Synthesis of chlorophyll <Emphasis Type="Italic">b</Emphasis>: Localization of chlorophyllide <Emphasis Type="Italic">a</Emphasis>oxygenase and discovery of a stable radical in the catalytic subunit

Background  

Assembly of stable light-harvesting complexes (LHCs) in the chloroplast of green algae and plants requires synthesis of chlorophyll (Chl) b, a reaction that involves oxygenation of the 7-methyl group of Chl a to a formyl group. This reaction uses molecular oxygen and is catalyzed by chlorophyllide a oxygenase (CAO). The amino acid sequence of CAO predicts mononuclear iron and Rieske iron-sulfur centers in the protein. The mechanism of synthesis of Chl b and localization of this reaction in the chloroplast are essential steps toward understanding LHC assembly.     

Evolutionary Changes in Chlorophyllide a Oxygenase (CAO) Structure Contribute to the Acquisition of a New Light-harvesting Complex in Micromonas

Chlorophyll b is found in photosynthetic prokaryotes and primary and secondary endosymbionts, although their light-harvesting systems are quite different. Chlorophyll b is synthesized from chlorophyll a by chlorophyllide a oxygenase (CAO), which is a Rieske-mononuclear iron oxygenase. Comparison of the amino acid sequences of CAO among photosynthetic organisms elucidated changes in the domain structures of CAO during evolution. However, the evolutionary relationship between the light-harvesting system and the domain structure of CAO remains unclear. To elucidate this relationship, we investigated the CAO structure and the pigment composition of chlorophyll-protein complexes in the prasinophyte Micromonas. The Micromonas CAO is composed of two genes, MpCAO1 and MpCAO2, that possess Rieske and mononuclear iron-binding motifs, respectively. Only when both genes were introduced into the chlorophyll b-less Arabidopsis mutant (ch1-1) was chlorophyll b accumulated, indicating that cooperation between the two subunits is required to synthesize chlorophyll b. Although Micromonas has a characteristic light-harvesting system in which chlorophyll b is incorporated into the core antennas of reaction centers, chlorophyll b was also incorporated into the core antennas of reaction centers of the Arabidopsis transformants that contained the two Micromonas CAO proteins. Based on these results, we discuss the evolutionary relationship between the structures of CAO and light-harvesting systems.     

The Light-harvesting Chlorophyll a/b-Protein Complex of Chlamydomonas reinhardii

The molecular organization of chlorophyll in Chlamydomonas reinhardii has been shown to be essentially similar to that in higher plants. Some 50% of the chlorophyll in Chlamydomonas reinhardii chloroplast membranes has been shown to be located in a chlorophyll a/b-protein complex. The complex was isolated in a homogeneous form by hydroxylapatite chromatography of sodium dodecyl sulfate extracts of the chloroplast membranes. Its absorption spectrum exhibits two maxima in the red region at 670 and 652 nm due to the presence of equimolar quantities of chlorophylls a and b in the complex. Preparations of the chlorophyll-protein also contain some of each of the carotenoids observed in the intact chloroplast membrane, but not in the same proportions. The native complex (S value = 2.3S) exhibits a molecular weight of 28,000 ± 2,000 on calibrated sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, on the basis of its amino acid composition and other data a more probable molecular weight of about 35,000 was calculated. Each 35,000 dalton unit contains three chlorophyll a and three chlorophyll b molecules, and on the average one carotenoid molecule conjugated with probably a single polypeptide of 29,000 daltons. Comparison of spectral and biochemical characteristics demonstrates that this algal chlorophyll-protein is homologous to the previously described major light-harvesting chlorophyll a/b-protein of higher plants. It is anticipated that the Chlamydomonas complex functions solely in a light-harvesting capacity in analogy to the function determined for the higher plant component.     

