The 55-kDa polypeptide released from spinach thylakoid membranes with 1 M LiCl is not the beta subunit of chloroplast F1

It was reported by Frasch et al. (Frasch, W. D., Green, J., Caguiat, J., and Mejia, A. (1989) J. Biol. Chem. 264, 5064-5069) that washing spinach thylakoid membranes with 1 M LiCl caused the release of the beta subunit of chloroplast F1 (CF1) which, existing as 180-kDa complexes of beta 3, retained considerable ATPase activity. We repeated their procedures and confirmed that a CF1 beta-like 55-kDa polypeptide was a major constituent of the 1 M LiCl-washed extract. However, the extract contained another polypeptide of which the Mr was 14,000, and these two polypeptides comprised a complex with approximate Mr 550,000 that had the same mobility in native polyacrylamide gel electrophoresis as that of ribulose-1,5-bisphosphate carboxylase. Only very low ATPase activity, less than 1% of the reported value, was detected for the extract and the purified complex. Antibody against the beta subunit of F1 from a thermophilic bacterium PS3 showed a clear cross-reactivity with the CF1 beta subunit but not with the 55-kDa polypeptide. Analysis of the N-terminal amino acid sequences of the 55- and 14-kDa polypeptides and the whole complex revealed that the complex was ribulose-1,5-bisphosphate carboxylase and that the 55- and 14-kDa polypeptides were its large and small subunits, respectively.     

Protein composition of the carboxysomes of Thiobacillus neapolitanus

For purifying carboxysomes of Thiobacillus neapolitanus an isolation procedure was developed which resulted in carboxysomes free from whole cells, protoplasts and cell fragments. These purified carboxysomes are composed of 8 proteins and at the most of 13 polypeptides. The two most abundant proteins which make up more than 60% of the carboxysomes, are ribulose-1,5-bisphosphate carboxylase and a glycoprotein with a molecular weight of 54,000. The shell of the carboxysomes consists of four glycoproteins, one also with a molecular weight of 54,000. The other proteins are present in minor quantities. Ribulose-1,5-bisphosphate carboxylase is the only enzyme which could be detected in the carboxysomes and 3-phosphoglycerate was the only product formed during incubation with ribulose-1,5-diphosphate and bicarbonate. The supernatant of a broken and centrifuged carboxysome suspension contained the large subunit of ribulose-1,5-bisphosphate carboxylase. The small subunit of ribulose-1,5-bisphosphate carboxylase was found in the pellet together with the shell proteins which indicates that the small subunit of ribulose-1,5-bisphosphate carboxylase is connected to the shell.Abbreviations RuBisCO ribulose-1,5-bisphosphate carboxylase - PMSF phenylmethylsulfonyl fluoride - PAA gelectrophoresis, polyacrylamide gelelectrophoresis - SDS sodium dodecyl sulphate - CIE crossed immunoelectrophoresis - IEF isoelectric focusing     

Ribulose 1,5-bisphosphate and activation of the carboxylase in the chloroplast

Ribulose 1,5-bisphosphate in the chloroplast has been suggested to regulate the activity of the ribulose bisphosphate carboxylase/oxygenase. To generate high levels of ribulose bisphosphate, isolated and intact spinach chloroplasts were illuminated in the absence of CO₂. Under these conditions, chloroplasts generate internally up to 300 nanomoles ribulose 1,5-bisphosphate per milligram chlorophyll if O₂ is also absent. This is equivalent to 12 millimolar ribulose bisphosphate, while the enzyme, ribulose bisphosphate carboxylase, offers up to 3.0 millimolar binding sites for the bisphosphate in the chloroplast stroma. During illumination, the ribulose bisphosphate carboxylase is deactivated, due mostly to the absence of CO₂ required for activation. The rate of deactivation of the ribulose bisphosphate carboxylase was not affected by the chloroplast ribulose bisphosphate levels. Upon addition of CO₂, the carboxylase in the chloroplast was completely reactivated. Of interest, addition of 3-phosphoglycerate stopped deactivation of the carboxylase in the chloroplast while ribulose bisphosphate accumulated. With intact chloroplasts in light, no correlation between deactivation of the carboxylase and ribulose bisphosphate levels could be shown.     

