Nucleotide sequence of two cDNAs encoding fucoxanthin chlorophyll a/c proteins in the diatom Odontella sinensis

Two cDNA clones encoding fucoxanthin chlorophyll a/c-binding proteins (FCP) in the diatom Odontella sinensis have been cloned and sequenced. The derived amino acid sequences of both clones are identical, comparison of the corresponding nucleic acids reveals differences only in the third codon position, suggesting a recent gene duplication. The derived proteins are similar to the chlorophyll a/b-binding proteins of higher plants. The presequences for plastid import resemble signal sequences for cotranslational import rather than transit peptides of higher plants. They are very similar to the presequences of FCP proteins in the diatom Phaeodactylum, but different from the presequences of the -subunit of CF₀CF₁ of Odontella and the peridinin chlorophyll a binding proteins (PCP) of the dinoflagellate Symbiodinium.Abbreviations CAB chlorophyll a/b-binding protein - FCP fucoxanthin chlorophyll a/c-binding protein - fcp the respective FCP genes - LHC light-harvesting complex - PCP peridinin chlorophyll a-binding protein - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

An amino-proximal hydrophobic domain in the major light-harvesting chlorophyll a/b-protein is essential for membrane integration and protein stability

The major light-harvesting chlorophyll a/b-protein (LHCP) of higher plant chloroplasts is a nuclearencoded, integral thylakoid membrane protein that binds photosynthetic pigments and occurs in situ in an oligomeric form. We have previously examined structural and functional domains of the mature apoprotein by use of mutant LHCPs and in vitro assays for uptake and insertion. Results presented here demonstrate the effects of several mutations in the amino terminal domain of the mature apoprotein. Deletion of amino acid residues 12–58 greatly affected import into chloroplasts, while deletion or alteration of the hydrophobic region E⁶⁵VIHARWAM⁷³ led to rapid degradation of the mutant LHCP. We suggest that this amino-proximal region is essential for the stability of the LHCP and its ability to integrate into the thylakoid membranes. A structural/functional relationship of this region to a previously examined hydrophobic carboxy-proximal domain [Kohorn and Tobin (1989), The Plant Cell 1, 159–166] is proposed.Abbreviations BSA bovine serum albumin faction V - ELIPs early light-inducible proteins - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - LHCP light-harvesting chlorophyll a/b-protein - LHC IIb light-harvesting complex associated with Photosystem II - pLHCP precursor to LHCP - Rubisco ribulose 1,5-biphosphate carboxylase-oxygenase - SDS-PAGE sodium dodecyl sulfate-poly-acrylamide gel electrophoresis     

Cloning and nucleotide sequence of a cDNA encoding a major fucoxanthin-, chlorophylla/c-containing protein from the chrysophyteIsochrysis galbana: implications for evolution of thecab gene family

We investigated the primary structure of a cDNA encoding a light-harvesting protein from the marine chrysophyteIsochrysis galbana. Antibodies raised against the major fucoxanthin, chlorophylla/c-binding light-harvesting protein (FCP) ofI. galbana were used to select a cDNA clone encoding one of the FCP apoproteins. The nucleic acid and deduced amino acid sequences reveal conserved regions within the first and third transmembrane spans with Chla/b-binding proteins and with FCPs of another chromophyte. However, the amino acid identity betweenI. galbana FCP and othercab genes of FCPs is only ca. 30%. Phylogenetic analyses demonstrated that the FCP genes of both diatoms and chrysophytes sequenced to date are more closely related tocab genes encoding LHC I, CP 29, and CP 24 of higher plants than tocab genes encoding LHC II of chlorophytes. We propose that LHC I, CP 24 and CP 29 and FCP might have originated from a common ancestral chl binding protein and that the major LHC II of Chla/b-containing organisms arose after the divergence between the chromophytes and the chlorophytes.     

