J. Philip Thornber

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.

The P700-chlorophyll a-protein. Isolation and some characteristics of the complex in higher plants.

Abstract A P700-chlorophyll a-protein complex has been purified from several higher plants by hydroxylapatite chromatography of Triton X-100-dissociated chloroplast membranes. The isolated material exhibits a red wavelength maximum at 677 nm, major spectral forms of chlorophyll a at 662, 669, 677, and 686 nm, a chlorophyll/P700 ratio of 40–45 1 , and contains only chlorophyll a and β-carotene of the photosynthetic pigments present in the chloroplast. The spectral characteristics and composition of the higher plant material are homologous to those of the P700-chlorophyll a-protein previously isolated from blue-green algae; however, unlike the blue-green algal component, cytochromes f and b6 are associated with the higher plant material. Evidence is presented that a chlorophyll a-protein termed “Complex I” which can be isolated from sodium dodecyl sulfate extracts of chloroplast membranes is a spectrally altered form of the eucaryotic P700-chlorophyll a-protein. The isolation procedure described in this paper is a more rapid technique for obtaining the heart of photosystem I than presently exists; furthermore, the P700 photooxidation and reduction kinetics in the fraction are improved over those in other isolated components showing the same enrichment of P700. It appears very probable that the heart of photosystem I is organized in the same manner in all chlorophyll a-containing organisms.

PLANT CHLOROPHYLL-PROTEIN COMPLEXES: RECENT ADVANCES

This article will review papers that have recently appeared on plant chlorophyll-protein complexes. Because of space limitations, it cannot be exhaustive or critical. We emphasize data that have appeared since the reviews of Anderson [8], Thornber [126], Thornber et al. [130], and Boardman et al. [31], but do not completely exclude some ealier pertinent results. Other recent reviews [35, 114, 127,213 and symposia (Brookhaven Symposium in Biology, Vol. 28, 1976 and ClBA Symposium, New Series 61, 1978) tangentially consider this topic. We will not use the term ‘chlorophyll protein’ in this review, since this term has been used ambiguously by researchers and sometimes misinterpreted to mean either the holocomplex or the apoprotein. Instead, we will use an old term, chlorophyllin, to refer to a holocomplex. No universally accepted nomenclature for chlorophyllins has been established. This and the fact that each group discovering what they believe to be a new chlorophyllin, generally gives the component some unique cryptic designation make it difficult for a reader to realize that 2 different laboratories may be describing the same chlorophyllin. Difficulty also arises because these components, solubilized by detergent solutions, are more difficult to purify than are water-soluble proteins, and hence the quoted characteristics of a given chlorophyllin can vary. Furthermore, most of them so far do not exhibit a distinguishing characteristic that readily permits unequivocal identification of the chlorophyllin. Absorption and emission spectra are not only very similar for each plant chlorophyllin but also the exact position of wavelength maxima for a chlorophyllin varies with the extent of denaturation of that chlorophyllin. In addition, techniques are frequently not identical between different laboratories for determining another frequently quoted characteristic, apparent molecular sizes of chlorophyllins and apoproteins (if the latter can be identified as such following denaturation of the complex). Confusion can also arise because some groups use the same designation for both a specific chlorophyllin and for a larger complex in which this chlorophyllin may be the principal, but not always the only, chlorophyllin present. It would be valuable if a session at the next International Congress on Photosynthesis in 1980 could be organized to establish a generally acceptable nomenclature. The problem grows increasingly acute in view of the large number of apparently different chlorophyllins now being reported,

Structure and biogenesis of Chlamydomonas reinhardtii photosystem I

The photosystem I complex of the green alga Chlamydomonas reinhardtii was isolated and fractionated into its two subcomplex components: the core complex (CC I), which contained the reaction center (P-700) and had four polypeptide subunits, and the light-harvesting complex (LHC I) which contained four polypeptides of about 22, 25, 26 and 27 kDa. The 22-kDa apoprotein was isolated as a chlorophyll a and b binding protein. In the isolated photosystem I holocomplex, about ten copies of the 22-kDa LHC I apoprotein are present for each CC I unit. The 22-kDa polypeptide as well as the other three polypeptides of this complex and the subunit II of CC I are translated on 80S cytoplasmic ribosomes, and therefore are coded in the nucleus. During the greening process of the Chlamydomonas reinhardtii y-1 mutant the 22-kDa LHC I polypeptide, which cross-reacts with polyclonal antibodies raised against the Lemna gibba 20-kDa LHC I apoprotein, accumulates in thylakoids at a late stage of their development, and about 2-3 h after the LHC II and CC I subunit II polypeptides have accumulated. Accumulation of the 22-kDa protein during greening is inhibited by cycloheximide but not by chloramphenicol.