Analysis of pea chloroplast inner and outer envelope membrane proteins by two-dimensional gel electrophoresis and their comparison with stromal proteins

Analysis of inner and outer pea (Pisum sativum var. Laxtons Progress No. 9) chloroplast envelope membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that, although the two membranes have distinct polypeptide compositions, there are several comigrating polypeptides in the two membrane fractions. To determine whether these comigrating polypeptides were identical by criteria other than molecular weight, the membrane proteins were analyzed by two-dimensional gel electrophoresis. The results demonstrated that an 86-kilodalton band found in both membranes represents at least two different polypeptides, one an outer membrane protein and the other an inner membrane protein. Several other polypeptide bands found in both membranes appear to be of stromal origin. Two of these polypeptides were shown to be the large and small subunits of ribulose 1,5-bisphosphate carboxylase. The large subunit was identified by two-dimensional electrophoresis of envelope membranes to which stromal proteins were added. Additionally, the large and small subunits of ribulose 1,5-bisphosphate carboxylase were immunologically identified using an electrophoretic transfer procedure coupled with an enzyme-linked immunosorbent assay. Various treatments, including sonication, resulted in no significant loss of the stromal polypeptides from the outer envelope membranes. Based on these results, it is suggested that the stromal proteins are not simply bound to the outer surface of the vesicles.     

Ribulose bisphosphate carboxylase in algae: synthesis,enzymology and evolution

Studies demonstrating differences in chloroplast structure and biochemistry have been used to formulate hypotheses concerning the origin of algal plastids. Genetic and biochemical experiments indicate that significant variation occurs in ribulose-1,5-bisphosphate carboxylase (Rubisco) when supertaxa of eukaryotic algae are compared. These differences include variations in the organelle location of the genes and their arrangement, mechanism of Rubisco synthesis, polypeptide immunological reactivity and sequence, as well as efficacy of substrate (ribulose bisphosphate and CO₂) binding and inhibitor (6-phosphogluconate) action. The structure-function relationships observed among chromophytic, rhodophytic, chlorophytic and prokaryotic Rubisco demonstrate that: (a) similarities among chromophytic and rhodophytic Rubisco exist in substrate/inhibitor binding and polypeptide sequence, (b) characteristic differences in enzyme kinetics and subunit polypeptide structure occur among chlorophytes, prokaryotes and chromophytes/rhodophytes, and (c) there is structural variability among chlorophytic plant small subunit polypeptides, in contrast to the conservation of this polypeptide in chromophytes and rhodophytes. Taxa-specific differences among algal Rubisco enzymes most likely reflect the evolutionary history of the plastid, the functional requirements of each polypeptide, and the consequences of encoding the large and small subunit genes in the same or different organelles.     

Protein synthesis in chloroplasts. VIII. Differential synthesis of chloroplast proteins during spinach leaf development

Excised primary leaves of spinach (Spinacia oleracea) incorporate [35S]-methionine into a number of chloroplast polypeptides. The ratio of incorporation of isotope into the large subunit of ribulose bisphosphate carboxylase relative to a thylakoid polypeptide (peak D) decreases during leaf development in whole leaves; this changing pattern of incorporation is also observed in isolated chloroplasts where these two polypeptides are the major products of protein synthesis. Chloroplast RNA prepared from developing leaves was translated in a reticulocyte lysate extract to yield full-length carboxylase large subunit and peak D polypeptides. The fidelity of translation of these two polypeptides was checked by partial protease digestion. Changes in the synthesis of the large subunit of the carboxylase and peak D in developing leaves are reflected in changes in the amount of translatable mRNA for these two polypeptides.     

Chloroplast gene for Mr 32000 polypeptide of photosystem II in Euglena gracilis is interrupted by four introns with conserved boundary sequences

The gene for the Mr 32000 herbicide binding polypeptide of photosystem II has previously been mapped to the 5 kbp EcoRI fragment Eco I of Euglena gracilis chloroplast DNA. The nucleotide sequence of 3324 bp of Eco I, containing the psbA locus, has been determined. This locus encodes a polypeptide of 345 amino acids which is co-linear with, and has 86% derived amino acid sequence homology to sequences derived from four higher plants chloroplast psbA loci. The Euglena psbA gene contains four introns of size 435, 443, 434, and 617 bp. The four introns have conserved boundary sequences of the type previously described in the Euglena chloroplast gene (rbcL) for the large subunit of ribulose-1,5-bisphosphate carboxylase (Koller et al., Cell 36, 545-553, 1984).     

