Identification of type 1 and type 2 light-harvesting chlorophyll a/b-binding proteins using monospecific antibodies (original) (raw)
Related papers
Photochemistry and Photobiology, 1993
Polyclonal antibodies against four different apoproteins of either the chlorophyll (Chl) d b light-harvesting antenna of photosystem I or 11, or a chlorophyll-protein complex homologous to CP26 from Chlamydomonas reinhardtii, crossreact with 1 1-1 3 thylakoid proteins of Chlamydomonas, Euglena gracilis and higher plants. The number of antigenically-related proteins correlates with the quantity of light-harvesting chlorophyll-protein complex (LHC) gene types that have been sequenced in higher plants. The antibodies also react specifically with Chl d cbinding proteins of three diatoms and Coccolithophora sp. as determined by immunoblot and Ouchterlony assays. Four to six crossreacting proteins are observed in each chromophyte species and a functional role for some can be deduced by antibody reactivity. It appears that despite major differences in the structures of their pigment ligands, at least some domains of Chl-binding LHC apoproteins have been conserved during their evolution, possibly functioning in protein : protein, as opposed to pigment : protein, interactions in photosynthetic membranes.
A nomenclature for the genes encoding the chlorophylla/b-binding proteins of higher plants
Plant Molecular Biology Reporter, 1992
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 named Lhcal through Lhca4 and Lhcbl through Lhcb6, 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.
Journal of Biological Chemistry, 2001
The high selectivity offered by reversed-phase highperformance liquid chromatography on-line coupled to electrospray ionization mass spectrometry has been utilized to characterize the major and minor light-harvesting proteins of photosystem II (Lhcb). Isomeric forms of the proteins, revealed either on the basis of different hydrophobicity enabling their chromatographic separation or on the basis of different molecular masses identified within one single chromatographic peak, were readily identified in a number of monocot and dicot species. The presence of several Lhcb1 isoforms (preferably in dicots) can explain the tendency of dicot Lhcb1 to form trimeric aggregates. The Lhcb1 molecular masses ranged from 24,680 to 25,014 among different species, whereas within the same species, the isoforms differed by 14 -280 mass units. All Lhcb1 proteins appear to be highly conserved among different species such that they belong to a single gene group that has several different gene family members. In all species examined, the number of isoforms corresponded more or less to the genes cloned previously. Two isoforms of Lhcb3 were found in petunia and tomato. For Lhcb6, the most divergent of all light-harvesting proteins, the greatest number of isoforms was found in petunia, tobacco, tomato, and rice. Lhcb2, Lhcb4, and Lhcb5 were present in only one form. The isoforms are assumed to play an important role in the adaptation of plants to environmental changes.
Journal of Photochemistry and Photobiology B: Biology, 1999
The major light-harvesting complex of photosystem II in higher plants is known as LHCII. It is composed of a number of chlorophyllbinding proteins sharing epitopes with each other. The number of apoproteins resolved by fully denaturing sodium dodecylsulfate polyacrylamide gel electrophoresis varies in different species. In order to know if this heterogeneity is caused by the expression of a number of homologous genes or if it is the product of post-translational modifications, we have resolved the six major apoproteins of Zeu mays LHCII.