Insertion of the precursor of the light-harvesting chlorophylla/b-protein into the thylakoids requires the presence of a developmentally regulated stromal factor.

The precursor of the major light-harvesting chlorophylla/b-proteins of photosystem II was synthesizedin vitro from a gene fromLemna gibba. When the labelled precursor was incubated with developing barley plastids, the precursor and the processed polypeptide were incorporated in the thylakoids in proportions that varied depending on the developmental stage of plastids. At early stages of development most of the precursor associated with the thylakoids could be removed by washing with 0.1 M NaOH, while in more mature plastids most of its was resistant to a NaOH wash. Insertion of the precursor into thylakoids required the presence of a stromal factor and Mg-ATP. The stromal factor is probably a protein. The insertion reaction has an optimal temperature of 25°C and a pH of 8. The appearance of the stromal factor and the thylakoid membranes receptivity for the insertion of the precursor depended on the stage of plastid development. These observations are consistent with the hypothesis that the insertion of the precursor into the thylakoid prior to its proteolytic processing, is one of the steps involved in the assembly of the light-harvesting complex of photosystem II.

Solubilization and two-dimensional electrophoretic procedures for studying the organization and composition of photosynthetic membrane polypeptides

We describe the optimum conditions for extraction of pigment-proteins from photosynthetic membranes with glycosidic surfactants and their subsequent fractionation by nondenaturing PAGE to purify them in a state as close as is currently possible to their native state. The amount and type of surfactant used to extract the pigment-proteins play a critical role, and surfactants and electrophoretic conditions can be found to purify the pigment-proteins in monomeric, oligomeric, or multiprotein complexes. These extraction and separation conditions are robust and can be applied to photosynthetic membranes of higher plants, algae, cyanobacteria, and purple bacteria. Two different second-dimension PAGE systems are used to analyze the composition of the pigmented regions from the first dimension. One is a nondenaturing gel configuration such that no stacking gel is required. This permits the use of lower ionic strength buffers, which is necesary so that the pigments are not stripped from the proteins during electrophoresis. This two-dimensional system enables multi-pigment-protein complexes obtained in the first dimension to be resolved into their individual pigment-proteins in the second dimension. The second system is an optimized fully denaturing SDS-PAGE method which differs from the standard conditions by the use of urea and a higher ionic strength buffer in the separating gel. The denaturing SDS-PAGE method is highly reproducible and resolves thylakoid membrane proteins from 70 to 5 kDa. This two-dimensional system is used to determine the subunit composition of each multiprotein complex in the first dimension.

Chlorophyll-protein complexes from higher plants: a procedure for improved stability and fractionation.

Abstract An improved procedure for the electrophoretic fractionation of higher plant chlorophyllprotein complexes is described. Compared with currently used systems, it greatly reduces the amount of chlorophyll that is found unassociated with protein after electrophoresis and resolves four chlorophyll-protein complexes. The slowest migrating band has a red adsorption maximum at 674 nm or greater, contains chlorophyll a but not chlorophyll b, and has a molecular weight equivalency of 110,000. These properties are similar to the previously described CPI or P700-chlorophyll a-protein complex. The amount of the total chlorophyll in this material is increased by two to three fold over that present in the equivalent complex fractionated by previous procedures. The other three chlorophyll-protein complexes contain both chlorophylls a and b, and have molecular weight equivalencies of 80,000, 60,000, and 46,000. None of these complexes seems to correspond directly to the previously characterized light-harvesting chlorophyll a b -protein complex.