Nucleotide sequence analysis of the C.AKR Lyt-2 agene: structural polymorphism in alleles encoding the Lyt-2.1 T-cell surface alloantigen

The Lyt-2 ^aallele of the C.AKR strain of mice (genotype Lyt-2 ^a, Lyt-3 ^a) was cloned, and its complete nucleotide sequence as well as that of 2 kb of 5 flanking DNA was determined. The sequence was comapred with the partial sequence of the Lyt-2 ^aallele of DBA/2 (genotype Lyt-2 ^a, Lyt-3 ^b) and the nearly complete sequence of the B10.CAS2 Lyt-2 ^ballele reported by Liaw and coworkers (1986). The coding regions of the two Lyt-2 ^aalleles differ from each other by two nucleotide substitutions in the three exons over which they could be compared, resulting in two amino acid substitutions in the leader and transmembrane segments. The coding region of the C.AKR Lyt-2 ^aallele differs from the Lyt-2 ^ballele by two nucleotide substitutions in the extracellular V-like domain, one of which is silent and the second of which leads to substitution of valine for methionine at amino acid position 78 giving rise to the Lyt-2.1 allotypic specificity. The coding region of the DBA/2 Lyt-2 ^aallele shares with C.AKR the allotypic substitution at position 78 and differs from Lyt-2 ^bby three additional nucleotide substitutions in the coding regions, two of which lead to amino acid substitutions in the leader and transmembrane segments. It would therefore appear that the Lyt-2 alleles of the three strains analyzed are distinct, and the nomenclature Lyt-2 ^a1 and Lyt-2 ^a2 is suggested to distinguish the alleles of C.AKR and DBA/2, respectively. These alleles share a common difference from the Lyt-2 ^bgene product at position 78, and since the amino acid substitutions which distinguish them from each other are in the leader and transmembrane segments, their mature Lyt-2 gene products appear antigenically identical.     

Domain structures of chlorophyllide<Emphasis Type="Italic"> a</Emphasis> oxygenase of green plants and<Emphasis Type="Italic"> Prochlorothrix hollandica</Emphasis> in relation to catalytic functions

Chlorophyll b is a photosynthetic antenna pigment found in prochlorophytes and chlorophytes. In chlorophytes, its biosynthesis regulates the photosynthetic antenna size. Chlorophyll b is synthesized from chlorophyll a in a two-step oxygenation reaction by chlorophyllide a oxygenase (CAO). In this study, we first identified the entire sequence of a prochlorophyte CAO gene from Prochlorothrix hollandica to compare it with those from chlorophytes, and we examined the catalytic activity of the gene product. Southern blot analysis showed that the CAO gene is presented in one copy in the P. hollandica genome. The P. hollandica CAO gene (PhCAO) has a coding capacity for 367 amino acids, which is much smaller than that of Arabidopsis thaliana (537 amino acids) and Oryza sativa (542 amino acids) CAO genes. In spite of the small size, PhCAO catalyzed the formation of chlorophyll b. By comparing these sequences, we classified the land-plant sequences into four parts: the N-terminal sequence predicted to be a transit peptide, the successive conserved sequence unique in land plants (A-domain, 134 amino acids), a less-conserved sequence (B-domain, 30 amino acids) and the C-terminal conserved sequence common in chlorophytes and prochlorophytes (C-domain, 337 to 344 amino acids). We demonstrated that the C-domain is sufficient for catalytic activity by transforming the cyanobacterium Synechocystis sp. PCC6803 with the C-domain from A. thaliana. In this paper, the role of the A-domain is discussed in relation to the formation of light-harvesting chlorophyll a/b–protein complexes in land plants.Abbreviations CAO Chlorophyllide a oxygenase - CP Chlorophyll protein - HPLC High-performance liquid chromatography - LHC Light-harvesting complex - PCR Polymerase chain reaction - PS Photosystem     

Nucleotide sequence of a mouse Tcp-1 pseudogene: A nucleotide record for a t complex gene carried by an ancestor of the mouse

We have isolated clones of a processed pseudogene of mouse t complex polypeptide 1 (Tcp-1) and determined the nucleotide sequence of the pseudogene. The pseudogene was 1363 bp long and had no intron. The Tcp-1 pseudogene had 88.4% or 88.3% nucleotide identity to the mouse Tcp-1 cDNA of wild-type (Tcp-1)^bor t haplotype (Tcp-1)^a, and 87.5% identity to the rat Tcp-1 cDNA. On 12 nucleotide positions where the open reading frames (ORFs) of mouse Tcp-1 ^band Tcp-1 ^acDNAs have bp substitutions, the Tcp-1 pseudogene had 6 bp identical to Tcp-1 ^b, 5 bp identical to Tcp-1 ^aand 1 bp not identical to neither. On ten amino acid positions where TCP-1B and TCP-1A polypeptides have substitutions, deduced amino acids of the Tcp-1 pseudogene had four amino acids identical to TCP-1B, five amino acids identical to TCP-1A and one amino acid identical to neither. These results suggest that the ancestral mouse Tcp-1 gene would have had no significant difference between the resemblance to Tcp-1 ^band that to Tcp-1 ^abefore they were diverged and that amino acids of TCP-1B and TCP-1A would have been substituted in similar high rates.The nucleotide sequence data reported in this paper have been submitted to GenBank and have been assigned the accession number D00851.     

