Molecular architecture of a light-harvesting antenna. Isolation and characterization of phycobilisome subassembly particles

Synechococcus 6301 mutant, strain AN112, produces phycobilisomes containing two major biliproteins, phycocyanin and allophycocyanin, and two major linker polypeptides of 27 and 75 kilodaltons (27K and 75K). These phycobilisomes have a molecular weight of approximately 2.5 X 10(6) and are the smallest of these particles known to date. Sucrose density gradient centrifugation of AN112 phycobilisomes partially dissociated in 50 mM N-[tris(hydroxymethyl)methyl]glycine, 5 mM CaCl2, 10% (w/v) glycerol, pH 7.8, separated three distinct fractions: (1) free trimeric biliproteins, (2) hexameric complexes of phycocyanin with 27K (11 S particles), and (3) phycobilisome subassemblies equivalent in mass to approximately 25% of the intact phycobilisome (18 S particles). The 18 S particles contained equimolar amounts of phycocyanin and allophycocyanin, which represented approximately 30 and 50%, respectively, of the content of these biliproteins in the AN112 phycobilisome. The 18 S particles also contained 75% and 100%, respectively, of 27K and 75K polypeptides; i.e. 75K was present in a 2-fold higher amount than in the intact phycobilisome. The absorption spectrum (lambda max 648 nm) of the 18 S particles was similar to that of allophycocyanin. Upon excitation at 580 nm, these particles exhibited a fluorescence emission spectrum consisting of 680 and 660 nm components, identical with that of intact phycobilisomes. The circular dichroism spectra of AN112 phycobilisomes and of the 18 S particles, in the region between 650 and 700 nm, were also very similar. Allophycocyanin B, which fluoresces at 680 nm, was found in fraction 1, and was totally absent from the 18 S particle. Thus, the long wavelength emission of the 18 S particle must have arisen from another terminal energy acceptor. The most probable candidate is the 75K polypeptide, which has been shown to carry a bilin chromophore and emit near 680 nm (Lundell, D. J., Yamanaka, G., and Glazer, A. N. (1980) J. Cell Biol. 91, 315-319). The 27K polypeptide, present in both fractions 2 and 3, was a component of different complexes in the two fractions. Fraction 2 displayed the physical and spectroscopic properties characteristic of the phycocyanin-linker complex, (alpha beta)6.27K. However, in the 18 S particle, 27K functioned in the assembly and attachment of phycocyanin trimers to a core domain. Based on the analysis of the components in fractions 1-3, a model is proposed which describes the structure of the AN112 phycobilisome, with emphasis on the roles of the linker polypeptides in the assembly of the core.     

Phycobilisome Structure of Porphyridium cruentum: POLYPEPTIDE COMPOSITION

Purified phycobilisomes of Porphyridium cruentum were solubilized in sodium dodecyl sulfate and resolved by sodium dodecyl sulfate-acrylamide gel electrophoresis into nine colored and nine colorless polypeptides. The colored polypeptides accounted for about 84% of the total stainable protein, and the colorless polypeptides accounted for the remaining 16%. Five of the colored polypeptides ranging in molecular weight from 13,300 to 19,500 were identified as the α and β subunits of allophycocyanin, R-phycocyanin, and phycoerythrin. Three others (29,000-30,500) were orange and are probably related to the γ subunit of phycoerythrin. Another colored polypeptide had a molecular weight of 95,000 and the characteristics of long wavelength-emitting allophycocyanin. Sequential dissociation of phycobilisomes, and analysis of the polypeptides in each fraction, revealed the association of a 32,500 molecular weight colorless polypeptide with a phycoerythrin fraction. The remaining eight colorless polypeptides were in the core fraction of the phycobilisome, which also was enriched in allophycocyanin. In addition, the core fraction was enriched in a colored 95,000 dalton polypeptide. Inasmuch as a polypeptide with the same molecular weight is found in thylakoid membranes (free of phycobilisomes), it is suggested that this polypeptide is involved in anchoring phycobilisomes to thylakoid membranes.     

