Enhanced thermal tolerance of photosynthesis and altered chloroplast ultrastructure in a mutant of Arabidopsis deficient in lipid desaturation

A mutant of Arabidopsis thaliana, deficient in activity of the chloroplast n-6 desaturase, accumulated high levels of C_16:1 and C_18:1 lipids and had correspondingly reduced levels of polyunsaturated lipids. The altered lipid composition of the mutant had pronounced effects on chloroplast ultrastructure, thylakoid membrane protein and chlorophyll content, electron transport rates, and the thermal stability of the photosynthetic membranes. The change in chloroplast ultrastructure was due to a 48% decrease in the amount of appressed membranes that was not compensated for by an increased amount of nonappressed membrane. This resulted in a net loss of 36% of the thylakoid membrane per chloroplast and a corresponding reduction in chlorophyll and protein content. Electrophoretic analysis of the chlorophyll-protein complexes further revealed a small decrease in the amount of light-harvesting complex. Relative levels of whole chain and protosystem II electron transport rates were also reduced in the mutant. In addition, the mutation resulted in enhanced thermal stability of photosynthetic electron transport. These observations suggest a central role of polyunsaturated lipids in determining chloroplast structure and maintaining normal photosynthetic function and demonstrate that lipid unsaturation directly affects the thermal stability of photosynthetic membranes.     

Nitrate-induced changes in protein synthesis and translation of RNA in maize roots

Nitrate regulation of protein synthesis and RNA translation in maize (Zea mays L. var B73) roots was examined, using in vivo labeling with [³⁵S]methionine and in vitro translation. Nitrate enhanced the synthesis of a 31 kilodalton membrane polypeptide which was localized in a fraction enriched in tonoplast and/or endoplasmic reticulum membrane vesicles. The nitrate-enhanced synthesis was correlated with an acceleration of net nitrate uptake by seedlings during initial exposure to nitrate. Nitrate did not consistently enhance protein synthesis in other membrane fractions. Synthesis of up to four soluble polypeptides (21, 40, 90, and 168 kilodaltons) was also enhanced by nitrate. The most consistent enhancement was that of the 40 kilodalton polypeptide. No consistent nitrate-induced changes were noted in the organellar fraction (14,000g pellet of root homogenates). When roots were treated with nitrate, the amount of [³⁵S]methionine increased in six in vitro translation products (21, 24, 41, 56, 66, and 90 kilodaltons). Nitrate treatment did not enhance accumulation of label in translation products with a molecular weight of 31,000 (corresponding to the identified nitrate-inducible membrane polypeptide). Incubation of in vitro translation products with root membranes caused changes in the SDS-PAGE profiles in the vicinity of 31 kilodaltons. The results suggest that the nitrate-inducible, 31 kilodalton polypeptide from a fraction enriched in tonoplast and/or endoplasmic reticulum may be involved in regulating nitrate accumulation by maize roots.     

Studies on Freezing Injury of Plant Cells: I. Relation between Thermotropic Properties of Isolated Plasma Membrane Vesicles and Freezing Injury

The thermotropic transition of plasma membrane of Dactylis glomerata was studied by using fluorescence polarization of embedded fluorophore, 1,6-diphenyl-1,3,5-hexatriene. Under the presence of 35% ethylene glycol, reversible thermotropic transitions were observed in isolated plasma membrane vesicles in nearly the same temperature range as the temperature of freezing injury to cells. In liposomes prepared from isolated plasma membranes, however, the thermotropic transitions occurred at much lower temperatures in comparison with those of intact membrane vesicles. Following treatment with pronase, the thermotropic transition also shifted downward.

Thus, the thermotropic properties of plasma membranes appeared to be dependent on the membrane proteins. In vitro freezing of the isolated plasma membrane vesicles without addition of any cryoprotectant, such as sorbitol, resulted in an irreversible alteration both in the fluorescence anisotropy values and the temperatures for the thermotropic transition, suggesting an irreversible alteration in the membrane structure, presumably changes in lipid-protein interactions and protein conformation.