Current Genetics, 1984
A 2,900 base pair DNA segment of the spinach plastid chromosome which encodes the genes for the 44 kd chlorophyll a apoprotein and a "32 kd"-like protein of the photosystem II reaction center has been subjected to sequence and Northern blot analysis. The genes are located almost centrally in the large single-copy segment of the chromosome adjacent to the two genes for the P7oo chlorophyll a apoproteins of the photosystem I reaction center. The DNA sequence reveals two uninterrupted protein-coding regions of 473 (44 kd chlorophyll a apoprotein) and 353 triplets ("32 kd"-like protein). The latter gene is strikingly similar to the gene for the herbicide-binding '32 kd" protein which maps some 30 kbp distant on the plastid chromosome. The two genes overlap by 50 base pairs but are read in different phases. They may be contranscribed and the RNA modified to give several discrete species ranging in size from 1.6 to 4.6 kb. A presumptive promoter site was only identified for the "32 kd"-like protein, while potential ribosome binding and transcription termination sites are found preceding and following both genes, respectively. The polypeptides possess a high content of hydrophobic amino acids, most of which appear to be clustered in transmembrane spans. The molecular weights of 51,785 (44 kd chlorophyll a apoprotein) and 39,465 ("32 kd"like protein) derived from the deduced amino acid sequences are higher than the experimentally determined protein sizes. Amino acid codon usage for both genes is highly selective. Comparison of the chlorophyll a apoproteins of spinach reveals regions of sequence homology.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1998
Mutation analysis of higher plants light harvesting proteins has been prevented for a long time by the lack of a suitable expression system providing chromophores essential for the folding of these membrane-intrinsic pigment-protein complexes. Early work on in vitro reconstitution of the major light harvesting complex of photosystem II (LHCII) indicated an alternative way to mutation analysis of these proteins. A new procedure for in vitro refolding of the four light harvesting complexes of photosystem II, namely CP24, CP29, CP26 and LHCII yields recombinant pigment-proteins indistinguishable from the native proteins isolated from leaves. This method allows both the performing of single point mutations on protein sequence and the exchange of the chromophores bound to the protein scaffold. We review here recent results obtained by this method on the pigment-binding properties, on the chlorophyll-binding residues, on the identification of proton-binding sites and on the role of xanthophylls in the regulation of light harvesting function.
Photosynthesis Research, 1988
The current state of knowledge concerning the expression of the nuclear genes that code the light-harvesting chlorophyll a/b-binding polypeptides of photosystem II is presented. This review covers the structure of these genes, the complex multistep pathway involved in their expression, and the environmental and other factors which regulate their expression. Some of the effects of these factors are mediated, at least in part, at the level of transcription, but other effects can be explained only by the existence of multiple posttranscriptional regulatory steps. Abbreviations: bp-base (nucleotide) pairs, BSC-bundle sheath cells of maize leaves, CATchloramphenicol acetyl transferase, eDNA-complementary DNA, kb-kilobases, LF-low fluence illumination, LHC-mature light-harvesting complex, LHC-II-mature lightharvesting complex of Photosystem II, LHCP-II-light-harvesting Chl a/b-binding protein of Photosystem II, MC-mesophyll cells of maize leaves, mRNA-messenger RNA, NPT
P700 Chlorophyll a-Protein : Purification, Characterization, and Antibody Preparation
PLANT PHYSIOLOGY, 1983
The P700 chlorophyll a-protein was purified by preparative sodium dodecyl sulfate (SDS) gel electrophoresis from SDS-solubilized barley (Hordewm vulgare L., cv Himalaya) chloroplast membranes. After elution from the gel in the presence of 0.05 to 0.1% Triton X-100, the recovered protein had a chlorophyll/P70. ratio of 50 to 60/1 and contained no chlorophyll b or cytochromes. Analysis of the polypeptide composition of the chlorophyll-protein revealed a 58 to 62 kilodalton (kD) polypeptide component but no lower molecular weight polypeptides. The 58 to 62 kD component was further resolved into two distinct polypeptide bands which were subsequently mapped by partial cyanogen bromide digestion and Staphylococcus aureus proteolysis. Based on results from the mapping experiments and other data, we suggest that the two components are conformational variants of a single