Molecular architecture of a light-harvesting antenna. Comparison of wild type and mutant Synechococcus 6301 phycobilisomes

Phycobilisomes of the cyanobacterium Synechococcus 6301 contain C-phycocyanin and allophycocyanin in a molar ratio of approximately 3.8:1, a minor biliprotein, allophycocyanin B, and nonpigmented polypeptides of 75, 33, 30, and 27 kilodaltons. A nitrosoguanidine-induced mutant AN112 produces altered phycobilisomes with the molar ratio of C-phycocyanin to allophycocyanin reduced to approximately 1.4:1 and without any of the 33- and 30-kilodalton polypeptides. The mutant and wild type phycobilisomes contain the same molar amount of the 75- and 27-kilodalton polypeptides relative to allophycocyanin. As seen by electron microscopy, the allophycocyanin-containing core of the mutant and of the wild type phycobilisomes appears the same. In some views of the core, each of the two core units in the mutant particles can be seen to consist of four discs approximately 3 nm thick. In wild type phycobilisomes five or six rods, made up of two to six stacked discs (11.5 X 6 nm) are attached to the core. In the mutant, no such rods are seen; rather, single disc-shaped elements, ranging from two to six in number, are found attached. Spectroscopic measurements show that the assembly form of phycocyanin in the mutant phycobilisomes differs from that in the wild type particles but reveal no difference in the organization of the core elements. These results indicate that the portions of the rod substructures of wild type phycobilisomes, beyond the disc proximal to the core, are made up of phycocyanin and the 33- and 30-kilodalton polypeptides. Emission from phycocyanin is a significant component in the fluorescence from isolated Synechococcus 6301 phycobilisomes and indicates an upper limit of 94% for the efficiency of energy transfer from phycocyanin to allophycocyanin and allophycocyanin B in these particles.     

Three-dimensional reconstruction of a light-harvesting complex I-photosystem I (LHCI-PSI) supercomplex from the green alga Chlamydomonas reinhardtii. Insights into light harvesting for PSI

A supercomplex containing the photosystem I (PSI) and chlorophyll a/b light-harvesting complex I (LHCI) has been isolated using a His-tagged mutant of Chlamydomonas reinhardtii. This LHCI-PSI supercomplex contained approximately 215 chlorophyll molecules of which 175 were estimated to be chlorophyll a and 40 to be chlorophyll b, based on P700 oxidation and chlorophyll a/b ratio measurements. Its room temperature long wavelength absorption peak was at 680 nm, and it emitted chlorophyll fluorescence maximally at 715 nm (77 K). The LHCI was composed of four or more different types of Lhca polypeptides including Lhca3. No LHCII proteins or other phosphoproteins were detected in the LHCI-PSI supercomplexes suggesting that the cells from which they were isolated were in State 1. Electron microscopy of negatively stained samples followed by image analysis revealed the LHCI-PSI supercomplex to have maximal dimensions of 220 A by 180 A and to be approximately 105 A thick. An averaged top view was used to model in x-ray and electron crystallographic data for PSI and Lhca proteins respectively. We conclude that the supercomplex consists of a PSI reaction center monomer with 11 Lhca proteins arranged along the side where the PSI proteins, PsaK, PsaJ, PsaF, and PsaG are located. The estimated molecular mass for the complex is 700 kDa including the bound chlorophyll molecules. The assignment of 11 Lhca proteins is consistent with a total chlorophyll level of 215 assuming that the PSI reaction center core binds approximately 100 chlorophylls and that each Lhca subunit binds 10 chlorophylls. There was no evidence for oligomerization of Chlamydomonas PSI in contrast to the trimerization of PSI in cyanobacteria.     

Identification of a protein mediating respiratory supercomplex stability

The complexes of the electron transport chain associate into large macromolecular assemblies, which are believed to facilitate efficient electron flow. We have identified a conserved mitochondrial protein, named respiratory supercomplex factor 1 (Rcf1-Yml030w), that is required for the normal assembly of respiratory supercomplexes. We demonstrate that Rcf1 stably and independently associates with both Complex III and Complex IV of the electron transport chain. Deletion of the RCF1 gene caused impaired respiration, probably as a result of destabilization of respiratory supercomplexes. Consistent with the hypothetical function of these respiratory assemblies, loss of RCF1 caused elevated mitochondrial oxidative stress and damage. Finally, we show that knockdown of HIG2A, a mammalian homolog of RCF1, causes impaired supercomplex formation. We suggest that Rcf1 is a member of an evolutionarily conserved protein family that acts to promote respiratory supercomplex assembly and activity.     