Hypothesis for the evolution of three-helix Chl a/b and Chl a/c light-harvesting antenna proteins from two-helix and four-helix ancestors

The nuclear-encoded Chl a/b and Chl a/c antenna proteins of photosynthetic eukaryotes are part of an extended family of proteins that also includes the early light-induced proteins (ELIPs) and the 22 kDa intrinsic protein of PS II (encoded by psbS gene). All members of this family have three transmembrane helices except for the psbS protein, which has four. The amino acid sequences of these proteins are compared and related to the three-dimensional structure of pea LHC II Type I (Kühlbrandt and Wang, Nature 350: 130–134, 1991). The similarity of psbS to the three-helix members of the family suggests that the latter arose from a four-helix ancestor that lost its C-terminal helix by deletion. Strong internal similarity between the two halves of the psbS protein suggests that it in turn arose as the result of the duplication of a gene encoding a two-helix protein. Since psbS is reported to be present in at least one cyanobacterium, the ancestral four-helix protein may have been present prior to the endosymbiotic event or events that gave rise to the photosynthetic eukaryotes. The Chl a/b and Chl a/c antenna proteins, and the immunologically-related proteins in the rhodophytes may have had a common ancestor which was present in the early photosynthetic eukaryotes, and predated their division into rhodophyte, chromophyte and chlorophyte lineages. The LHC I-LHC II divergence probably occurred before the separation of higher plants from chlorophyte algae and euglenophytes, and the different Types of LHC I and LHC II proteins arose prior to the separation of angiosperms and gymnosperms.Abbreviations CAB Chl a/b-binding - ELIP early light-induced protein - FCP fucoxanthin-Chl a/c protein - PCR polymerase chain reaction - TMH trans-membrane helix     

Nucleotide sequence of a cDNA clone encoding the precursor of the peridinin-chlorophyll a-binding protein from the dinoflagellate Symbiodinium sp.

mRNA from the dinoflagellate Symbiodinium sp. isolated from the staghorn coral Acropora formosa was used for the construction of cDNA libraries. A cDNA clone was identified which encoded the precursor of peridinin-chlorophyll a-binding protein (PCP), including a 52 amino acid transit peptide and the 313 amino acid mature protein. The deduced amino acid sequence clearly contains an internal duplication, implying that amongst dinoflagellates the M _r 35 000 form of PCP has arisen by duplication and fusion of genes encoding the M _r 15 000 form. This is the first reported sequence of a dinoflagellate light-harvesting protein. The anatomy of the mature protein and the transit peptide are discussed.Abbreviations PCP peridinin-chlorophyll a-binding protein; cab, chlorophyll a/b-binding protein - LHC light-harvesting complex - FCP fucoxanthin-chlorophyll a/c-binding protein     

Assembly of the barley light-harvesting chlorophyll a/b proteins in barley etiochloroplasts involves processing of the precursor on thylakoids

A barley gene encoding the major light-harvesting chlorophyll a/b-binding protein (LHCP) has been sequenced and then expressed in vitro to produce a labelled LHCP precursor (pLHCP). When barley etiochloroplasts are incubated with this pLHCP, both labelled pLHCP and LHCP are found as integral thylakoid membrane proteins, incorporated into the major pigment-protein complex of the thylakoids. The presence of pLHCP in thylakoids and its proportion with respect to labelled LHCP depends on the developmental stage of the plastids used to study the import of pLHCP. The reduced amounts of chlorophyll in a chlorophyll b-less mutant of barley does not affect the proportion of pLHCP to LHCP found in the thylakoids when import of pLHCP into plastids isolated from the mutant plants is examined. Therefore, insufficient chlorophyll during early stages of plastid development does not seem to be responsible for their relative inefficiency in assembling pLHCP. A chase of labelled pLHCP that has been incorporated into the thylakoids of intact plastids, by further incubation of the plastids with unlabelled pLHCP, reveals that the pLHCP incorporated into the thylakoids can be processed to its mature size. Our observations strongly support the hypothesis that after import into plastids, pLHCP is inserted into thylakoids and then processed to its mature size under in vivo conditions.     

