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     

Structure,function and assembly of Photosystem II and its light-harvesting proteins

Photosystem II (PSII) is a multisubunit chlorophyll–protein complex that drives electron transfer from water to plastoquinone using energy derived from light. In green plants, the native form of PSII is surrounded by the light-harvesting complex (LHCII complex) and thus it is called the PSII–LHCII supercomplex. Over the past several years, understanding of the structure, function, and assembly of PSII and LHCII complexes has increased considerably. The unicellular green alga Chlamydomonas reinhardtii has been an excellent model organism to study PSII and LHCII complexes, because this organism grows heterotrophically and photoautotrophically and it is amenable to biochemical, genetic, molecular biological and recombinant DNA methodology. Here, the genes encoding and regulating components of the C. reinhardtii PSII–LHCII supercomplex have been thoroughly catalogued: they include 15 chloroplast and 20 nuclear structural genes as well as 13 nuclear genes coding for regulatory factors. This review discusses these molecular genetic data and presents an overview of the structure, function and assembly of PSII and LHCII complexes.     

Assembly and composition of the chlorophyll a-b light-harvesting complex of barley (Hordeum vulgare L.): Immunochemical analysis of chlorophyll b-less and chlorophyll b-deficient mutants

The chlorina-f2 mutant of barley (Hordeum vulgare L.) contains no chlorophyll b in its light-harvesting antenna, whereas the chlorina-103 mutant contains approximately 10% of the chlorophyll b found in wild-type. The absolute chlorophyll antenna size for Photosystem-II in wild-type, chlorina-103 and chlorina-f2 mutant was 250, 58 and 50 chlorophyll molecules, respectively. The absolute chlorophyll antenna size for Photosystem-I in wild-type, chlorina-103 and chlorina-f2 mutant was 210, 137 and 150 chlorophyll molecules, respoectively. In spite of the smaller PS I antenna size in the chlorina mutants, immunochemical analysis showed the presence of polypeptide components of the LHC-I auxiliary antenna with molecular masses of 25, 19.5 and 19 kDa. The chlorophyll a-b-binding LHC-II auxiliary antenna of PS II contained five polypeptide subunits in wild-type barley, termed a, b, c, d and e, with molecular masses of 30, 28, 27, 24 and 21 kDa, respectively. The polypeptide composition of the LHC-II auxiliary antenna of PS II was found to be identical in the two mutants, with only the 24 kDa subunit d present at an equal copy number per PS II in each of the mutants and in the wild-type barley. This d subunit assembles stably in the thylakoid membrane even in the absence of chlorophyll b and exhibits flexibility in its complement of bound chlorophylls. We suggest that polypeptide subunit d binds most of the chlorophyll associated with the residual PS II antenna in the chlorina mutants and that is proximal to the PS II-core complex.Abbreviations CP chlorophyll-protein - LHC the chlorophyll a-b binding light-harvesting complex - LHC-II subunit a the Lhcb4/5 gene product - subunit b the Lhcb1 gene product - subunit c Lhcb2 the gene product - subunit d the Lhcb3 gene product - subunit e the Lhcb6 gene product - PMSF phenylmethane sulphonyl fluoride - RC reaction center - Q_A the primary quinone electron acceptor of Photosystem-II - P700 the reaction center of PS I     

The molecular biology of self-incompatibility systems in flowering plants

Self-incompatibility is a common mechanism by which flowering plants can exert some control over the process of fertilization. Typically, the self-incompatibility response involves the recognition and rejection of self-incompatible pollen which leads to a block in self-fertilization and, as a consequence, promotes outcrossing. In recent years, considerable progress has been made in the molecular understanding of several self-incompatibility systems. Interestingly, a common mechanism for self-incompatibility is not employed by all flowering plants, but in fact quite diverse mechanisms have been recruited for the rejection of self-incompatible pollen. In this review, the recent advances in the self-incompatibility systems of the Solanaceae, Papaveraceae, and Brassicaceae will be described as well as some of the molecular work that is emerging for the Poaceae and the heteromorphic self-incompatibility systems.     