Molecular architecture of a light-harvesting antenna. Core substructure in Synechococcus 6301 phycobilisomes: two new allophycocyanin and allophycocyanin B complexes

Two new allophycocyanin-containing complexes were found among the products of partial dissociation of the phycobilisomes of Synechococcus 6301 strain AN112. These complexes were purified to homogeneity and characterized with respect to composition, stability, and spectroscopic properties. The structures of the complexes were established to be (alpha AP beta AP)3 . 10.5K and (alpha 1APB alpha 2AP beta 3AP) . 10.5 K, where alpha AP and beta AP are subunits of allophycocyanin, and alpha APB is the subunit of allophycocyanin B (see Lundell, D. J., and Glazer, A. N. (1981) J. Biol. Chem. 256, 12600-12606), and 10.5K is an uncolored polypeptide of 10.5-kilodaltons. These complexes are derived from the core substructure of the phycobilisome. Electron microscopic studies of the morphology of the core of strain AN112 phycobilisomes (Yamanaka, G., Glazer, A. N., and Williams, R. C. (1980) J. Biol. Chem. 255, 11004-11010) as well as structural studies of an 18 S subassembly derived from the phycobilisomes by partial dissociation (Yamanaka, G., Lundell, D. J., and Glazer, A. N. (1982) J. Biol. Chem. 257, 4077-4086) indicated that the core assembly consisted of two cylindrical elements each made up of the same four distinct "trimeric" biliprotein-containing complexes. Two such core components, (alpha AP beta AP)3 and alpha 2AP beta 2AP. 18.3K . 75K (where 18.3K and 75K are polypeptides of 18.3- and 75-kilodaltons), were shown to be contained within the 18 S subassembly (Lundell, D. J., and Glazer, A. N. (1983) J. Biol. Chem. 258, 894-901). The isolation of the two allophycocyanin-containing complexes described here completes the characterization of the four types of components in the Synechococcus 6301 phycobilisome core. Two lines of evidence indicate that each of the four complexes is present twice in the core: comparison of the compositions (and yields) of the complexes with that of the intact AN112 phycobilisome, and near-coincidence of the molar absorption spectrum of the phycobilisome with that generated by summing the spectra of the constituent complexes taken in appropriate molar proportions.     

Molecular architecture of a light-harvesting antenna. Structure of the 18 S core-rod subassembly of the Synechococcus 6301 phycobilisome

The 18 S subassembly particles obtained by partial dissociation of phycobilisomes from Synechococcus 6301 (Anacystis nidulans) strain AN 112 contain approximately one-half of the mass of the phycobilisome and include core-rod junctions (Yamanaka, G., Lundell, D. J., and Glazer, A. N. (1982) J. Biol. Chem. 257, 4077-4086). The polypeptide composition of 18 S complexes, determined by analysis of uniformly 14C-labeled phycobilisomes, gave the following stoichiometry: 75K:27K:18.3K:alpha beta allophycocyanin monomer: alpha beta phycocyanin monomer of 1:2:1:5:6; where 75K, 27K, etc. represent polypeptides of 75, 27 kilodaltons, etc. The 18.3K polypeptide is a hitherto underscribed biliprotein bearing a single phycocyanobilin. The NH2-terminal sequence of this subunit was determined to be homologous to that of the beta subunit of allophycocyanin. Chromatography of products resulting from limited trypsin treatment of the 18 S complex led to the isolation of three subcomplexes: a mixture of (alpha beta)3 . 22K and (alpha beta)3 . 24K phycocyanin complexes, an (alpha beta)3 allophycocyanin trimer, and an (alpha beta)2 . 18.3K.40K.11K allophycocyanin-containing complex. The 22K and 24K components were products of the degradation of the 27K polypeptides, whereas the 40K and 11K components were derived from the 75K polypeptide. The subcomplexes accounted for the composition of the 18 S complex. Determination of the composition, stoichiometry, and spectroscopic properties of the subcomplexes has led to a model of the polypeptide arrangement within the 18 S complex and of the pathway of energy transfer among these polypeptides.     