A Phylogenetic Assessment of the Eukaryotic Light-Harvesting Antenna Proteins, with Implications for Plastid Evolution

The light-harvesting complexes (LHCs) are a superfamily of chlorophyll-binding proteins present in all photosynthetic eukaryotes. The Lhc genes are nuclear-encoded, yet the pigment–protein complexes are localized to the thylakoid membrane and provide a marker to follow the evolutionary paths of plastids with different pigmentation. The LHCs are divided into the chlorophyll a/b-binding proteins of the green algae, euglenoids, and higher plants and the chlorophyll a/c-binding proteins of various algal taxa. This work examines the phylogenetic position of the LHCs from three additional taxa: the rhodophytes, the cryptophytes, and the chlorarachniophytes. Phylogenetic analysis of the LHC sequences provides strong statistical support for the clustering of the rhodophyte and cryptomonad LHC sequences within the chlorophyll a/c-binding protein lineage, which includes the fucoxanthin–chlorophyll proteins (FCP) of the heterokonts and the intrinsic peridinin–chlorophyll proteins (iPCP) of the dinoflagellates. These associations suggest that plastids from the heterokonts, haptophytes, cryptomonads, and the dinoflagellate, Amphidinium, evolved from a red algal-like ancestor. The Chlorarachnion LHC is part of the chlorophyll a/b-binding protein assemblage, consistent with pigmentation, providing further evidence that its plastid evolved from a green algal secondary endosymbiosis. The Chlorarachnion LHC sequences cluster with the green algal LHCs that are predominantly associated with photosystem II (LHCII). This suggests that the green algal endosymbiont that evolved into the Chlorarachnion plastid was acquired following the emergence of distinct LHCI and LHCII complexes. Received: 25 February 1998 / Accepted: 13 May 1998     

Pigment and protein composition of reconstituted light-harvesting complexes and effects of some protein modifications

The structure and heterogeneity of LHC II were studied by in vitro reconstitution of apoproteins with pigments (Plumley and Schmidt 1987, Proc Natl Acad Sci 84: 146–150). Reconstituted CP 2 complexes purified by LDS-PAGE were subsequently characterized and shown to have spectroscopic properties and pigment-protein compositions and stoichiometries similar to those of authentic complexes. Heterologous reconstitutions utilizing pigments and light-harvesting proteins from spinach, pea and Chlamydomonas reinhardtii reveal no evidence of specialized binding sites for the unique C. reinhardtii xanthophyll loroxanthin: lutein and loroxanthin are interchangeable for in vitro reconstitution. Proteins modified by the presence of a transit peptide, phosphorylation, or proteolytic removal of the NH₂-terminus could be reconstituted. Evidence suggests that post-translational modification are not responsible for the presence of six electrophoretic variants of C. reinhardtii CP 2. Reconstitution is blocked by iodoacetamide pre-treatment of the apoproteins suggesting a role for cysteine in pigment ligation and/or proper folding of the pigment-protein complex. Finally, no effect of divalent cations on pigment reassembly could be detected.Abbreviations cab chlorophyll a/b-binding protein genes - Chl chlorophyll - CP2 light-harvesting chlorophyll A+b-protein complex fractionated by mildly denaturing LDS-PAGE from Photosystem II in thylakoids - CP 43 and CP 47 chlorophyll a-antenna complexes fractionated from Photosystem II in thylakoids by mildly denaturing LDS-PAGE at 4°C - IgG gamma immunoglobulin - LDS lithium dodecyl sulfate - LDS-PAGE lithium dodecyl sulfate polyacrylamide gel electrophoresis at 4°C - LHC I and LHC II thylakoid light-harvesting chlorophyll a+b-protein holocomplexes associated with Photosystems I and II, respectively - PS II Photosystem II - TX100 Triton X-100 - TX100-derived LHC light-harvesting complexes enriched in LHC II following fractionation of thylakoids by TX100     