Characterization of genes encoding the light-harvesting proteins in diatoms: biogenesis of the fucoxanthin chlorophylla/c protein complex

In chromophytic algae the major light-harvesting complex is the fucoxanthin chlorophylla/c protein complex. Recently, we have cloned several highly related cDNA and genomic sequences encoding the fucoxanthin chlorophylla/c proteins from the diatomPhaeodactylum tricornutum. These genes are clustered on the nuclear genome. The sequences of the fucoxanthin chlorophylla/c proteins as deduced from the gene sequences have some similarity to the chlorophylla/b proteins associated with light-harvesting complexes of higher plants and green algae. Like the chlorophylla/b proteins of higher plants, the fucoxanthin chlorophylla/c proteins are synthesized as higher-molecular weight precursors in the cytoplasm of the cell and are transported into the plastids. However, the mode of transport into diatom plastids is very different from the mechanism involved in transporting proteins into the chloroplasts of higher plants and green algae. We focus here on the characteristics of the fucoxanthin chlorophylla/c proteins, the mode of transport of these proteins into plastids, the arrangement of the genes encoding these proteins, and efforts to utilize these genes to develop a DNA transformation system for diatoms.     

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 atypical chlorophyll a/b/c light-harvesting complex of Mantoniella squamata: molecular cloning and sequence analysis

cDNA species encoding precursor polypeptides of the chlorophyll a/b/c light-harvesting complex (LHC) of Mantoniella squamata were cloned and sequenced. The precursor polypeptides have molecular weights of 24.2 kDa and are related to the major chlorophyll a/b polypeptides of higher plants. Southern analysis showed that their genes belong to the nuclear encoded Lhc multigene family; the investigated genes most probably do not contain introns. The chlorophyll a/b/c polypeptides contain two highly conserved regions common to all LHC polypeptides and three hydrophobic -helices, which span the thylakoid membrane. The first membrane-spanning helix, however, is not detected by predictive methods: its atypical hydrophilic domains may bind the chlorophyll c molecules within the hydrophobic membrane environment. Homology to LHC 11 of higher plants and green algae is specifically evident in the C-terminal region comprising helix III and the preceding stroma-exposed domain. The N-terminal region of 29 amino acids resembles the structure of a transit sequence, which shows only minor similarities to those of LHC II sequences. Strikingly, the mature light-harvesting polypeptides of M. squamata lack an N-terminal domain of 30 amino acids, which, in higher plants, contains the phosphorylation site of LHC 11 and simultaneously mediates membrane stacking. Therefore, the chlorophyll a/b/c polypeptides of M. squamata do not exhibit any light-dependent preference for photosystem I or 11. The lack of this domain also indicates that the attractive forces between stacked thylakoids are weak.This study is dedicated to Prof. Dr. W Rüdiger on the occasion of his 60th birthday     

Cloning of a pea cDNA encoding a polypeptide of the light-harvesting complex associated with photosystem I using a monoclonal antibody

A monoclonal antibody (MAb UB42) is described that binds to thylakoids in pea chloroplasts, as shown by EM-immunogold labelling. The antibody recognised proteins of ca. 23–29 kDa in western blots of a pea leaf homogenate. A cDNA library was prepared from pea epidermal cells in the vector ZAP II, and immunoscreening of the library with UB42 led to the isolation of a clone, pUB42. This was sequenced and had an open reading frame of 269 codons encoding a predicted polypeptide of 28.9 kDa. The sequence showed extensive homology with three closely related polypeptides belonging to a family of chlorophyll a/b-binding proteins from the light harvesting complex of photosytem I (LHCI). Collectively, the results suggest that MAb UB42 recognises an epitope on the type II chlorophyll a/b-binding protein from LHCI and that clone pUB42 encodes this protein.     

Nucleotide sequence and expression of the two genes encoding D2 protein and the single gene encoding the CP43 protein of Photosystem II in the cyanobacterium synechococcus sp. PCC 7002

The unicellular photoheterotrophic cyanobacterium Synechococcus sp. PCC 7002 was shown to encode two genes for the Photosystem II reaction center core protein D2 and one gene for the reaction center chlorophyhll-binding protein CP43. These three genes were cloned and their DNA sequences determined along with their flanking DNA sequences. Northern hybridization experiments show that both genes which encode D2, psbD1 and psbD2, are expressed at roughly equivalent levels. For each of the two psbD genes, there are 18 nucleotide differences among the 1059 nucleotides which are translated. The DNA sequences surrounding the coding sequences are nearly 70% divergent. Despite the DNA sequence differences in the genes, the proteins encoded by the two genes are predicted to be identical. The proteins encoded by psbD1 and psbD2 are 92% homologous to other sequenced cyanobacterial psbD genes and 86% homologous to sequenced chloroplast-encoded psbD genes.The single gene for CP43, psbC, overlaps the 3 end of psbD1 and is co-transcribed with it. Results from previous sequencing of psbC genes encoded by chloroplasts suggest that the 5 end of the psbC gene overlaps the 3 end of the coding sequence of psbD by 50 nucleotides. In Synechococcus sp. PCC 7002, the methionine codon previously proposed to be the start codon for psbC is replaced by an ACG (threonine) codon. We propose an alternative start for the psbC gene at a GTG codon 36 nucleotides downstream from the threonine codon. This GTG codon is preceded by a consensus E. coli-like ribosome binding sequence. Both the GTG start codon and its preceding ribosome binding sequence are conserved in all psbC genes sequenced from cyanobacteria and chloroplasts. This suggests that all psbC genes start at this alternative GTG codon. Based on this alternative start codon, the gene product is 85% identical to other cyanobacterial psbC gene products and 77% identical to eucaryotic chloroplast-encoded psbC gene products.     