Phenylmethylsulphonyl fluoride Inhibits the Formation of Jasmonate-Induced Proteins in Cotyledons of Cucurbita pepo (zucchini)

Phenylmethylsulphonyl fluoride (PMSF), a well known inhibitor of both thiol- and serine-type proteases, in aqueous solutions either alone or with the plant growth regulators, methyl ester of jasmonic acid (MeJA) and N⁶-benzyl-aminopurine (BAP), significantly inhibited the growth of excised Cucurbita pepo L. (zucchini) cotyledons. SDS-PAGE analysis of the protein profiles showed that PMSF suppressed the gradual decline of the main 20 – 25 kDa polypeptide group and the low molecular mass polypeptides (below 15 kDa) while leupeptine was not able to affect the electrophoretic pattern of cotyledon proteins. On the other hand, in the presence of PMSF, the content of the polypeptides with higher molecular mass including the 97.4 kDa polypeptide and the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (55 kDa) decreased. Besides, when applied together with MeJA, PMSF prevented the appearance of the jasmonate-induced polypeptides (JIPs; 69, 60 and 43 kDa) thus suggesting that JIPs are synthesized from aminoacids released during the breakdown of cotyledon storage proteins. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characterization of the pyrenoid isolated from unicellular green algaChlamydomonas reinhardtii: Particulate form of RuBisCO protein

Summary The pyrenoid is a protein complex in the chloroplast stroma of eukaryotic algae. After the treatment with mercury chloride, pyrenoids were isolated by sucrose density gradient centrifugation from cell-wall less mutant cells, CW-15, as well as wild type cells, C-9, of unicellular green algaChlamydomonas reinhardtii. Pyrenoids were characterized as a fraction whose protein/chlorophyll ratio was very high, and also examined by Nomarski differential interference microscopy. Most of the components consisted of 55 kDa and 16 kDa polypeptides (11) which were immunologically identified as the large and small subunit of RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) protein, respectively. Some minor polypeptides were also detected. Substantial amount of RuBisCO protein is present as a particulate form in the pyrenoid in addition to the soluble form in algal chloroplast stroma.Abbreviations BPB bromophenol blue - DAB 3,3-diaminobenzidine - DTT dithiothreitol - ELISA enzyme-linked immunosorbent assay - High-CO₂ cells cells grown under air enriched with 4% CO₂ - Low-CO₂ cells cells grown under ordinary air (containing 0.04% CO₂) - NP-40 nonionic detergent (Nonidet) P-40 - PAGE polyacrylamide gel electrophoresis - PAP peroxidase-antiperoxidase conjugate - RuBisCO ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - SDS sodium dodecylsulfate     

Preliminary structural studies of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) from Rhodospirillum rubrum has been crystallized in a form that is suitable for structural studies by x-ray diffraction. The asymmetric unit of the crystal contains one dimeric enzyme molecule of molecular mass 101,000 Da. The enzyme was activated prior to crystallization and is presumed to be in the CO2-activated state in the crystal. The method of hydrophobicity correlation has been used to compare the amino acid sequence of this molecule (466 residues) to that of the large subunit of a higher plant ribulose-1,5-bisphosphate carboxylase/oxygenase (477 residues in Nicotiana tabacum). The pattern of residue hydrophobicities is similar along the two polypeptides. This suggests that the three-dimensional folding of the large polypeptide chains may be similar in plant and bacterial enzymes. If this is so, knowing the structure of either the plant or bacterial ribulose-1,5-bisphosphate carboxylase/oxygenase should aid in learning the structure of the other.     

Measurement of ribulose 1,5-bisphosphate from spinach chloroplasts

A technique has been developed for the rapid and simple measurement of ribulose 1,5-bisphosphate from isolated spinach chloroplasts. The endogenous ribulose bisphosphate was detected enzymically using (14)CO(2) and ribulose bisphosphate carboxylase/oxygenase released from the chloroplasts. Ribulose 5-phosphate kinase was inhibited with 0.4 to 0.6 millimolar 2,6-dichlorophenol-indophenol and 4 micromolar carbonyl cyanide m-chlorophenylhydrazone. Phosphoenolpyruvate carboxylase activity was low with washed chloroplasts and its labeled product, [(14)C]oxalacetate, was destroyed by heating with 1.0 n HCl at 90 C. The assay method was linear from 0.05 to 0.87 nanomoles ribulose bisphosphate per milliliter. The latter value was determined with chloroplast material having 44 micrograms of chlorophyll per milliliter. This technique was simple and direct, used less chloroplast material, yet provided results comparable to a previously described enzymic technique in which ribulose bisphosphate was determined after the precipitation of chloroplast proteins by perchloric acid.     