     

Localization of the glucocorticoid receptor in rat liver cells: Evidence for plasma membrane bound receptor

• 1.1. The cytoplasmic glucocorticoid receptor of rat liver cells is in part recovered in the plasma membrane fraction.
• 2.2. After in vivo administration of [³H]dexamethasone, 0.35% of the radioactivity recovered is bound on plasma membranes.
• 3.3. Dexamethasone also binds in vitro specifically to plasma membranes. Expressed as fmol/mg protein, binding of dexamethasone to plasma membranes is comparable to binding to the soluble cytoplasmic fraction (cytosol).
• 4.4. Using polyclonal antibody to the glucocorticoid receptor and the indirect immunofluorescence technic, an intense decoration of the plasma membranes is observed, denoting a high concentration of glucocorticoid receptor on plasma membranes.
• 5.5. The localization of the receptor on plasma membranes could be of potential importance for its interaction with agents (mitogens, growth factors) initially acting on the cell membrane, regulating subsequent cell proliferation and growth at the level of the cell nucleus.

     

Neutron scattering studies of photosynthetic membranes in aqueous dispersion

A method for structural analysis of biological membranes using neutron scattering from suspensions is described and applied to photosynthetic membranes from bacteria. The variation of scattering density across the membrane is analysed using small-angle scattering and contrast variation with H₂O/²H₂O mixtures. Effects due to membrane curvature and scattering density variation in the plane of the membrane are evaluated. Thickness parameters (D) are obtained from the small-angle scattering data, which are the one-dimensional analogues of radii of gyration. The formalism of contrast variation is used to describe the change of intensity and thickness parameter with H₂O/²H₂O mixture. The results are expressed in terms of a thickness parameter at infinite contrast, which is directly related to the physical thickness of the membrane, and a measure of the variation of the scattering density across the membrane, produced, for example, by the higher scattering densities of the polar surfaces relative to a hydrocarbon interior of the membrane. Asymmetry in the membrane scattering density is also evaluated.The results for photosynthetic membranes demonstrate a lipid hydrocarbon core in the membrane. About two-thirds of the protein is closely associated with the lipid layer, and no substantial amounts of protein project more than short distances from the lipid layer. There is a contribution to the variation in scattering density across the membrane that cannot be attributed to lipid, and may involve scattering density heterogeneity within the protein, giving a high proportion of hydrophobic protein segments at the interior of the membrane that have lower scattering densities than the hydrophilic segments at the surfaces of the membrane. The membrane scattering density is not markedly asymmetric. Several alternative structures previously proposed for photosynthetic membranes are incompatible with these results.     

Effects of light intensity on membrane differentiation in Rhodopseudomonas capsulata

Cells of Rhodopseudomonas capsulata, strain 37b4, leu^?, precultivated anaerobically under low light intensity, were exposed to high light intensity (2000 W · m^?2). The cells grew with a mass doubling time of 3 h. The synthesis of bacteriochlorophyll (BChl) began after two doublings of cell mass. Reaction center and light-harvesting BChl I (B-875) were the main constituents of the photosynthetic apparatus incorporated into the membrane. The size of the photosynthetic unit (total BChl/reaction center) decreased and light-harvesting BChl I became the dominating BChl species. Concomitant with the appearance of the different spectral forms of BChl the respective proteins were incorporated into the membrane, i.e. the three reaction center polypeptides, the polypeptide associated with light-harvesting BChl I, the two polypeptides associated with BChl II. A polypeptide of an apparent molecular weight of 45 000 was also incorporated. A lowering of the light intensity to 7 W · m^?2 resulted in a lag phase of growth for 6 h. Afterwards, the time for doubling of cell mass was 11 h. The concentration of all three BChl complexes (reaction center, light-harvesting BChl I and II complexes)/cell and per membrane protein increased immediately. Also the size of the photosynthetic unit and the amount of intracytoplasmic membranes/cell increased.The activities of photophosphorylation, succinate dehydrogenase, NADH dehydrogenase and NADH oxidation (respiratory chain)/membrane protein are higher in membrane preparations isolated from cells grown at high light intensities than in such preparations from cells grown at low light intensities.     

Properties of reaction center depleted membranes of Rhodospirillum rubrum

Intracytoplasmic membranes isolated from Rhodospirillum rubrum, mutant strain VI, were extracted with the detergent lauryl dimethyl amine oxide. Subsequently two fractions were isolated, one of which contained reaction centers and the other contained light-harvesting bacteriochlorophyll of the photosynthetic apparatus. The two fractions are compared with unextracted membranes on the basis of protein patterns obtained by different methods of polyacrylamide gelelectrophoresis. Electron micrographs of the light-harvesting bacteriochlorophyll fraction reveal the presence of vesicular membrane structures. The only difference between such membranes and unextracted membranes is identified after freeze etching. While unextracted membrane surfaces are studded with particles extracted membranes exhibit a smooth surface.     