Fluorescence studies on interaction between phospho-LHC II and subchloroplast Photosystem 1 preparations

Chloroplast proteins were phosphorylated under two test conditions: white light irradiance alone and white light irradiance with the addition of glucose and glucose oxidase, used to produce an anaerobic medium. The interaction of phospho-LHC II with Photosystem 1 (PS 1) was studied for two types of PS I preparation. Changes in the chlorophyll a/b ratio and the ratio of 650 and 680 nm band intensities (E650/E680) in fluorescence excitation spectra were used in calculating the phospho-LHC II portion which became associated with PS 1. It is shown that the associated portion of phospho-LHC II varies for each of the PS 1 preparations and phosphorylation procedures. Possible conclusions as regards the transfer of various sets of LHC II subpopulations under different phosphorylation procedures and the differences of interaction with PS 1 are discussed.Abbreviations PS 1 Photosystem 1 - PS 2 Photosystem 2 - LHC II light-harvesting chlorophyll a/b protein complex II - Chl chlorophyll - fluorescence quantum yield - _f life time of fluorescence at =685 nm - F735 fluorescence band with a maximum at 735 nm - F685 fluorescence band with a maximum at 685 nm - E650/E680 ratio of amplitudes in excitation fluorescence spectrum at 650 and 680 nm     

Evolutionary conservation of the chlorophyll a/b-binding proteins: cDNAs encoding type I, II and III LHC I polypeptides from the gymnosperm Scots pine

Summary cDNAs encoding three different LHC I polypeptides (Type I, Type II and Type III) from the gymnosperm Scots pine (Pinus sylvestris L.) were isolated and sequenced. Comparisons of the deduced amino acid sequences with the corresponding tomato sequences showed that all three proteins were highly conserved although less so than the LHC II proteins. The similarities between mature Scots pine and tomato Types I, II and III LHC I proteins were 80%, 87% and 85%, respectively. Two of the five His residues that are found in AXXXH sequences, which have been identified as putative chlorophyll ligands in the Type I and Type II proteins, were not conserved. The same two regions of high homology between the different LHC proteins, which have been identified in tomato, were also found in the Scots pine proteins. Within the conserved regions, the Type I and Type II proteins had the highest similarity; however, the Type II and Type III proteins also showed a similarity in the central region. The results suggest that all flowering plants (gymnosperms and angiosperms) probably have the same set of LHC polypeptides. A new nomenclature for the genes encoding LHC polypeptides (formerly cab genes) is proposed. The names lha and lhb are suggested for genes encoding LHC I and LHC II proteins, respectively, analogous to the nomenclature for the genes encoding other photosynthetic proteins.     

The apoprotein precursor of the major light-harvesting complex of photosystem II (LHCIIb) is inserted primarily into stromal lamellae and subsequently migrates to the grana

The formation of the lateral distribution of the major antenna complex of photosystem II (LHCIIb) between the granal and stromal lamellae was studied. Specifically, the localization of the insertion and the assembly of the precursor of the apoprotein of LHCIIb (pLHCP) were studied with isolated thylakoids. After insertion of pLHCP into isolated thylakoids, fractionation of the latter into granal and stromal lamellar was performed. At 25 °C most of the precursor was located in the granal lamellae, although both highly purified granal and stromal lamellar fractions demonstrated a similar capability to insert pLHCP. When the insertion reaction to the thylakoids was performed at 10 °C, followed by their separation into stromal and granal lamellae, the labelled pLHCP was localized in the stromal ones. To examine whether pLHCP inserts into both granal and stromal lamellae, or preferentially into stromal lamellae and subsequently migrating to granal lamellae, a chase experiment was performed. Insertion of pLHCP at 10 °C was followed by chase of the radioactive precursor with excess of non-radioactive pLHCP at 25 °C. From the results presented it is evident that the level of pLHCP in stromal lamellae was gradually reduced, while it gradually accumulated in the granal lamellae. Furthermore, the pLHCP in the stromal lamellae was found to be in a free form, while after migrating to the granal lamellae it assembled into the pigmented LHCIIb.     