Chloroplast Dedifferentiation in Mechanically Isolated Asparagus Cells during Culture Initiation

Mechanically isolated asparagus (Asparagus officinalis) mesophyll cells dedifferentiate and divide when cultured in the dark in a medium containing sucrose. A strong correlation was observed between the onset of cell division and a loss of photosynthetic capacity. For the first 8 to 9 d of culture, there was no change in chloroplast size or morphology. However, following this period, the chloroplasts divided to form smaller proplastid-like structures. The gross chlorophyll content of the cell population did not change, suggesting that the loss of photosynthetic potential was not by senescence. Northern analysis showed that mRNA of the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase was undetectable within 1 d postisolation, which was quicker than in dark-treated plants. The mRNA of the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase decreased to low levels within 2 d of cell isolation. Both the large and small subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase protein showed a gradual reduction in abundance, falling to basal levels by days 6 to 7, which coincided with the onset of rapid cell division. A similar trend was observed with chloroplast rRNA molecules, which decreased to basal levels by day 6 in culture.     

Synthesis of proteins by chloroplasts from iron-deficient Euglena gracilis

Chloroplasts isolated from Euglena gracilis made iron deficient by growth on 0.5 μm iron show distinct qualitative and quantitative changes in their polypeptide composition in comparison with iron-sufficient (40 μm) chloroplasts. These changes were noted in the stromal, thylakoid, and envelope subfractions. Iron-deficient chloroplasts have a sedimentation behavior similar to that of iron-sufficient chloroplasts and also contain substantial amounts of ribulose-1,5-bisphosphate carboxylase. In addition, iron-deficient chloroplasts incorporate [³H]leucine into polypeptides at rates about one-third of those from control chloroplasts (40 μm Fe) on a per-microgram-chlorophyll basis. Incorporation of [³H]leucine into specific polypeptides, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, shows relatively normal synthesis of the large subunit of ribulose-1,5-bisphosphate carboxylase and two of the three major chloroplast-derived polypeptides of the thylakoids. No incorporation was detected, however, into a polypeptide of ca. 33 kd which is synthesized by normal plastids. Iron-deficient chloroplasts also synthesize a stromal polypeptide of ca. 85 kd not seen in chloroplasts from normal cells. This evidence is consistent with a direct or indirect role for iron in the regulation of synthesis of specific proteins in the chloroplast.     

香蕉rbcS基因启动子的克隆及序列分析

以巴西香蕉为材料,根据已经获得的香蕉1,5-二磷酸核酮糖羧化/加氧酶小亚基基因的全长cDNA序列设计1对专一引物,通过PCR扩增得到了香蕉1,5-二磷酸核酮糖羧化/加氧酶小亚基的基因组全长,序列长811 bp,含有2个内含子。根据其基因组序列设计引物,采用SEFA-PCR方法,以总DNA为模板克隆了香蕉1,5-二磷酸核酮糖羧化/加氧酶小亚基基因的启动子序列,长1 681 bp。用PLACE软件分析发现该序列具有启动子的基本元件TATA-box、CAAT-box,包含多个胁迫诱导元件,如光诱导元件、赤霉素、低温诱导元件、昼夜节律调控元件等。该序列的克隆与分析为进一步研究香蕉1,5-二磷酸核酮糖羧化/加氧酶小亚基基因的表达调控奠定了基础。     

Suitable conditions for characterization,identification, and isolation of the mRNA of the small subunit of ribulose-1,5-bisphosphate carboxylase from Nicotiana sylvestris