polypeptide. Measurement of the chlorophyll to protein ratio by quantitative amino acid analysis and consideration of the yield of P700 in the protein isolate suggest that, contrary to previous models (Bengis and Nelson, 1975, 1977), P700 in vivo is associated with a minimum of four subunits of approximately 60 kD. Antibodies raised against the photochemically active chlorophyll-protein complex from barley reacted specifically with the 58 to 62 kD apoprotein. The same preparative electrophoresis procedure was used to isolate photochemically active P700 chlorophyll a-protein from soybean (Glycine max L.), tobacco (Nicotiana tabacum L.), petunia (Petunia x hybrida), tomato (Lycopersicum esculentwm), and Chiamydomonas reinhardti. The isolated complex from all species exhibited identical polypeptide compositions and chlorophyll/P7.. ratios. Antibodies to the barley protein cross reacted with all species tested demonstrating the highly conserved structure of the apoprotein. The P700 Chl a-protein is defined as the Chl-protein which binds the reaction center Chl of PSI (P700) and approximately 40 molecules of light-harvesting Chl a (32). Current knowledge of the structure and function of this important protein has been derived from the analyses of P700-enriched chloroplast membrane fractions prepared by several methods (5, 6, 12, 25, 30). Although there is general agreement that the P700 Chl a-protein is the structural and functional center of PSI, there is conflicting evidence regarding the number and mol wt of the polypeptides which comprise the apoprotein component of this complex. Several workers have concluded that the P700 Chl a-protein is composed of a single 60 ' Supported by National Institutes of Health grant GM 23944. 2 Supported by National Institutes of Health predoctoral training grant GM 07183 and a Hutchinson Foundation Fellowship.
The Journal of Cell Biology, 1987
A processing activity has been identified in higher plant chloroplasts that cleaves the precursor of the light-harvesting chlorophyll a/b-binding protein (LHCP). A wheat LHCP gene previously characterized (Lamppa, G.K., G. Morelli, and N.-H. Chua, 1985. Mol. Cell Biol. 5:1370-1378) was used to synthesize RNA and subsequently the labeled precursor polypeptide in vitro. Incubation of the LHCP precursors with a soluble extract from lysed chloroplasts, after removal of the thylakoids and membrane vesicles, resulted in the release of a single 25-kD peptide. In contrast, when the LHCP precursors were used in an import reaction with intact pea or wheat chloroplasts, two forms (25 and 26 kD) of mature LHCP were found. The peptide released by the processing activity in the organelle-free assay comigrated with the lower molecular mass form of mature LHCP produced during import. Properties of the processing activity suggest that it is an endopeptidase. Chloroplasts from both pea and wheat, two...
Plant Physiology, 2002
The light-harvesting proteins (Lhca) of photosystem I (PSI) from four monocot and five dicot species were extracted from plant material, separated by reversed-phase high-performance liquid chromatography (HPLC) and subsequently identified on the basis of their intact molecular masses upon on-line hyphenation with electrospray ionization mass spectrometry. Although their migration behavior in gel electrophoresis was very similar, the elution times among the four antenna types in reversed-phase-HPLC differed significantly, even more than those observed for the light-harvesting proteins of photosystem II. Identification of proteins is based on the good agreement between the measured intact molecular masses and the values calculated on the basis of their nucleotide-derived amino acid sequences, which makes the intact molecular masses applicable as intact mass tags. These values match excellently for Arabidopsis, most probably because of the availability of high-quality DNA sequence data. In all species examined, the four antennae eluted in the same order, namely Lhca1 Ͼ Lhca3 Ͼ Lhca4 Ͼ Lhca2. These characteristic patterns enabled an unequivocal assignment of the proteins in preparations from different species. Interestingly, in all species examined, Lhca1 and Lhca2 were present in two or three isoforms. A fifth antenna protein, corresponding to the Lhca6 gene, was found in tomato (Lycopersicon esculentum). However PSI showed a lower heterogeneity than photosystem II. In most plant species, Lhca2 and Lhca4 proteins are the most abundant PSI antenna proteins. The HPLC method used in this study was found to be highly reproducible, and the chromatograms may serve as a highly confident fingerprint for comparison within a single and among different species for future studies of the PSI antenna.