Adhesion between liposomes mediated by the chlorophyll a/b light- harvesting complex isolated from chloroplast membranes

A highly purified chlorophyll a/b light-harvesting complex (chl a/b LHC; chl a/b ratio 1.2) was obtained from Triton-solubilized chloroplast membranes of pea and barley according to the method of Burke et al. (1978, Arch. Biochem. Biophys. 187: 252--263). Gel electrophoresis of the cation-precipitated chl a/b LHC from peas reveals the presence of four polypeptides in the 23- to 28-kdalton size range. Three of these peptides appear to be identical to those derived from re-electrophoresed CPII and CPII* bands. In freeze-fracture replicas, the cation-precipitated chl a/b LHC appears as a semicrystalline aggregate of membranous sheets containing closely spaced granules. Upon removal of the cations by dialysis, the aggregates break up into their constituent membranous sheets without changing their granular substructure. These membranous sheets can be resolubilized in 1.5% Triton X-100, and the chl a/b LHC particles then reconstituted into soybean lecithin liposomes. Freeze-fracture micrographs of the reconstituted chl a/b LHC vesicles suspended in a low salt medium reveal randomly dispersed approximately 80-A particles on both concave and convex fracture faces as well as some crystalline particle arrays, presumably resulting from incompletely solubilized fragments of the membranous sheets. Based on the approximately 80-A diameter of the particles, and on the assumption that one freeze- fracture particle represents the structural unit of one chl a/b LHC aggregate, a theoretical mol wt of approximately 200 kdalton has been calculated for the chl a/b LHC. Deep-etching and negative-staining techniques reveal that the chl a/b LHC particles are also exposed on the surface of the bilayer membranes. Addition of greater than or equal to 2 mM MgCl2 or greater than or equal to 60 mM NaCl to the reconstituted vesicles leads to their aggregation and, with divalent cations, to the formation of extensive membrane stacks. At the same time, the chl a/b LHC particles become clustered into the adhering membrane regions. Under these conditions the particles in adjacent membranes usually become precisely aligned. Evidence is presented to aupport the hypothesis that adhesion between the chl a/b LHC particles is mediated by hydrophobic interactions, and that the cations are needed to neutralize surface charges on the particles.     

Oceanic picophytoplankton having a high abundance of chlorophyll b in the major light harvesting chlorophyll protein complex

Chl a-containing, very small unicellular, eukaryotic phytoplankton (picophytoplankton) often become the dominant organisms near the bottom of the euphotic zone in the ocean, where light is limited, not only in intensity (about 0.5% of the surface irradiance), but also in quality (dominant in blue to green wavelengths). We have isolated picophytoplankton from subsurface waters (from 75 to 150 m in depth) of the Kuroshio area near Japan. EM observations showed that a single chloroplast occupies a large part of the cytoplasm. Some of the isolates have a flagellum. The major photosynthetic pigments found in these isolates were chlorophyll a and b. The light-harvesting chlorophyll a/b complex (LHCP) was isolated from three clones of picophytoplankton, one flagellated form (NIBB8001) and two coccoid forms (94B8100A and 94B5100C) . More than 50% of the total chlorophylls were recovered in the major LHCP fraction. A common feature of the major LHCPs isolated from the three picophytoplankton clones was a high abundance of chlorophyll b: the ratios of chlorophyll a to b were about 0.8, 0.7 and 0.6 for the clones NIBB8001, 94B8100A and 94B5100C, respectively. These values were very low compared with those in chlorophyll a/b-binding LHCIIs in higher plants and in the major chlorophyll a/b-binding LHCPs in microalgae (higher than 1.0). The major LHCP apoproteins of NIBB8001 and 94B5100C contained one major polypeptide; the apparent molecular masses analyzed with SDS-PAGE were about 22 kDa and 27 kDa, respectively. The major LHCP apoprotein of 94B8100A had two major polypeptides having apparent molecular masses of about 23 and 25 kDa. None of the thylakoid proteins cross-reacted with an antibody raised against the LHC II apoprotein of spinach. It is suggested that the high abundance of chlorophyll b in picophytoplankton, together with a large chloroplast in a small cell, enable them to utilize the reduced light in their habitat.     