Structure of a TOC-TIC supercomplex spanning two chloroplast envelope membranes

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The architecture and function of the light-harvesting apparatus of purple bacteria: from single molecules to in vivo membranes

This review describes the structures of the two major integral membrane pigment complexes, the RC-LH1 'core' and LH2 complexes, which together make up the light-harvesting system present in typical purple photosynthetic bacteria. The antenna complexes serve to absorb incident solar radiation and to transfer it to the reaction centres, where it is used to 'power' the photosynthetic redox reaction and ultimately leads to the synthesis of ATP. Our current understanding of the biosynthesis and assembly of the LH and RC complexes is described, with special emphasis on the roles of the newly described bacteriophytochromes. Using both the structural information and that obtained from a wide variety of biophysical techniques, the details of each of the different energy-transfer reactions that occur, between the absorption of a photon and the charge separation in the RC, are described. Special emphasis is given to show how the use of single-molecule spectroscopy has provided a more detailed understanding of the molecular mechanisms involved in the energy-transfer processes. We have tried, with the help of an Appendix, to make the details of the quantum mechanics that are required to appreciate these molecular mechanisms, accessible to mathematically illiterate biologists. The elegance of the purple bacterial light-harvesting system lies in the way in which it has cleverly exploited quantum mechanics.     

Structure of a large photosystem II supercomplex from Acaryochloris marina

Acaryochloris marina is a prochlorophyte-like cyanobacterium containing both phycobilins and chlorophyll d as light harvesting pigments. We show that the chlorophyll d light harvesting system, composed of Pcb proteins, functionally associates with the photosystem II (PSII) reaction center (RC) core to form a giant supercomplex. This supercomplex has a molecular mass of about 2300 kDa and dimensions of 385 A x 240 A. It is composed of two PSII-RC core dimers arranged end-to-end, flanked by eight symmetrically related Pcb proteins on each side. Thus each PSII-RC monomer has four Pcb subunits acting as a light harvesting system which increases the absorption cross section of the PSII-RC core by almost 200%.     

Evidence of the supercomplex organization of photosystem II and light-harvesting complexes in <Emphasis Type="Italic">Nannochloropsis granulata</Emphasis>

Diverse light-harvesting complexes (LHCs) have been found in photosynthetic microalgae that originated from secondary endosymbiosis involving primary red algae. However, the associations between LHCs and photosystem I (PSI) and photosystem II (PSII) in these microalgae are not fully understood. Eustigmatophyta is a red algal lineage that appears to have a unique organization in its photosynthetic machinery, consisting of only chlorophyll a and carotenoids that are atypical compared with other closely related groups. In this study, the supramolecular organization of pigment–protein complexes in the eustigmatophyte alga, Nannochloropsis granulata was investigated using Clear Native (CN) PAGE coupled with two-dimensional (2D) SDS-PAGE. Our results showed two slowly migrating green bands that corresponded to PSII supercomplexes, which consisted of reaction centers and LHCs. These green bands were also characterized as PSII complexes by their low temperature fluorescence emission spectra. The protein subunits of the PSII–LHC resolved by 2D CN/SDS-PAGE were analyzed by mass spectrometry, and four different LHC proteins were identified. Phylogenetic analysis of the identified LHC protein sequences revealed that they belonged to four different Lhc groups; (1) stress-related Lhcx proteins, (2) fucoxanthin chlorophyll a/c-binding Lhcf proteins, (3) red-shifted Chromera light-harvesting proteins (Red-CLH), and (4) Lhcr proteins, which are commonly found in organisms possessing red algal plastids. This is the first report showing evidence of a pigment–protein supercomplex consisting of PSII and LHCs, and to identify PSII-associated LHC proteins in Nannochloropsis.     

Phycobilisome model with novel skeleton-like structures in a glaucocystophyte Cyanophora paradoxa