The cell biology of ion pumps: sorting and regulation

The physiologic function of an ion pump is determined, in part, by its subcellular localization and by the cellular mechanisms that modulate its activity. The Na,K-ATPase and the gastric H,K-ATPase are two closely related members of the P-type family of ion transporting ATPases. Despite their homology, these pumps are sorted to different domains in polarized epithelial cells and their enzymatic activities are subject to distinct regulatory pathways. The molecular signals responsible for these properties have begun to be elucidated. It appears that a complex array of inter- and intra-molecular interactions govern these proteins' trafficking, distribution and catalytic capacity.     

Molecular characterization of the virulence gene virA of the Agrobacterium tumefaciens octopine Ti plasmid

The virulence loci play an essential role in tumor formation by Agrobacterium tumefaciens. Induction of vir gene expression by plant signal molecules is solely dependent on the virulence loci virA and virG. This study focused on the virA locus of the octopine type Ti plasmid pTi15955. The nucleic acid sequence of a 5.7-kilobase fragment encompassing virA was determined. Genetic analysis of this region revealed that virA contains one open reading frame coding for a protein of 91 639 daltons. Immunodetection with antibodies raised against a 35-kDa VirA fusion protein produced in E. coli identified by the VirA product in wild-type Agrobacterium cells. Moreover, it is shown that the VirA protein is located in the cytoplasmic membrane fraction of Agrobacterium. These data confirm the proposed regulatory function of VirA whereby VirA acts as a membrane sensor protein to identify plant signal molecules in the environment. The proposed sensory function of VirA strikingly resembles the function of the chemotaxis receptor proteins of E. coli.     

Peculiar properties of the PsaF photosystem I protein from the green alga Chlamydomonas reinhardtii: presequence independent import of the PsaF protein into both chloroplasts and mitochondria

It has previously been shown that presequences of nuclear-encoded chloroplast proteins from the green alga Chlamydomonas reinhardtii contain a region that may form an amphiphilic -helix, a structure characteristic of mitochondrial presequences. We have tested two precursors of chloroplast proteins (the PsaF and PsaK photosystem I subunits) from C. reinhardtii for the ability to be imported into spinach leaf mitochondria in vitro. Both precursors bound to spinach mitochondria. The PsaF protein was converted into a protease-protected form with high efficiency in a membrane potential-dependent manner, indicating that the protein had been imported, whereas the PsaK protein was not protease protected. The protease protection of PsaF was not inhibited by a synthetic peptide derived from the presequence of the N. plumbaginifolia mitochondrial F₁ subunit. Furthermore, if the presequence of PsaF was truncated or deleted by in vitro mutagenesis, the protein was still protease-protected with approximately the same efficiency as the full-length precursor. These results indicate that PsaF can be imported by spinach mitochondria in a presequence-independent manner. However, even in the absence of the presequence, this process was membrane potential-dependent. Interestingly, the presequence-truncated PsaF proteins were also protease-protected upon incubation with C. reinhardtii chloroplasts. Our results indicate that the C. reinhardtii chloroplast PsaF protein has peculiar properties and may be imported not only into chloroplasts but also into higher-plant mitochondria. This finding indicates that additional control mechanisms in the cytosol that are independent of the presequence are required to achieve sorting between chloroplasts and mitochondria in vivo.Abbreviations cTP chloroplast transit peptide - mTP mitochondrial targeting peptide - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - pF₁(1,25) a synthetic peptide derived from the first 25 residues of the Nicotiana plumbaginifolia mitochondrial ATP synthase F₁ subunit - PsaF(2–30) and PsaF(2–61) mutant proteins lacking regions corresponding to residues 2–30 and 2–61 in the PsaF precursor protein, respectively     

Molecular characterization of the virulence gene virA of the Agrobacterium tumefaciens octopine Ti plasmid

The virulence loci play an essential role in tumor formation by Agrobacterium tumefaciens. Induction of vir gene expression by plant signal molecules is solely dependent on the virulence loci virA and virG. This study focused on the virA locus of the octopine type Ti plasmid pTi15955. The nucleic acid sequence of a 5.7-kilobase fragment encompassing virA was determined. Genetic analysis of this region revealed that virA contains one open reading frame coding for a protein of 91 639 daltons. Immunodetection with antibodies raised against a 35-kDa VirA fusion protein produced in E. coli identified the VirA product in wild-type Agrobacterium cells. Moreover, it is shown that the VirA protein is located in the cytoplasmic membrane fraction of Agrobacterium. These data confirm the proposed regulatory function of VirA whereby VirA acts as a membrane sensor protein to identify plant signal molecules in the environment. The proposed sensory function of VirA strikingly resembles the function of the chemotaxis receptor proteins of E. coli.     