The products synthesized in vitro by messenger RNA (mRNA) extracted from Nicotiana sylvestris were analyzed by electrophoresis on polyacrylamide slab gels. Only three of the major polypeptides synthesized are considered here: P₅₅, P₃₂, and P₂₀. P₅₅ and P₃₂ were translated from chloroplast mRNA. P₅₅ corresponds to the large subunit of ribulose-1,5-bisphosphate (RuP₂) carboxylase; P₃₂ is probably a chloroplast membrane protein. P₂₀, the polypeptide synthesized from cytoplasmic poly(A)⁺ RNA, is the precursor of the small subunit of RuP₂ carboxylase. The balance between P₂₀ and P₃₂, in which their relative proportions varied inversely, was regulated by the age of the leaves and the time of illumination; we took advantage of this phenomenon to isolate the mRNA from the small subunit in relatively large amounts. This mRNA has a molecular weight of 350,000.Abbreviations RuP₂ ribulose-1,5-bisphosphate - mRNA messenger RNA - SDS sodium dodecyl sulfate     

Stromal protein phosphorylation in spinach (Spinacia oleracea) chloroplasts.

When intact spinach chloroplasts were supplied with [32P]Pi, stromal protein phosphorylation was found to occur in the dark. On illumination the thylakoid protein kinase was activated and the amount of label found in thylakoid proteins quickly exceeded that incorporated into stromal protein, such that the latter was found to account for only 10-15% of the total radioactivity bound to chloroplast proteins after 5 min illumination. The rate of phosphorylation of stromal polypeptides was unchanged by light. After SDS/polyacrylamide-gel electrophoresis, more than 15 labelled polypeptides of stromal origin were observed. A polypeptide with an Mr of approx. 70 000 had the highest specific activity of labelling. Both the large and small subunits of the ribulose-1,5-bisphosphate carboxylase were phosphorylated. The level of phosphorylation of stromal protein was increased by CO2 fixation in intact chloroplasts. This increase was not observed in the absence of NaHCO3 or in the presence of the phosphoribulokinase inhibitor DL-glyceraldehyde. These effects appeared to be largely due to changes in the phosphorylation state of the large and small subunits of ribulose-1,5-bisphosphate carboxylase. Studies with the reconstituted chloroplast system showed that the thylakoid protein kinase(s) played no part in the phosphorylation of stromal protein. The rate and level of phosphorylation of stromal protein was unaffected by the activation state of the thylakoid protein kinase and was unchanged when thylakoids were omitted from the reaction medium. The phosphorylation of stromal proteins is therefore catalysed by a discrete soluble protein kinase.     

Immunological determination of phosphoenolpyruvate carboxylase and the large and small subunits of ribulose 1,5-bisphosphate carboxylase in leaves of the c(4) plant pearl millet

The light-dependent development of the photosynthetic apparatus in the first leaf of the C₄ plant pearl millet (Pennisetum americanum) was monitored by immunologically determining the concentration of phospho-enolpyruvate carboxylase and ribulose 1,5-bisphosphate carboxylase. A competitive enzyme-linked immunosorbent assay procedure using antibodies to the monomeric subunit of phosphoenolpyruvate carboxylase and the large and small subunit of ribulose 1,5-bisphosphate carboxylase was used to quantitate the amounts of these polypeptides in the first leaf of etiolated seedlings and etiolated seedlings exposed to light for varying periods of time. Phosphoenolpyruvate carboxylase was present in etiolated tissue; however, light stimulated its synthesis nearly 23-fold. Maximum accumulation of phosphoenolpyruvate carboxylase occurred approximately 4 days after etiolated plants were placed in the light. Both the large subunit and the small subunit of ribulose 1,5-bisphosphate carboxylase were present in leaves of etiolated seedlings. Light also stimulated the synthesis of both of these polypeptides, but at different rates. In etiolated leaves there was approximately a 3-fold molar excess of the small subunit to large subunit. Exposure of the etiolated leaves to light resulted in the molar ratio of the large subunit to the small subunit increasing to approximately 0.72. These data indicate that the net synthesis of these two polypeptides is not coordinately regulated at all times.     

Fraction I protein analysis of parasexual hybrid plants Arabidopsis thaliana + Brassica campestris

The polypeptide composition of Fraction I protein (ribulose-1,5-bisphosphate carboxylase) prepared from leaves of two clones of the parasexual hybrid plant Arabidopsis thaliana + Brassica campestris as well as their parents was analyzed by isoelectric focusing. The protein in hybrid plants contained a heterogenous population of small subunits resulting from the expression of both Arbabidopsis and Brassica nuclear genes, whereas the large subunit polypeptides, and hence the functional chloroplast DNA, were from the Brassica parent.