Photosynthetic phosphorylation by a membrane preparation of the cyanobacterium Spirulina Maxima

A new method for the isolation of photosynthetic membranes from the cyanobacterium Spirulina maxima has been developed. When illuminated, these membranes evolve oxygen in the presence of ferricyanide (Hill reaction) and consume oxygen in the presence of methyl viologen (Mehler reaction). When the membranes are left to stand at 4°C for 30 min, they develop the ability to consume oxygen in the light without an added, artificial electron acceptor. The Hill and Mehler reactions are not affected by the presence of ADP or uncouplers, but are inhibited by triphenyltin chloride. We have detected a cryptic ATPase activity stimulated by trypsin in the 2000×g supernatant fraction of the membrane preparation. In addition, the membrane vesicles contain an ATPase activity which is enhanced by treatment with dithiothreitol in the presence of light. These observations of ATPase led us to try a careful titration of the membrane vesicles with both triphenyltin chloride and N,N′-dicyclohexylcarbodiimide. When the vesicles were sealed with these reagents, we could observe both cyclic and stoichiometric photosynthetic phosphorylation.     

Rapid Changes in Plasma Membrane Protein Phosphorylation during Initiation of Cell Wall Digestion

Plasma membrane vesicles from wild carrot cells grown in suspension culture were isolated by aqueous two-phase partitioning, and ATP-dependent phosphorylation was measured with [γ-³²P]ATP in the presence and absence of calcium. Treatment of the carrot cells with the cell wall digestion enzymes, driselase, in a sorbitol osmoticum for 1.5 min altered the protein phosphorylation pattern compared to that of cells treated with sorbitol alone. Driselase treatment resulted in decreased phosphorylation of a band of M_r 80,000 which showed almost complete calcium dependence in the osmoticum treated cells; decreased phosphorylation of a band of M_r 15,000 which showed little calcium activation, and appearance of a new band of calcium-dependent phosphorylation at M_r 22,000. These effects appeared not to be due to nonspecific protease activity and neither in vivo nor in vitro exposure to driselase caused a significant loss of Coomassie blue-staining bands on the gels of the isolated plasma membranes. However, protein phosphorylation was decreased. Adding driselase to the in vitro reaction mixture caused a general decrease in the membrane protein phosphorylation either in the presence or absence of calcium which did not mimic the in vivo response. Cells labeled in vivo with inorganic ³²P also showed a response to the Driselase treatment. An enzymically active driselase preparation was required for the observed responses.     

Localisation and interactions of the Vipp1 protein in cyanobacteria

The Vipp1 protein is essential in cyanobacteria and chloroplasts for the maintenance of photosynthetic function and thylakoid membrane architecture. To investigate its mode of action we generated strains of the cyanobacteria Synechocystis sp. PCC6803 and Synechococcus sp. PCC7942 in which Vipp1 was tagged with green fluorescent protein at the C‐terminus and expressed from the native chromosomal locus. There was little perturbation of function. Live‐cell fluorescence imaging shows dramatic relocalisation of Vipp1 under high light. Under low light, Vipp1 is predominantly dispersed in the cytoplasm with occasional concentrations at the outer periphery of the thylakoid membranes. High light induces Vipp1 coalescence into localised puncta within minutes, with net relocation of Vipp1 to the vicinity of the cytoplasmic membrane and the thylakoid membranes. Pull‐downs and mass spectrometry identify an extensive collection of proteins that are directly or indirectly associated with Vipp1 only after high‐light exposure. These include not only photosynthetic and stress‐related proteins but also RNA‐processing, translation and protein assembly factors. This suggests that the Vipp1 puncta could be involved in protein assembly. One possibility is that Vipp1 is involved in the formation of stress‐induced localised protein assembly centres, enabling enhanced protein synthesis and delivery to membranes under stress conditions.     