Probing the structure of the core light-harvesting complex (LH1) of Rhodopseudomonas viridis by dissociation and reconstitution methodology

A subunit complex was formed from the core light-harvesting complex (LH1) of bacteriochlorophyll(BChl)-b-containing Rhodopseudomonas viridis. The addition of octyl glucoside to a carotenoid-depleted Rps. viridis membrane preparation resulted in a subunit complex absorbing at 895 nm, which could be quantitatively dissociated to free BChl b and then reassociated to the subunit. When carotenoid was added back, the subunit could be reassociated to LH1 with a 25% yield. Additionally, the Rps. viridis - and -polypeptides were isolated, purified, and then reconstituted with BChl b. They formed a subunit absorbing near 895 nm, similar to the subunit formed by titration of the carotenoid depleted membrane, but did not form an LH1-type complex at 1015 nm. The same results were obtained with the -polypeptide alone and BChl b. Isolated polypeptides were also tested for their interaction with BChl a. They formed subunit and LH1-type complexes similar to those formed using polypeptides isolated from BChl-a-containing bacteria but displayed 6–10 nm smaller red shifts in their long-wavelength absorption maxima. Thus, the larger red shift of BChl-b-containing Rps. viridis is not attributable solely to the protein structure. The -polypeptide of Rps. viridis differed from the other -polypeptides tested in that it could form an LH1-type complex with BChl a in the absence of the - and -polypeptides. It apparently contains the necessary information required to assemble into an LH1-type complex. When the -polypeptide was tested in reconstitution with BChl a and BChl b with the - and -polypeptides, it had no effect; its role remains undetermined.Abbreviations B820 the subunit form of the core light-harvesting complex in BChl-a-containing bacteria which has an absorption maximum at or near 820 nm - B875 the core light-harvesting complex of Rhodobacter sphaeroides which has an absorption maximum at 875 nm - B881 the core light-harvesting complex of wild-type Rhodospirillum rubrum which has an absorption maximum at 881 nm - B895 the subunit form of the core light-harvesting complex in Rps. viridis which has an absorption maximum near 888–895 nm - B1015 the core light-harvesting complex of Rps. viridis which has an absorption maximum at 1015 nm - CD circular dichroism - LH1 the core light-harvesting complex - OG n-octyl -d-glucopyranoside     

Type I and Type II genes for the chlorophyll a/b-binding protein in the gymnosperm Pinus sylvestris (Scots pine): cDNA cloning and sequence analysis

A cDNA library was constructed from mRNA prepared from light-treated seedlings of Scots pine (Pinus sylvestris L.) and cDNAs for the chlorophyll a/b-binding protein LHC-II were identified using a pea gene as the heterologous probe. Three cDNA clones were sequenced. The deduced amino acid sequences of two of the genes corresponded to Type I and one to Type II LHC-II proteins which were ca. 90% homologous to their angiosperm counterparts. The transit peptides of the Scots pine preLHC-II showed features common to angiosperm transit peptides. The three cDNAs had a 70 to 75% preference for G+C in the third base position. CpG and GpC profiles and degenerate codon position bias suggested that two of the corresponding genes lie within CpG islands.     

The Chlamydomonas reinhardtii LI818 gene represents a distant relative of the cabI/II genes that is regulated during the cell cycle and in response to illumination

In the green unicellular alga Chlamydomonas reinhardtii, as in higher plants, the expression of the genes encoding the chlorophyll a/b-binding (CAB) polypeptides associated with photosystem I (PSI) and photosystem II (PSII) is regulated by endogenous (circadian clock) and exogenous signals (light and temperature). The circadian clock ensures that the oscillation in the levels of the different cab mRNAs is continuously kept in phase with light/dark (LD) cycles and is maximal by the middle of the day. On the other hand, light controls the amplitude of the oscillations. We report here the cloning and characterization of the C. reinhardtii LI818 gene, which identifies a CAB-related polypeptide and whose expression is regulated quite differently from the cabI/II genes. We show: (1) that in LD synchronized Chlamydomonas cells LI818 mRNA accumulation is subject to dual regulation that involves separable regulation by light and an endogenous oscillator; (2) that LI818 mRNA is fully expressed several hours before the cab I/II mRNAs and that the latter accumulate concomitantly; (3) that blocking the electron flow through PSII using DCMU prevents cells from accumulating cab I/II mRNAs but not LI818 mRNA and (4) that the accumulation of LI818 mRNA is abolished by blocking cytoplasmic protein synthesis, suggesting that these regulatory mechanisms are mediated by labile proteins.