Nuclear pea mutants deficient in chlorophyll b and the major polypeptide of the light-harvesting chlorophyll a/b protein of photosystem II

Eight chlorophyll b deficient nuclear mutants of pea (Pisum sativum L.) have been characterized by low temperature fluorescence emission spectra of their leaves and by the ultrastructure, photochemical activities and polypeptide compositions of the thylakoid membranes. The room temperature fluorescence induction kinetics of leaves and isolated thylakoids have also been recorded. In addition, the effects of Mg²⁺ on the fluorescence kinetics of the membranes have been investigated. The mutants are all deficient in the major polypeptide of the light-harvesting chlorophyll a/b protein of photosystem II. The low temperature fluorescence emission spectra of aurea-5106, xantha-5371 and –5820 show little or no fluorescence around 730 nm (photosystem I fluorescence), but possess maxima at 685 and 695 nm (photosystem II fluorescence). These three mutants have low photosystem II activities, but significant photosystem I activities. The long-wavelength fluorescence maximum is reduced for three other mutants. The Mg²⁺ effect on the variable component of the room temperature fluorescence (685 nm) induction kinetics is reduced in all mutants, and completely absent in aurea-5106 and xantha-5820. The thylakoid membranes of these 2 mutants are appressed pairwise in 2-disc grana of large diameter. Chlorotica-1-206A and–130A have significant long-wavelength maxima in the fluorescence spectra and show the largest Mg²⁺ enhancement of the variable part of the fluorescence kinetics. These two mutants have rather normally structured chloroplast membranes, though the stroma regions are reduced. The four remaining mutants are in several respects of an intermediate type.Abbreviations Chl chlorophyll - CPI Chi-protein complex I, F_o, F_v - F_m parameters of room temperature chlorophyll fluorescence induction kinetics - F685, F695 and F-1 components of low temperature chlorophyll emission with maximum at 685, 695 and ca 735 nm, respectively - PSI photosystem I - PSII photosystem II - LHCI and LHCII light-harvesting chlorophyll a/b complexes associated with PSI and PSII, respectively - SDS sodium dodecyl sulfate     

Light-induced fluorescence quenching in the light-harvesting chlorophyll a/b protein complex

Summary Irradiation of the principal photosystem II light-harvesting chlorophyll-protein antenna complex, LHC II, with high light intensities brings about a pronounced quenching of the chlorophyll fluorescence. Illumination of isolated thylakoids with high light intensities generates the formation of quenching centres within LHC II in vivo, as demonstrated by fluorescence excitation spectroscopy. In the isolated complex it is demonstrated that the light-induced fluorescence quenching: a) shows a partial, biphasic reversibility in the dark; b) is approximately proportional to the light intensity; c) is almost independent of temperature in the range 0–30°C; d) is substantially insensitive to protein modifying reagents and treatments; e) occurs in the absence of oxygen. A possible physiological importance of the phenomenon is discussed in terms of a mechanism capable of dissipating excess excitation energy within the photosystem II antenna.Abbreviations chla chlorophyll a - chlb chlorophyll b - F₀ fluorescence yield with reaction centers open - F_m fluorescence yield with reaction centres closed - F_i fluorescence at the plateau level of the fast induction phase - LHC II light-harvesting chlorophyll a/b protein complex II - PS II photosystem II - PSI photosystem I - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine     

Immunological identification and quantification of light harvesting chlorophyll a/b protein of Chlorella protothecoides

The alteration of photosynthetic membrane proteins in relation to the disappearance of pigments during the heterotrophic growth of Chlorella protothecoides was investigated. Chlorophylls and certain polypeptides associated with the LHC II disappeared after 50 hr of heterotrophic growth but the 24 kDa apoprotein constituting LHC II was not affected. Immunological analysis indicated that the chlorophylls and the light harvesting complex proteins of the thylakoid membranes are not tightly coupled and the latter is retained in its native form irrespective of the presence or absence of the former. The circumstantial evidence that the other photosynthetic membrane polypeptides are degraded along with the pigments due to increased proteolytic activity in the rapidly dividing heterotrophic cells indicate that chlorophyll synthesis is not a pre-requisite for the synthesis of the LHC II apoprotein.     

The structure of membrane crystals of the light-harvesting chlorophyll a/b protein complex

Membrane crystals of the light-harvesting chlorophyll a/b protein complex from pea chloroplasts were investigated using electron microscopy and image analysis. The membrane crystals formed upon precipitation of the detergent-solubilized complex with mono- and divalent cations in the presence of small amounts of Triton X-100. The crystalline fraction contained two polypeptides of 25,000 and 27,000 mol wt. Freeze-dried and freeze-etched specimens showed a periodic honeycomb structure on the surface of membrane crystals. Double replicas of freeze-fractured sheets showed a hexagonal lattice of particles on both fracture faces. Image analysis of negatively stained membrane crystals suggested that they had threefold rather than sixfold symmetry in projection. A projection map at 20-A resolution revealed two triangular structural units of opposite handedness per crystallographic unit cell. The structural units appeared to be inserted bidirectionally into the membrane, alternating in orientation perpendicular to the membrane plane.     