Deletions and Mutations in the Acidic Lipid-binding Region of the Plasma Membrane Ca2+ Pump: A STUDY ON DIFFERENT SPLICING VARIANTS OF ISOFORM 2*

Acidic phospholipids increase the affinity of the plasma membrane Ca²⁺-ATPase pump for Ca²⁺. They interact with the C-terminal region of the pump and with a domain in the loop connecting transmembrane domains 2 and 3 (A_L region) next to site A of alternative splicing. The contribution of the two phospholipid-binding sites and the possible interference of splicing inserts at site A with the regulation of the ATPase activity of isoform 2 of the pump by phospholipids have been analyzed. The activity of the full-length z/b variant (no insert at site A), the w/b (with insert at site A), and the w/a variant, containing both the 45-amino acid A-site insert and a C-site insert that truncates the pump in the calmodulin binding domain, has been analyzed in microsomal membranes of overexpressing CHO cells. The A-site insertion did not modify the phospholipid sensitivity of the pump, but the doubly inserted w/a variant became insensitive to acidic phospholipids, even if containing the intact A_L phospholipid binding domain. Pump mutants in which 12 amino acids had been deleted, or single lysine mutations introduced, in the A_L region were studied by monitoring agonist-induced Ca²⁺ transients in overexpressing CHO cells. The 12-residue deletion completely abolished the ATPase activity of the w/a variant but only reduced that of the z/b variant, which was also affected by the single lysine substitutions in the same domain. A structural interpretation of the interplay of the pump with phospholipids, and of the mechanism of their activation, is proposed on the basis of molecular modeling studies.     

Functional analysis of N-terminal domains of Arabidopsis chlorophyllide a oxygenase.

Higher plants acclimate to various light environments by changing the antenna size of a light-harvesting photosystem. The antenna size of a photosystem is partly determined by the amount of chlorophyll b in the light-harvesting complexes. Chlorophyllide a oxygenase (CAO) converts chlorophyll a to chlorophyll b in a two-step oxygenation reaction. In our previous study, we demonstrated that the cellular level of the CAO protein controls accumulation of chlorophyll b. We found that the amino acids sequences of CAO in higher plants consist of three domains (A, B, and C domains). The C domain exhibits a catalytic function, and we demonstrated that the combination of the A and B domains regulates the cellular level of CAO. However, the individual function of each of A and B domain has not been determined yet. Therefore, in the present study we constructed a series of deleted CAO sequences that were fused with green fluorescent protein and overexpressed in a chlorophyll b-less mutant of Arabidopsis thaliana, ch1-1, to further dissect functions of A and B domains. Subsequent comparative analyses of the transgenic plants overexpressing B domain containing proteins and those lacking the B domain determined that there was no significant difference in CAO protein levels. These results indicate that the B domain is not involved in the regulation of the CAO protein levels. Taken together, we concluded that the A domain alone is involved in the regulatory mechanism of the CAO protein levels.     

Structure and expression of the gene encoding ribosomal protein S1 from the cyanobacterium Synechococcus sp. strain PCC 6301: striking sequence similarity to the chloroplast ribosomal protein CS1

We isolated a 38 kDa ssDNA-binding protein from the unicellular cyanobacterium Synechococcus sp. strain PCC 6301 and determined its N-terminal amino acid sequence. A genomic clone encoding the 38 kDa protein was isolated by using a degenerate oligonucleotide probe based on the amino acid sequence. The nucleotide sequence and predicted amino acid sequence revealed that the 38 kDa protein is 306 amino acids long and homologous to the nuclear-encoded 370 amino acid chloroplast ribosomal protein CS1 of spinach (48% identity), therefore identifying it as ribosomal protein (r-protein) S1. Cyanobacterial and chloroplast S1 proteins differ in size from Escherichia coli r-protein S1 (557 amino acids). This provides an additional evidence that cyanobacteria are closely related to chloroplasts. The Synechococcus gene rps1 encoding S1 is located 1.1 kb downstream from psbB, which encodes the photosystem 11 P680 chlorophyll a apoprotein. An open reading frame encoding a potential protein of 168 amino acids is present between psbB and rps1 and its deduced amino acid sequence is similar to that of E. coli hypothetical 17.2 kDa protein. Northern blot analysis showed that rps1 is transcribed as a monocistronic mRNA.     