In vitro Unfolding and Refolding of the Small Multidrug Transporter EmrE

The composition of the lipid bilayer is increasingly being recognised as important for the regulation of integral membrane protein folding and function, both in vivo and in vitro. The folding of only a few membrane proteins, however, has been characterised in different lipid environments. We have refolded the small multidrug transporter EmrE in vitro from a denatured state to a functional protein and monitored the influence of lipids on the folding process. EmrE is part of a multidrug resistance protein family that is highly conserved amongst bacteria and is responsible for bacterial resistance to toxic substances. We find that the secondary structure of EmrE is very stable and only small amounts are denatured even in the presence of unusually high denaturant concentrations involving a combination of 10 M urea and 5% SDS. Substrate binding by EmrE is recovered after refolding this denatured protein into dodecylmaltoside detergent micelles or into lipid vesicles. The yield of refolded EmrE decreases with lipid bilayer compositional changes that increase the lateral chain pressure within the bilayer, whilst conversely, the apparent rate of folding seems to increase. These results add further weight to the hypothesis that an increased lateral chain pressure hinders protein insertion across the bilayer. Once the protein is inserted, however, the greater pressure on the transmembrane helices accelerates correct packing and final folding. This work augments the relatively small number of biophysical folding studies in vitro on helical membrane proteins.     

In vitro translation of epidermal RNA from different anatomical regions and metamorphic stages of Hyalophora cecropia

Epidermal RNA isolated from different anatomical regions and metamorphic stages of Hyalophora cecropia was translated in vitro with commercial wheat germ and rabbit reticulocyte systems. The translation products were analyzed by 2D gel electrophoresis. The two systems yielded identical products if canine microsomal membranes were added to remove signal peptides from the reticulocyte products. The endogenous processing by the wheat germ extract occurred even in the presence of protease inhibitors. Some of the processed translation products co-migrated with unlabeled cuticular protein standards. All of the major cuticular proteins could be identified, but only when translations were carried out with RNA from epidermis underlying cuticle containing these proteins. Hence, cuticular protein distribution is due to differential synthesis and not to differential extractability. For larval abdominal RNA, most of the major translation products did not co-migrate with known larval cuticular proteins or with proteins synthesized and retained by the epidermis. These unknown products were lower in apparent molecular weight than the cuticular proteins. Their identity remains unknown; they may be premature translation products, but altering translation conditions did not reduce their abundance.     

A comparative approach towards thylakoid membrane proteome analysis of unicellular green alga Scenedesmus obliquus

The chlorophyll (Chl)-containing membrane protein complexes from the green alga Scenedesmus obliquus have been isolated from the thylakoid membranes by solubilization with dodecyl-β-maltoside and fractionation using a sucrose density gradient. The Chl-containing protein fractions were characterized by absorption spectroscopy, tricine SDS PAGE, BN-PAGE, and dynamic light scattering (DLS). BN-PAGE showed the presence of seven protein complexes with molecular weights in the range of 68, 118, 157, 320, 494, 828 and 955 kDa, respectively. Furthermore, light scattering reveals the simultaneous presence of particles of different sizes in the 3-4 nm and 6.0-7.5 nm range, respectively. The smaller size is related to the hydrodynamic radius of the trimer Light Harvesting Complex (LHCII), whereas the larger size is associated with the presence of photosystem I and photosystem II reaction centers. Additionally, functional information regarding protein-protein interactions was deconvoluted using coupling 2-D BN-PAGE, MALDI-TOF MS and a detailed mapping of S. obliquus photosynthetic proteome of the solubilized thylakoid membranes is therefore presented.     

Synthesis, transport and localization of a nuclear coded 22-kd heat-shock protein in the chloroplast membranes of peas and Chlamydomonas reinhardi