Monoclonal antibodies to the light-harvesting chlorophyll a/b protein complex of photosystem II

A collection of 17 monoclonal antibodies elicited against the light-harvesting chlorophyll a/b protein complex which serves photosystem II (LHC-II) of Pisum sativum shows six classes of binding specificity. Antibodies of two of the classes recognize a single polypeptide (the 28- or the 26- kD polypeptides), thereby suggesting that the two proteins are not derived from a common precursor. Other classes of antibodies cross-react with several polypeptides of LHC-II or with polypeptides of both LHC-II and the light-harvesting chlorophyll a/b polypeptides of photosystem I (LHC-I), indicating that there are structural similarities among the polypeptides of LHC-II and LHC-I. The evidence for protein processing by which the 26-, 25.5-, and 24.5-kD polypeptides are derived from a common precursor polypeptide is discussed. Binding studies using antibodies specific for individual LHC-II polypeptides were used to quantify the number of antigenic polypeptides in the thylakoid membrane. 27 copies of the 26-kD polypeptide and two copies of the 28-kD polypeptide were found per 400 chlorophylls. In the chlorina f2 mutant of barley, and in intermittent light-treated barley seedlings, the amount of the 26-kD polypeptide in the thylakoid membranes was greatly reduced, while the amount of 28-kD polypeptide was apparently not affected. We propose that stable insertion and assembly of the 28-kD polypeptide, unlike the 26-kD polypeptide, is not regulated by the presence of chlorophyll b.     

Three-dimensional crystals of the light-harvesting chlorophyll a/b protein complex from pea chloroplasts

Two forms of three-dimensional crystals of the light-harvesting chlorophyll a/b protein complex from pea have been obtained. Crystals of one form grew as hexagonal plates measuring up to 150 micron across and 2 to 3 micron in thickness. Electron diffraction patterns of thin hexagonal plates showed sharp reflections to a resolution of 3.7 A on a hexagonal reciprocal lattice. The unit cell in projection (a = 127.0 A) and the symmetry of the diffraction pattern (6 mm) suggested that the hexagonal plates were highly ordered stacks of two-dimensional crystals suitable for structure analysis by electron microscopy and image processing. Crystals of a second form grew as dark green octahedra measuring roughly 0.5 mm across. Low-resolution X-ray diffraction patterns suggested a large cubic unit cell (a = 390 A). SDS/polyacrylamide gel electrophoresis of single octahedral crystals showed the same polypeptide composition as the starting solution, one major band at 24,000 apparent molecular weight and two satellite bands of 23,000 and 23,500 apparent molecular weight.     

Cytokinins modulate the expression of genes encoding the protein of the light-harvesting chlorophyll a/b complex

Summary Tobacco cell suspension cultures responded to cytokinins (for instance kinetin) by full chloroplast differentiation. The hormone had the effect of stimulating the appearance of a few prominent plastid proteins. Synthesis of the light-harvesting chlorophyl a/b-binding protein (LHCP) in response to kinetin was noteworthy (Axelos M. et al.: Plant Sci Lett 33:201–212, 1984).Poly(A)⁺RNAs were prepared from cells grown in the presence of or without added kinetin. Poly(A)⁺RNA recovery and translation activity were not quantitatively altered by the hormone treatment. In vitro translation of polyadenylated mRNA into precursor polypeptides of LHCP (pLHCP) was quantified by immunoprecipitation and SDS-PAGE fractionation of pLHCP immunoprecipitates: pLHCP-mRNA translating activity was found to be stimulated in parallel to mature LHCP accumulation by kinetin-induced cells.Dot-blot and northern-blot hybridizations of poly(A)⁺RNA were carried out, using as a probe a pea LHCP-cDNA clone (Broglie R. et al.: Proc Natl Acad Sci USA 78: 7304–7308, 1981). A ten-fold increase of the level of pLHCP-encoding sequences was observed in poly(A)⁺RNA prepared from 9-d kinetin-stimulated cells, compared to control cells. Oligo(dT)-cellulose-excluded RNA fractions exhibited very low hybridization levels, in the same ratios as those obtained with poly(A)⁺RNA.Thus, the expression of LHCP-gene activity, in response to kinetin addition to tobacco cell suspension cultures, is regulated by the level of pLHCP-encoding mRNA rather than by translational or post-translational controls. re]19850218 rv]19850605 ac]19850613     

Topographical studies of the polypeptide subunits of the thylakoid cytochrome b6-f complex

The orientation of specific polypeptides of the cytochrome b6-f complex with respect to the chloroplast stromal phase has been studied using trinitrobenzenesulfonate (TNBS) and pronase E as impermeant modifying reagents. Of the four polypeptides of the complex (33,23,20 and 17 kDa), only cytochrome f was labeled by 14C-TNBS in unfractionated membranes. However, to a varying degree, all of the constituent polypeptides were sensitive to pronase digestion and, in the case of cytochrome f, it was possible, by immunoblotting techniques to identify several degradation products. These results are discussed in relation to the organization of the cytochrome complex in thylakoid membranes and argue for an exposure to the stromal phase of all of the polypeptides, while functional considerations indicate that at least cytochrome f and the Rieske iron-sulfur protein have a possible transmembrane organization.     