Biogenesis of thylakoid membranes with emphasis on the process in Chlamydomonas

Recent results obtained by electron microscopic and biochemical analyses of greening Chlamydomonas reinhardtii y1 suggest that localized expansion of the plastid envelope is involved in thylakoid biogenesis. Kinetic analyses of the assembly of light-harvesting complexes and development of photosynthetic function when degreened cells of the alga are exposed to light suggest that proteins integrate into membrane at the level of the envelope. Current information, therefore, supports the earlier conclussion that the chloroplast envelope is a major biogenic structure, from which thylakoid membranes emerge. Chloroplast development in Chlamydomonas provides unique opportunities to examine in detail the biogenesis of thylakoids.Abbreviations Rubisco ribulose bisphosphate carboxylase/oxygenase - CAB Chl a/b-binding (proteins) - Chlide chlorophyllide - LHC I light-harvesting complex of PS I - LHC II light-harvesting complex of PS II - Pchlide protochlorophyllide     

Nomenclature for membrane-bound light-harvesting complexes of cyanobacteria

Accessory chlorophyll-binding proteins (CBP) in cyanobacteria have six transmembrane helices and about 11 conserved His residues that might participate in chlorophyll binding. In various species of cyanobacteria, the CBP proteins bind different types of chlorophylls, including chlorophylls a, b, d and divinyl-chlorophyll a, b. The CBP proteins do not belong to the light-harvesting complexes (LHC) superfamily of plant and algae. The proposed new name of CBP for this class of proteins, which is a unique accessory light-harvesting superfamily in cyanobacteria, clarifies the confusion of names of prochlorophytes chlorophyll binding protein (Pcb), PSII-like light-harvesting proteins and iron-stress-induced protein A (IsiA). The CBP complexes are a member of a larger family that includes the chlorophyll a-binding proteins CP43 and CP47 that function as core antennas of photosystem II.     

A nomenclature for the genes encoding the chlorophylla/b-binding proteins of higher plants

We propose a nomenclature for the genes encoding the chlorophylla/b-binding proteins of the light-harvesting complexes of photosystem I and II. The genes encoding LHC I and LHC II polypeptides are namedLhca1 throughLhca4 andLhcb1 throughLhcb6, respectively. The proposal follows the general format recommended by the Commision on Plant Gene Nomenclature. We also present a table for the conversion of old gene names to the new nomenclature.     

Immunological characterization of chlorophyll a/b-binding proteins of barley thylakoids

Monoclonal antibodies have been raised against the light-harvesting chlorophyll a/b-binding proteins of photosystem I (LHCI) using a photosystem (PS) I preparation (PSI-200) wild-type from barley (Hordeum vulgare L. cv. Svaløf's Bonus) as the antigen. These antibodies cross-reacted with a minor light-harvesting chlorophyll a/b-protein of PSII (Chla/b-P1=CP29), but not with the major one, LHCII (=Chla/b-P2^**). Similarly, a monoclonal antibody to Chla/b-P1, elicited by a PSII preparation as the antigen, cross-reacted with LHCI, but not LHCII. This explains why an antigen consisting of LHCII, free of LHCI, but contaminated with Chla/b-P1, can elicit antibodies which cross-react with LHCI. Immunoblot assays showed that LHCI and Chla/b-P1 have at least two epitopes in common. Immunogold labelling of thin-sectioned wild-type thylakoids confirmed a preferential localisation of Chla/b-P1 in grana partition membranes and LHCI in stroma lamellae. The presence of LHCI was demonstrated in barley mutants lacking the PSI reaction centre (viridis-zb ⁶³) and chlorophyll b (chlorina-f2), and was correlated with the presence of long-wavelength (730 nm) fluorescence emission at 77 K. The mutant viridis-k ²³, which has a 77 K long-wavelength fluorescence peak at 720 nm, was shown by immune-blot assay to lack LHCI, although Chla/b-P1 was present.Abbreviations Chl-P chlorophyll-protein - CM Carlsberg Monoclonal - Da dalton - LHC light-harvesting complex - PAGE polyacrylamide gel electrophoresis - PSI, II photosystem I, II - PSI-200 PSI containing LHCI polypeptides - SDS sodium dodecyl sulphate     