Further Chemical and Morphological Characterization of Chloroplast Membranes from a Chlorophyll b-less Mutant of Hordeum vulgare

A comparative study of peptide composition and freeze-fracture morphology of chloroplast membranes from a chlorophyll b-less mutant and a normal barley plant (Hordeum vulgare L.) is reported in this work. Using a high resolution, discontinuous sodium dodecyl sulfate—acrylamide gel electrophoretic system, we show that the mutant chloroplast membranes not only completely lack the 25-kilodalton peak, which accounts for about 18% of the chloroplast membrane protein in the normal plant, but also exhibit gross reduction in other components at 27.5- and 20-kilodalton regions. Despite such extensive deletions in the peptide composition of the mutant chloroplast lamellae, no alteration could be detected in either density or size of the intramembranous particles, visualized by freeze-fracturing.     

Lack of Types 1 and 2A Protein Serine(P)/Threonine(P) Phosphatase Activities in Chloroplasts

Protein phosphatase activity in crude leaf extracts and in purified intact chloroplasts of wheat (Triticum aestivum) and pea (Pisum sativum) was analyzed using exogenously supplied phosphoproteins or endogenous thylakoid proteins. Leaf extracts contain readily detectable amounts of protein phosphatase activity measured with either phosphohistone or phosphorylase a, substrates of mammalian protein phosphatases. No significant chloroplast protein phosphatase activity was detected using these exogenous phosphoproteins. The dephosphorylation of endogenous thylakoid light-harvesting chlorophyll a/b binding proteins in situ was inhibited by fluoride, but not by microcystin-LR or okadaic acid, diagnostic inhibitors of mammalian types 1 and 2A protein phosphatases. Additionally, no evidence for a pea chloroplast alkaline phosphatase activity was found using β-glycerolphosphate or 4-methylum-belliferyl phosphate as substrates. From these results, we conclude that phosphohistone and phosphorylase a are not useful substrates for chloroplast thylakoid protein phosphatase activity and that the chloroplast enzymes may not fit into one of the canonical classifications currently used for protein phosphatases.     

Interaction of chloroplasts with inhibitors: induction of chlorosis by diuron during prolonged illumination in vitro

A primary symptom of diuron (DCMU) phytotoxicity in plants is the destruction of chlorophyll. To study this process in vitro, chloroplasts from pea leaves (Pisum sativum L.) have been incubated in the light with DCMU for periods of up to 34 hours. The sequence of photodestruction of chlorophylls and carotenoids has been followed to try and establish the nature of the chloroplast protection mechanisms that are destroyed by DCMU. β-Carotene decays most rapidly, followed by chlorophyll a and xanthophylls which are destroyed in a constant ratio, followed finally by chlorophyll b. Bypassing the DCMU block in the electron transport system with an artificial electron donor provides complete protection against chlorophyll and carotenoid photodestruction. The same protection by this electron donor system is afforded to stroma-free lamellae from which soluble reductants have been removed so that NADPH formation, which has been proposed as an essential part of a protective xanthophyll cycle, is not possible. Both this and the simultaneous loss of chlorophyll a and xanthophylls tend to preclude the breakdown of a xanthophyll cycle from the possible protective mechanisms inhibited or destroyed by DCMU.     

The light-harvesting chlorophyll a/b · protein complex of the green alga Acetabularia mediterranea isolation and characterization of two subunits

In the green alga Acetabularia mediterranea a light-harvesting chlorophyll a/b · protein complex of 67 000 daltons has been found which contains two polypeptide chains of 21 500 and 23 000 daltons. These two polypeptides were isolated on a preparative scale and were further characterized by several different methods. Both polypeptides proved to be very similar. While their amino acid and sugar compositions as well as their immunochemical properties were almost identical the tryptic peptides and the cyanogen bromide fragments of the two polypeptides revealed minor but significant differences. The 67 000-dalton chlorophyll a/b · protein complex and its two polypeptide components were compared to the light-harvesting chlorophyll a/b · protein of higher plants.     

