold good promise.     

Cytochrome b-560, a new component of thylakoid membranes

During a survey of a series of electron transport mutants of Chlamydomonas reinhardtii, we have observed a hitherto unrecognised component, cytochrome b-560, in a strain (F18) which entirely lacks the cytochrome bf complex. This component is present at approx. 1 nmol/mg chlorophyll, has a midpoint potential of −125 mV (n = 1) at pH 7.2 and is slowly reduced by dithionite. The cytochrome was also observed in thylakoid membranes prepared from wild-type Chlamydomonas and from higher plants after extraction of the cytochrome bf complex by detergent treatment. It could not be observed in thylakoid membrane fragments prepared from the cyanobacterium Phormidium laminosum.     

The control of transmembrane helix transverse position in membranes by hydrophilic residues

The ability of hydrophilic residues to shift the transverse position of transmembrane (TM) helices within bilayers was studied in model membrane vesicles. Transverse shifts were detected by fluorescence measurements of the membrane depth of a Trp residue at the center of a hydrophobic sequence. They were also estimated from the effective length of the TM-spanning sequence, derived from the stability of the TM configuration under conditions of negative hydrophobic mismatch. Hydrophilic residues (at the fifth position in a 21-residue hydrophobic sequence composed of alternating Leu and Ala residues and flanked on both ends by two Lys) induced transverse shifts that moved the hydrophilic residue closer to the membrane surface. At pH 7, the dependence of the extent of shift upon the identity of the hydrophilic residue increased in the order: L < GYT < RH < S < P < K < EQ < N < D. By varying pH, shifts with ionizable residues fully charged or uncharged were measured, and the extent of shift increased in the order: L < GYH^oT < E^oR < S < P < K⁺< QD^oH⁺ < NE^– < D^–. The dependence of transverse shifts upon hydrophilic residue identity was consistent with the hypothesis that shift magnitude is largely controlled by the combination of side chain hydrophilicity, ionization state, and ability to position polar groups near the bilayer surface (snorkeling). Additional experiments showed that shift was also modulated by the position of the hydrophilic residue in the sequence and the hydrophobicity of the sequence moved out of the bilayer core upon shifting. Combined, these studies show that the insertion boundaries of TM helices are very sensitive to sequence, and can be altered even by weakly hydrophilic residues. Thus, many TM helices may have the capacity to exist in more than one transverse position. Knowledge of the magnitudes of transverse shifts induced by different hydrophilic residues should be useful for design of mutagenesis studies measuring the effect of transverse TM helix position upon function.     

One of the protein components of lens fibre membranes is glyceraldehyde 3-phosphate dehydrogenase

The chloroplast envelope contains an acyl-CoA synthetase and an acyl-CoA thioesterase which are associated with the outer and inner membrane, respectively.     

Supramolecular organization of ATP synthase and respiratory chain in mitochondrial membranes

Mitochondrial ATP synthase is mostly isolated in monomeric form, but in the inner mitochondrial membrane it seems to dimerize and to form higher oligomeric structures from dimeric building blocks. Following a period of electron microscopic single particle analyses that revealed an angular orientation of the membrane parts of monomeric ATP synthases in the dimeric structures, and after extensive studies of the monomer-monomer interface, the focus now shifts to the potentially dynamic state of the oligomeric structures, their potential involvement in metabolic regulation of mitochondria and cells, and to newly identified interactions like physical associations of complexes IV and V. Similarly, larger structures like respiratory strings that have been postulated to form from individual respiratory complexes and their supercomplexes, the respirasomes, come into the focus. Progress by structural investigations is paralleled by insights into the functional roles of respirasomes including substrate channelling and stabilization of individual complexes. Cardiolipin was found to be important for the structural stability of respirasomes which in turn is required to maintain cells and tissues in a healthy state. Defects in cardiolipin remodeling cause devastating diseases like Barth syndrome. Novel species-specific roles of respirasomes for the stability of respiratory complexes have been identified, and potential additional roles may be deduced from newly observed interactions of respirasomes with components of the protein import machinery and with the ADP/ATP translocator.     

The formation of non-bilayer structures in total polar lipid extracts of chloroplast membranes

The structural organisation of aqueous dispersions of total membrane lipid extracts of broad bean (Vicia faba) chloroplasts is dependent on pH and the presence of cations. In the absence of inorganic salts, sonicated dispersions of lipid extract in distilled water form smooth, single-shell vesicles approximately 30–50 nm in diameter. Reducing the pH of the dispersions, to neutralise the acidic lipids present in the extract, or the addition of low concentrations of metal cations, leads to the fusion of the vesicles and a partial phase-separation of the non-bilayer forming lipid monogalactosyldiacylglycerol to form spherical inverted micelles similar to those previously reported for binary mixtures of monogalactosyl and digalactosyldiacylglycerol (Biochim. Biophys. Acta 685, 297–306). Increasing concentrations of polyvalent, but not monovalent, cations lead to further structural rearrangements involving the formation of para-crystalline arrays of tubular and spherical inverted micelles. The factors determining the formation of these different structures, and their possible relevance to the structural organisation of the native chloroplast membrane, are discussed.     

Integrating effects of contaminants across levels of biological organization: an overview

Effects of contaminants may occur at all levels oforganization, from molecular to ecosystem-levelresponses. While biochemical and physiologicalalterations in organisms may occur rapidly and areoften stressor-specific, the ecological relevance ofthese suborganismal indicators is uncertain.Alterations in populations and communities havegreater ecological relevance, but a firm mechanisticunderstanding of these responses is often lacking.Developing mechanistic linkages across levels ofbiological organization would greatly improve ourunderstanding of how organisms are affected bycontaminants in nature. The papers in this seriespresent several innovative approaches for integratingeffects of contaminants across levels of biologicalorganization. Authors were asked to describe theecological consequences of responses at lower levelsof organization (biochemical, physiological,individual) and to speculate on the underlyingmechanisms associated with population and communityalterations. The most consistent finding of the fivepapers in this series is that there is no singlespatiotemporal scale or level of biologicalorganization at which ecotoxicological investigationsshould be conducted.     

Effects of 2-hydroxyoleic acid on the structural properties of biological and model plasma membranes

Genetic hypertension is associated with alterations in lipid metabolism, membrane lipid composition and membrane-protein function. 2-Hydroxyoleic acid (2OHOA) is a new antihypertensive molecule that regulates the structure of model membranes and their interaction with certain peripheral signalling proteins in vitro. While the effect of 2OHOA on elevated blood pressure is thought to arise through its influence on signalling proteins, its effects on membrane lipid composition remain to be assessed. 2OHOA administration altered the lipid membrane composition of hypertensive and normotensive rat plasma membranes, and increased the fluidity of reconstituted liver membranes from hypertensive rats. In spontaneously hypertensive rats (SHR), treatment with 2OHOA increased the cholesterol and sphingomyelin content while decreasing that of phosphatidylserine-phosphatidylinositol lipids. In addition, monounsaturated fatty acid levels increased as well as the propensity of reconstituted membranes to form H_II-phases. These data suggest that 2OHOA regulates lipid metabolism that is altered in hypertensive animals, and that it affects the structural properties of liver plasma membranes in SHR. These changes in the structural properties of the plasma membrane may modulate the activity of signalling proteins that associate with the cell membrane such as the Gαq/11 protein and hence, signal transduction.