A barley cDNA clone encoding a type III chlorophyll a/b-binding polypeptide of the light-harvesting complex II

The nucleotide sequence of a leaf cDNA clone encoding a Type III chlorophyll a/b-binding (CAB) protein of light-harvesting complex II (LHCII) in barley is reported. Sequence comparisons and results from in vitro import into chloroplasts demonstrate that the cDNA clone encodes a functional transit peptide of 45 amino acid residues and a mature polypeptide of 223 residues with a predicted molecular mass of 24.3 kDa. After insertion into thylakoids, the mature protein is resistant to protease attack. Hybridization analysis using a gene-specific probe shows that the gene is expressed in dark-grown seedlings and that the amount of mRNA increases during illumination.     

The resolution and biochemical characterization of subcomplexes of the main light-harvesting chlorophyll a/b-protein complex of Photosystem II (LHC II)

LHC II isolated from carnation leaves has been solubilized and resolved by a newly developed, vertical-bed non-denaturing isoelectric focusing in polyacrylamide slab gels to yield three trimeric subcomplexes focusing at pH 4.52, 4.42 and 4.37 (designated a, b and c, respectively), comprising approximately 38%, 24% and 38% of the chlorophyll. The spectroscopic data demonstrated a close similarity among LHC II subcomplexes concerning their chlorophyll content and organization. The most alkaline and the most acidic subcomplex contained the 27 kDa polypeptide of LHC II while the intermediate pI fraction contained both LHC II polypeptides, i.e. 27 kDa and 26 kDa ones associated at 2:1 stoichiometry. The 27 kDa polypeptide could be resolved by denaturing isoelectrofocusing into 10 pI molecular isoforms covering 5.90–4.20 pH range. Three of the isoforms were found in the subcomplexes a and b and eight in the subcomplex c. The 26 kDa polypeptide comprised the unique pI molecular isoform focusing at pH 5.61.Abbreviations CBB G-250 Coomassie Brilliant Blue G-250 - chl chlorophyll - DM n-dodecyl--d-maltoside - EDTA ethylendiaminotetraacetic acid - IEF isoelectric focusing - LHC II the main light-harvesting chlorophyll a/b-protein complex of Photosystem II - LHCP II apoprotein of the main light-harvesting chlorophyll a/b-protein complex of Photosystem II - NP-40 polyethyleneglycol-p-isooctylphenyl ether - pI isoelectric point - OG octyl--d-glucopyranoside - PS II Photosystem II - SDS-PAGE sodium dodecylsulphate polyacrylamide gel electrophoresis - TCA trichlorooacetic acid     

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     

Sequence conservation of light-harvesting and stress-response proteins in relation to the three-dimensional molecular structure of LHCII

The structure of pea light-harvesting complex LHCII determined to 3.4 Å resolution by electron crystallography (Kühlbrandt, Wang and Fujiyoshi (1994) Nature 367: 614–621) was examined to determine the relationship between structural elements and sequence motifs conserved in the extended family of light-harvesting antennas (Chl a/b, fucoxanthin Chl a/c proteins) and membrane-intrinsic stress-induced proteins (ELIPs) to which LHCII belongs. It is predicted that the eukaryotic ELIPs can bind at least four molecules of Chl. The one-helix prokaryotic ELIP of Synechococcus was modelled as a homodimer based on the high degree of conservation of residues involved in the interactions of the first (B) and third (A) helices of LHCII.Abbreviations CAB Chl a/b-binding - ELIP early light-inducible protein - FCP fucoxanthin-Chl a/c protein - Lut1, Lut2 lutein molecules 1 and 2     

Light-harvesting proteins of diatoms: Their relationship to the chlorophyll a/b binding proteins of higher plants and their mode of transport into plastids