Phycobilisome (PBS) is a photosynthetic antenna supercomplex consisting of a central core subcomplex with several peripheral rods radiating from the core. Subunit structure of PBS was studied in a glaucocystophyte Cyanophora paradoxa strain NIES 547. Subunit composition of PBS was identified by N-terminal sequencing and genes for the subunits were determined by homology search of databases. They included rod linker proteins CpcK1 and CpcK2, rod-core linker proteins CpcG1 and CpcG2, and core linker proteins ApcC1 and ApcC2. Subfractionation by native polyacrylamide gel electrophoresis provided evidence for novel subcomplexes (ApcE/CpcK1/CpcG2/ApcA/ApcB/CpcD and ApcE/CpcK2/CpcG1/ApcA/ApcB), which connect rod and core subcomplexes. These skeleton-like structures may serve as a scaffold of the whole PBS assembly. Different roles of ApcC1 and ApcC2 were also suggested. Based on these findings, structural models for PBS were proposed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

p120-catenin is a key component of the cadherin-gamma-secretase supercomplex

In this work, we show several previously unknown features of p120-catenin in a cadherin–catenin complex that are critical for our understanding of cadherin-based adhesion and signaling. We show that in human epithelial A-431 cells, nearly all p120 molecules engage in high-affinity interaction with E-cadherin–catenin complexes located at the cellular surface. p120 is positioned in proximity to α-catenin in the complex with cadherin. These findings suggest a functional cooperation between p120 and α-catenin in cadherin-based adhesion. A low level of cadherin-free p120 molecules, in contrast, could facilitate p120-dependent signaling. Finally, we present compelling evidence that p120 is a key linker cementing the E-cadherin–catenin complex with the transmembrane protease γ-secretase. The cell–cell contact location of this supercomplex makes it an important candidate for conducting different signals that rely on γ-secretase proteolytic activity.     

Light Intensity Adaptation and Phycobilisome Composition of Microcystis aeruginosa

Phycobilisomes isolated from Microcystis aeruginosa grown to midlog at high light (270 microeinsteins per square meter per second) or at low light intensities (40 microeinsteins per square meter per second) were found to be identical. Electron micrographs established that they have a triangular central core apparently consisting of three allophycocyanin trimers surrounded by six rods, each composed of two hexameric phycocyanin molecules. The apparent mass of a phycobilisome obtained by gel filtration is 2.96 × 10⁶ daltons. The molar ratio of the phycobiliproteins per phycobilisome is 12 phycocyanin hexamers:9 allophycocyanin trimers. The electron microscopic observations combined with the phycobilisome apparent mass and the phycobiliprotein stoichiometry data indicate that M. aeruginosa phycobilisomes are composed of a triangular central core of three stacks of three allophycocyanin trimers and six rods each containing two phycocyanin hexamers. Adaptation of M. aeruginosa to high light intensity results in a decrease in the number of phycobilisomes per cell with no alteration in phycobilisome composition or structure.     

Reconstructing an ancestral mammalian immune supercomplex from a marsupial major histocompatibility complex

The first sequenced marsupial genome promises to reveal unparalleled insights into mammalian evolution. We have used theMonodelphis domestica (gray short-tailed opossum) sequence to construct the first map of a marsupial major histocompatibility complex (MHC). The MHC is the most gene-dense region of the mammalian genome and is critical to immunity and reproductive success. The marsupial MHC bridges the phylogenetic gap between the complex MHC of eutherian mammals and the minimal essential MHC of birds. Here we show that the opossum MHC is gene dense and complex, as in humans, but shares more organizational features with non-mammals. The Class I genes have amplified within the Class II region, resulting in a unique Class I/II region. We present a model of the organization of the MHC in ancestral mammals and its elaboration during mammalian evolution. The opossum genome, together with other extant genomes, reveals the existence of an ancestral “immune supercomplex” that contained genes of both types of natural killer receptors together with antigen processing genes and MHC genes.     

Identification of a cytochrome bc1-aa3 supercomplex in Rhodobacter sphaeroides

Respiration is carried out by a series of membrane-bound complexes in the inner mitochondrial membrane or in the cytoplasmic membrane of bacteria. Increasing evidence shows that these complexes organize into larger supercomplexes. In this work, we identified a supercomplex composed of cytochrome (cyt.) bc₁ and aa₃-type cyt. c oxidase in Rhodobacter sphaeroides. We purified the supercomplex using a His-tag on either of these complexes. The results from activity assays, native and denaturing PAGE, size exclusion chromatography, electron microscopy, optical absorption spectroscopy and kinetic studies on the purified samples support the formation and coupled quinol oxidation:O₂ reduction activity of the cyt. bc₁-aa₃ supercomplex. The potential role of the membrane-anchored cyt. c_y as a component in supercomplexes was also investigated.