The synthesis, transport and localization of a nuclear coded 22-kd heat-shock protein (HSP) in the chloroplast membranes was studied in pea plants and Chlamydomonas reinhardi. HSPs were detected in both systems by in vivo labeling and in vitro translation of poly(A)⁺RNA, using the wheat-germ and reticulocyte lysate systems. Heat-shock treatment of pea plants for 2 h at 42-45°C induces the expression of ˜10 nuclear coded proteins, among which several (18 kd, 19 kd, 22 kd) are predominant. A 22-kd protein is synthesized as a 26-kd precursor protein and is localized in a chloroplast membrane fraction in vivo. Following post-translational transport into intact chloroplasts in vitro of the 26-kd precursor, the protein is processed but the resulting 22-kd mature protein is localized in the chloroplast stroma. If, however, the in vitro transport is carried out with chloroplasts from heat-shocked plants, the 22-kd protein is preferentially transported to the chloroplast membrane fraction. In C. reinhardi the synthesis of poly(A)⁺RNAs coding for several HSPs is progressively and sequentially induced when raising the temperature for 1.5 h from 36°C to 42°C, while that of several preexisting RNAs is reduced. Various pre-existing poly(A)⁺RNAs endure in the cells at 42°C up to 5 h but are no longer translated in vivo, whereas some poly(A)⁻RNAs persist and are translated. As in pea, a poly(A)⁺RNA coded 22-kd HSP is localized in the chloroplast membranes in vivo, although it is translated as a 22-kd protein in vitro. The in vitro translated protein is not transported in isolated pea chloroplast which, however, processes and transports other nuclear coded chloroplast proteins of Chlamydomonas. The poly(A)⁺RNA coding for the 22-kd HSP appears after 1 h at 36°C. Its synthesis increases with the temperature of incubation up to 42°C, although it decreases after ˜2 h of heat treatment and the already synthesized RNA is rapidly degraded. The degradation is faster upon return of the cells to 26°C. None of the heat-induced proteins is identical to the light-inducible proteins of the chloroplast membranes.     

Wheat germ cell-free translation, purification, and assembly of a functional human stearoyl-CoA desaturase complex

A wheat germ cell-free extract was used to perform in vitro translation of human stearoyl-CoA desaturase in the presence of unilamelar liposomes, and near complete transfer of the expressed integral membrane protein into the liposome was observed. Moreover, co-translation of the desaturase along with human cytochrome b₅ led to transfer of both membrane proteins into the liposomes. A simple, single step purification via centrifugation in a density gradient yielded proteoliposomes with the desaturase in high purity as judged by capillary electrophoresis. After in vitro reconstitution of the non-heme iron and heme active sites, the function of the reconstituted enzyme complex was demonstrated by conversion of stearoyl-CoA to oleoyl-CoA. This simple translation approach obviates the use of detergents or other lipids to stabilize and isolate a catalytically active integral membrane enzyme. The applicability of cell-free translation to the assembly and purification of other integral membrane protein complexes is discussed.     

From ecophysiology to phenomics: some implications of photoprotection and shade–sun acclimation in situ for dynamics of thylakoids in vitro

Half a century of research into the physiology and biochemistry of sun–shade acclimation in diverse plants has provided reality checks for contemporary understanding of thylakoid membrane dynamics. This paper reviews recent insights into photosynthetic efficiency and photoprotection from studies of two xanthophyll cycles in old shade leaves from the inner canopy of the tropical trees Inga sapindoides and Persea americana (avocado). It then presents new physiological data from avocado on the time frames of the slow coordinated photosynthetic development of sink leaves in sunlight and on the slow renovation of photosynthetic properties in old leaves during sun to shade and shade to sun acclimation. In so doing, it grapples with issues in vivo that seem relevant to our increasingly sophisticated understanding of ΔpH-dependent, xanthophyll-pigment-stabilized non-photochemical quenching in the antenna of PSII in thylakoid membranes in vitro.     

Environmentally modulated phosphorylation and dynamics of proteins in photosynthetic membranes

Recent advances in vectorial proteomics of protein domains exposed to the surface of photosynthetic thylakoid membranes of plants and the green alga Chlamydomonas reinhardtii allowed mapping of in vivo phosphorylation sites in integral and peripheral membrane proteins. In plants, significant changes of thylakoid protein phosphorylation are observed in response to stress, particularly in photosystem II under high light or high temperature stress. Thylakoid protein phosphorylation in the algae is much more responsive to the ambient redox and light conditions, as well as to CO₂ availability. The light-dependent multiple and differential phosphorylation of CP29 linker protein in the green algae is suggested to control photosynthetic state transitions and uncoupling of light harvesting proteins from photosystem II under high light. The similar role for regulation of the dynamic distribution of light harvesting proteins in plants is proposed for the TSP9 protein, which together with other recently discovered peripheral proteins undergoes specific environment- and redox-dependent phosphorylation at the thylakoid surface. This review focuses on the environmentally modulated reversible phosphorylation of thylakoid proteins related to their membrane dynamics and affinity towards particular photosynthetic protein complexes.