Summary We have cloned and characterized members of a gene family encoding polypeptide constituents of the fucoxanthin, chlorophyll a/c protein complex, a light-harvesting complex associated with photosystem II of diatoms and brown algae. Three cDNA clones encoding proteins associated with this complex in the diatom Phaeodactylum tricornutum have been isolated. As deduced from the nucleotide sequences, these light-harvesting proteins show homology to the chlorophyll a/b binding polypeptides of higher plants. Specifically, the N-terminal regions of the fucoxanthin, chlorophyll a/c-binding proteins are homologous to the chlorophyll a/b binding proteins in both the third membrane-spanning domain and the stroma-exposed region between membrane-spanning domains 2 and 3. Like the chlorophyll a/b-binding proteins, the mature fucoxanthin, chlorophyll a/c polypeptides have three hydrophobic -helical domains which could span the membrane bilayer. The similarities between the two light-harvesting proteins might reflect the fact that both bind chlorophyll molecules and/or might be important for maintaining certain structural features of the complex. There is little similarity between the N-terminal sequences of the primary translation products of the fucoxanthin, chlorophyll a/c proteins and any transit sequences that have been characterized. Instead, the N-terminal sequences have features resembling those of signal sequences. Thus either transit peptides used in P. tricornutum show little resemblance to those of higher plants and green algae or the nuclear-encoded plastid proteins enter the organelle via a mechanism different from that used in higher plants.     

The fucoxanthin-chlorophyll proteins from a chromophyte alga are part of a large multigene family: structural and evolutionary relationships to other light harvesting antennae

 A fucoxanthin-chlorophyll protein (FCP) cDNA from the raphidophyte Heterosigma carterae encodes a 210-amino acid polypeptide that has similarity to other FCPs and to the chlorophyll a/b-binding proteins (CABs) of terrestrial plants and green algae. The putative transit sequence has characteristics that resemble a signal sequence. The Heterosigma fcp genes are part of a large multigene family which includes members encoding at least two significantly different polypeptides (Fcp1, Fcp2). Comparison of the FCP sequences to the recently determined three-dimensional structure of the pea LHC II complex indicates that many of the key amino acids thought to participate in the binding of chlorophyll and the formation of complex-stabilizing ionic interactions are well conserved. Phylogenetic analyses of sequences of light-harvesting proteins shows that the FCPs of several chromophyte phyla form a natural group separate from the intrinisic peridinin-chlorophyll proteins (iPCPs) of the dinoflagellates. Although the FCP and CAB genes shared a common ancestor, these lineages diverged from each other prior to the separation of the CAB LHC I and LHC II sequences in the green algae and terrestrial plants. Received: 8 July 1996 / Accepted: 21 August 1996     

Changes in lipid composition of Photosystem 1 particles from thylakoids phosphorylated under reductive or anaerobic conditions

Changes in lipid composition of Photosystem 1 (PS 1) particles isolated from thylakoids phosphorylated under reductive or anaerobic conditions have been studied. Under reductive conditions, there was an increase in monogalactosyldiacylglycerol containing highly saturated fatty acids and phosphatidylglycerol containing transhexadecenoic fatty acid. Under anaerobic conditions, the amount of all lipid classes was increased. As we have shown earlier (S. V. Manuilskaya, O. I. Volovik, A. I. Mikhno, A. I. Polischuk and S. M. Kochubey (1990) Photosynthetica 24: 419–423) these changes were due to a co-migration of some lipid species and light-harvesting chlorophyll a/b complex LHC II from PS 2 to PS 1. These data allow us to conclude that LHC II consists of the lipoproteins containing specific lipids. Different composition of lipids co-migrating with LHC II under various conditions of phosphorylation might be caused by the variety of LHC II subpopulations transferred under each reductive condition.Abbreviations PS 1 Photosystem 1 - PS 2 Photosystem 2 - LHC II light-harvesting chlorophyll a/b protein complex II - Chl chlorophyll - MGDG monogalactosyldiacylglycerol - DGDG digalactosyldiacylglycerol - PG phosphatidylglycerol - SQDG sulfoquinovosyldiacylglycerol