A study of the water-soluble proteins of spinach beet chloroplasts with particular reference to fraction I protein

Abstract 1. 1.|The rate of release of high molecular weight water-soluble components from isolated chloroplasts has been studied. 2. 2.|Chloroplasts have been broken by osmosis and successively washed with hypotonic buffer solutions, or ruptured in a needle-valve disintegrator to free lamellae particles of soluble constituents. The resulting solubilized material has been examined by ultracentrifugation, and by polyacrylamide gel electrophoresis in homogeneous buffer system. 3. 3.|Fraction I protein was more readily liberated from the chloroplasts than the Fraction II components. A component of s20, w = 12 S was observed in some of the extracts, but the bulk of this was only liberated after complete disruption of the lamellae, and it is thought to be situated within the lamellae ‘loculi’ and ‘fret-channels’. 4. 4.|A procedure for the purification of Fraction I protein using DEAE-cellulose chromatography and gel filtration is described. 5. 5.|An investigation of the nature of our preparation of Fraction I protein is reported; the protein has a partial specific volume of 0.744 ml/g, and an s°20, w = 18.30 S. A minimum molecular weight of 24427 has been calculated from the amino acid analysis, and the molecular weight of the macromolecule has been determined by ultracentrifugation (on two preparations) as 585 000 and 561 000. The protein was observed to dissociate into subunits of 2.6–4.8 S, in alkali (pH 11.5), acetic acid (70%), urea (8 M) and sodium dodecyl benzene sulphate (detergent-protein, 1:2, w/w). 6. 6.|The isolated Fraction I protein contains 84% protein; the other material present is thought to be accounted for by carbohydrate and material giving rise to ash. The predominant monosaccharides in the hydrolysates of the protein have been identified as glucose and xylose. 7. 7.|Fraction I protein has been shown to be identical with the enzyme, ribulose-1,5-diphosphate carboxylase (EC 4.1.1.39). Correspondence between the physical properties of Fraction I protein and those of protochlorophyll-protein complex of etiolated tissues indicates another possible identity.

Partial purification, subunit structure and thermal stability of the photochemical reaction center of the thermophilic green bacterium Chloroflexus aurantiacus

Abstract Spectrally pure reaction center preparations from Chloroflexus aurantiacus have been obtained in a stable form; however, the product contained several contaminating polypeptides. The reaction center pigment molecules (probably three bacteriochlorophyll a and three bacteriopheophytin a molecules) are associated with two polypeptides ( M r = 30000 and 28000) in a reaction center complex of M r = 52000. No carotenoid is present in the complex. These data together with previous spectral data suggest that the Chloroflexus reaction center represents a more primitive evolutionary form of the purple bacterial reaction center, and that it has little if any relationship to the green bacterial component. A reaction center preparation from Rhodopseudomonas sphaeroides R26 was fully denatured at 50°C while the Chloroflexus reaction center required higher temperatures (70–75°C) for complete denaturation. Thus, an intrinsic membrane protein of a photosynthetic thermophile has been demonstrated to have greater thermal stability than the equivalent component of a mesophile.

Isolation and spectral characteristics of the photochemical reaction center of Rhodopseudomonas viridis

A method is described for isolation of the Rhodopseudomonas viridis reaction center complex free of altered, 685 nm absorbing pigment. This improved preparation contains two c-type cytochromes in the ratio P-960: cytochrome c-558: cytochrome c-553 of 1:2:2 to 3. The near infrared spectral forms of the reduced preparation are located at 790, 832, 846, and 987 nm at 77 K; the oxidized complex absorbs at 790, 808, 829 and approx. 1310 nm. The 790 nm band is attributed to bacteriophaeophytin b and the other absorbances to bacteriochlorophyll b, The visible absorption bands may be assigned to these pigments and to the cytochromes present and, probably to a carotenoid. The presence of two bacteriochlorophyll b spectral forms in the P+-830 band suggests that exciton interactions occur among pigments in the oxidized, as well as the reduced, reaction center. Changes in the 790 and 544 nm bands upon illumination of the reaction center preparation at low redox potential may be indicative of a role for bacteriophaeophytin b in primary photochemical events.