Determination of relative chlorophyll binding affinities in the major light-harvesting chlorophyll a/b complex

The major light-harvesting complex (LHCIIb) of photosystem II can be reconstituted in vitro from its recombinant apoprotein in the presence of a mixture of carotenoids and chlorophylls a and b. By varying the chlorophyll a/b ratio in the reconstitution mixture, the relative amounts of chlorophyll a and chlorophyll b bound to LHCIIb can be changed. We have analyzed the chlorophyll stoichiometry in recombinant wild type and mutant LHCIIb reconstituted at different chlorophyll a/b ratios in order to assess relative affinities of the chlorophyll-binding sites. This approach reveals five sites that exclusively bind chlorophyll b. Another site exhibits a slight preference of chlorophyll b over chlorophyll a. The remaining six sites are filled preferentially with chlorophyll a but also tolerate chlorophyll b when this is offered at a large excess. Three of these chlorophyll a-affine sites could be assigned to distinct positions defined by the three-dimensional LHCIIb structure. Exclusive chlorophyll b sites complemented by chlorophyll a sites that are selective only to a certain extent are consistent with the observation that chlorophyll b but not chlorophyll a is essential for reconstituting stable LHCIIb. These data offer an explanation why a rather constant chlorophyll a/b ratio is observed in native LHCIIb despite the apparent promiscuity of some binding sites.     

Polyadenylated mRNA for the light-harvesting chlorophyll a/b protein

In thylakoid membranes isolated from green plants of parsley, pea, and barley, the light-harvesting chlorophyll a/b protein complex (LHCP, mol. weight: 25,000), is a major constituent. Poly(A)RNA isolated from these species was translated in a wheat germ, cell-free system. The in vitro translation products were treated with antibodies raised against the LHCP. This treatment resulted in the precipitation of a precursor protein (mol. weight: 29,000). Poly(A)RNA was also prepared from a cell culture ofPetroselinum that does not develop chloroplasts upon illumination. This poly(A)RNA is capable of stimulating amino acid incorporation in the in vitro translation system, however, it does not direct the synthesis of LHCP.     

Crystallization of the light-harvesting chlorophyll a/b complex within thylakoid membranes

We have found that treatment of the photosynthetic membranes of green plants, or thylakoids, with the nonionic detergent Triton X-114 at a 10:1 ratio has three effects: (a) photosystem I and coupling factor are solubilized, so that the membranes retain only photosystem II (PS II) and its associated light-harvesting apparatus (LHC-II); (b) LHC-II is crystallized, and so is removed from its normal association with PS II; and (c) LHC-II crystallization causes a characteristic red shift in the 77 degrees K fluorescence from LHC-II. Treatment of thylakoids with the same detergent at a 20:1 ratio results in an equivalent loss of photosystem I and coupling factor, with LHC-II and PS II being retained by the membranes. However, no LHC-II crystals are formed, nor is there a shift in fluorescence. Thus, isolation of a membrane protein is not required for its crystallization, but the conditions of detergent treatment are critical. Membranes with crystallized LHC-II retain tetrameric particles on their surface but have no recognizable stromal fracture face. We have proposed a model to explain these results: LHC-II is normally found within the stromal half of the membrane bilayer and is reoriented during the crystallization process. This reorientation causes the specific fluorescence changes associated with crystallization. Tetrameric particles, which are not changed in any way by the crystallization process, do not consist of LHC-II complexes. PS II appears to be the only other major complex retained by these membranes, which suggests that the tetramers consist of PS II.     