Spectral characterization of five chlorophyll-protein complexes

Sodium dodecyl sulfate-solubilized chloroplast internal membranes of higher plants (cowpea [Vigna unguiculata L. Walp], chinese cabbage [Brassica chinensi L.], and tobacco [Nicotiana tabacum L.]) are resolved by polyacrylamide gel electrophoresis into two chlorophyll a- and three chlorophyll a,b-proteins. A small portion (about 15%) of the membrane chlorophyll migrates as a component of high electrophoretic mobility and presumably consists of detergent-complexed, protein-free pigment.

One of the chlorophyll a-proteins is qualitatively similar to the P₇₀₀ chlorophyll a-protein but contains a much larger proportion of total chlorophyll (about 30%) than previously reported. The second chlorophyll a-protein is a recently discovered component of the membrane and accounts for about 7% of the total chlorophyll. The absorption and fluorescence emission spectra of these two chlorophyll a-proteins differ.

The three chlorophyll a,b-proteins are components of the chloroplast membrane chlorophyll a,b-light-harvesting complex which was previously resolved as a single chlorophyll-protein band. The two additional chlorophyll a,b-proteins observed in our work probably represent larger aggregates contained within that membrane complex which are preserved under the solubilization and electrophoretic conditions used here.

     

Clp protease controls chlorophyll b synthesis by regulating the level of chlorophyllide a oxygenase

Chlorophyll b is one of the major light-harvesting pigments in green plants and it is essential for optimal light harvesting. Chlorophyll b is synthesized from chlorophyll a by chlorophyllide a oxygenase (CAO) which consists of A, B and C domains. Previously, we demonstrated that the C domain alone has a catalytic function, while the A and B domains control the level of CAO protein in response to chlorophyll b accumulation. We hypothesized that the accumulation of chlorophyll b triggers the proteolytic degradation of CAO. In this study, in order to gain further insight into this regulatory mechanism we screened for mutants that have defects in the control of CAO accumulation. Seeds from a transgenic line of Arabidopsis which overexpressed a CAO-GFP fusion were mutagenized and their progenies were screened by laser-scanning confocal microscopy for mutants showing an elevated level of GFP fluorescence. One particular mutant (dca1) exhibited stronger GFP fluorescence and accumulated a GFP-CAO fusion protein at a higher level. Concomitantly, the chlorophyll a to b ratio decreased in this mutant. The mutation in the dca1 mutant was mapped to the ClpC1 gene, thereby indicating that a chloroplast Clp protease is involved in regulating chlorophyll b biosynthesis through the destabilization of CAO protein in response to the accumulation of chlorophyll b.     

Identification and characterization of a locus (RTM1) that restricts long‐distance movement of tobacco etch virus in Arabidopsis thaliana

The chloroplast homolog of the 54 kDa subunit of signal recognition particle is required for the in vitro targeting of chlorophyll a/b binding proteins (LHCP) to the thylakoid membrane. To explore the function of cpSRP54 in vivo, plants that are mutated in cpSRP54 function were generated. Dominant negative forms of cpSRP54 altered in single amino acids within the conserved guanine nucleotide binding domain were expressed in Arabidopsis. Transformed plants contained less than 30% of the wild-type level of cpSRP54 protein. As a consequence of the reduced cpSRP54 protein content, the first emerging leaves were yellow and contained immature chloroplasts. Although the chlorophyll (chl) content of the leaves was reduced by 75%, the chl a/b ratio was unaffected, indicating a role of cpSRP54 in the biogenesis of proteins besides LHCP. Many chloroplast proteins were less abundant in the first emerging leaves, including non-pigmented proteins, thylakoid proteins known to be targeted by alternative pathways, and soluble proteins. These observations indicate that the cpSRP54 mutation also has a pleiotropic effect on chloroplast biogenesis. Whereas the level of cpSRP54 remained low as the plants aged, leaves emerging subsequently had a wild-type appearance, suggesting that the adult plants compensated for the reduction in cpSRP54 protein.     

Biochemical studies on the H-2K mutant B6.C-H-2 bm10

The H-2K glycoprotein from the MHC mutant bm10 was analyzed biochemically to determine where primary structural differences distinguished it from the parental standard molecule, K^b. Comparative peptide maps showed differences in two peptides known to be part of the parental CNBr fragment spanning amino acids 139 to 228. Partial sequence analyses of CNBr fragments and tryptic peptides identified two tightly clustered amino acid substitutions at amino acids 165 (Val to Met) and 173 (Lys to unknown). The substitutions in bm10 represent the most carboxy-terminal substitutions characterized in the K^b molecules of the spontaneous, histogenically active H-2 mutants.