The rapidly phosphorylated 25 kDa polypeptide of the light- harvesting complex of photosystem II is encoded by the type 2 cab-II genes

The main light-harvesting complex of Photosystem II (LHC II) in higher plants consists of two sub-populations. The 'inner' pool consists only of a 27 kDa polypeptide, whereas in the 'outer' pool both the 27 kDa and a 25 kDa polypeptide are found. We purified the 25 and the 27 kDa LHC II polypeptides from Scots pine and 25 kDa LHC II polypeptide from spinach. Protein sequencing after cleavage with endoproteinase Lys-C showed that the 25 kDa polypeptide is encoded by the Type 2 cab-II genes and the 27 kDa polypeptide by the Type I cab-II genes. A fatty acid was not covalently attached to the peptides assembled into the pigment-protein complex. Our results show that the different polypeptides seen on a gel are different gene products, and not the result of different processing.     

Isolation of two different subpopulations of the light-harvesting chlorophyll a/b complex of photosystem II (LHCII)

Isolated LHCII from spinach has been solubilized and fractionated by non-denaturing isoelectric focusing to yield two subpopulations with different polypeptide but equal chlorophyll composition. One LHCII subpopulation contains only a 27 kDa polypeptide while the other contains the 27 and 25 kDa polypeptides in about equal amounts. The polypeptide patterns of the two subpopulations closely correspond to those suggested previously for the inner LHCII and peripheral LHCII, respectively.     

Effects of chlorophyll a, chlorophyll b, and xanthophylls on the in vitro assembly kinetics of the major light-harvesting chlorophyll a/b complex, LHCIIb

The major light-harvesting chlorophyll a/b complex (LHCIIb) of photosystem II in higher plants can be reconstituted with pigments in lipid-detergent micelles. The pigment-protein complexes formed are functional in that they perform efficient internal energy transfer from chlorophyll b to chlorophyll a. LHCIIb formation in vitro, can be monitored by the appearance of energy transfer from chlorophyll b to chlorophyll a in time-resolved fluorescence measurements. LHCIIb is found to form in two apparent kinetic steps with time constants of about 30 and 200 seconds. Here we report on the dependence of the LHCIIb formation kinetics on the composition of the pigment mixture used in the reconstitution. Both kinetic steps slow down when the concentration of either chlorophylls or carotenoids is reduced. This suggests that the slower 200 seconds formation of functional LHCIIb still includes binding of both chlorophylls and carotenoids. LHCIIb formation is accelerated when the chlorophylls in the reconstitution mixture consist predominantly of chlorophyll a although the complexes formed are thermally less stable than those reconstituted with a chlorophyll a:b ratio < or = 1. This indicates that although chlorophyll a binding is more dominant in the observed rate of LHCIIb formation, the occupation of (some) chlorophyll binding sites with chlorophyll b is essential for complex stability. The accelerating effect of various carotenoids (lutein, zeaxanthin, violaxanthin, neoxanthin) on LHCIIb formation correlates with their affinity to two lutein-specific binding sites. We conclude that the occupation of these two carotenoid binding sites but not of the third (neoxanthin-specific) binding site is an essential step in the assembly of LHCIIb in vitro.     

Effect of papain digestion on polypeptide subunits and electron-transfer pathways in mitochondrial b-c1 complex

Papain digestion of subunits of mitochondrial b-c1 complex (ubiquinol-cytochrome-c reductase) isolated from bovine heart and its impact on redox and proton-motive activity of the whole complex were investigated. A 5-min incubation of the oxidized enzyme with papain resulted in digestion of core protein II and the 14-kDa subunit, and limited digestion of the iron-sulfur protein. This was accompanied by a small inhibition of the rate of electron flow and a marked inhibition of proton translocation with decrease of the H+/e- ratio for proton pumping. When papain treatment was performed on the b-c1 complex pre-reduced with ascorbate, partial proteolysis of the iron-sulfur protein and the 14-kDa subunit was greatly accelerated and the electron transfer activity was more markedly inhibited. In all the conditions tested, digestion of the Rieske iron-sulfur protein paralleled the inhibition of reductase activity. Under ascorbate-reduced conditions, papain digestion of the complex gave rise to an alteration of the EPR line shape of the iron-sulfur cluster, namely a broadening and shift of the gx negative peak and destabilization of the protein-bound antimycin-sensitive semiquinone. The latter paralleled the decrease in electron transfer activity and inhibition of antimycin-sensitive cytochrome-b reduction. The results obtained indicate the following. 1. Core protein II and the 14-kDa protein may contribute to the proton-conducting pathway(s) from the matrix aqueous phase to the primary protolytic redox center (protein-bound semiquinone/quinone couple). 2. The iron-sulfur protein contributes, together with other protein(s) (the 14-kDa subunit), to the stabilization of the protein-bound antimycin-sensitive semiquinone species in a protein pocket in the complex. 3. Reduction of the high-potential redox centers induces a change in the quaternary structure of the complex which results in an enhanced surface exposure of segments of the 14-kDa protein and the iron-sulfur protein.