Primary structure of the maize NADP-dependent malic enzyme

Chloroplast-localized NADP-dependent malic enzyme (EC 1.1.1.40) (NADP-ME) provides a key activity for the carbon 4 fixation pathway. In maize, nuclear encoded NADP-ME is synthesized in the cytoplasm as a precursor with a transit peptide that is removed upon transport into the chloroplast stroma. We present here the complete nucleotide sequence for a 2184-base pair full-length maize NADP-ME cDNA. The predicted precursor protein is 636 amino acids long with a Mr of 69,800. There is a strong codon bias found in the amino-terminal portion of NADP-ME that is present in genes for the other enzymes of the C-4 photosynthetic pathway. The NADP-ME transit peptide has general features common to other known chloroplast stroma transit peptides. Comparison of mature maize NADP-ME to the amino acid sequences of known malic enzymes shows two conserved dinucleotide-binding sites. There is a third highly conserved region of unknown function. On the basis of amino acid sequence similarity, the maize chloroplastic enzyme is more closely related to eukaryotic cytosolic isoforms of malic enzyme than to prokaryotic isoforms. We discuss the functional and evolutionary relationship between the chloroplastic and cytosolic forms of NADP-ME.     

Purification and cDNA cloning of chloroplastic monodehydroascorbate reductase from spinach

The chloroplastic isoform of monodehydroascorbate (MDA) radical reductase was purified from spinach chloroplasts and leaves. The cDNA of chloroplastic MDA reductase was cloned, and its deduced amino acid sequence, consisting of 497 residues, showed high homology with those of putative organellar MDA reductases deduced from cDNAs of several plants. The amino acid sequence of the amino terminal of the purified enzyme suggested that the chloroplastic enzyme has a transit peptide consisting of 53 residues. A southern blot analysis suggested the occurrence of a gene encoding another isoform homologous to the chloroplastic isoform in spinach. The recombinant enzyme was highly expressed in Eschericia coli using the cDNA, and purified to a homogeneous state with high specific activity. The enzyme properties of the chloroplastic isoform are presented in comparison with those of the cytosolic form.     

Molecular characterization and physiological role of ascorbate peroxidase from halotolerant Chlamydomonas sp. W80 strain

A cDNA clone encoding an ascorbate peroxidase was isolated from the cDNA library from halotolerant Chlamydomonas W80 by a simple screening method based on the bacterial expression system. The cDNA clone contained an open reading frame encoding a mature protein of 282 amino acids with a calculated molecular mass of 30,031 Da, preceded by the chloroplast transit peptide consisting of 37 amino acids. In fact, ascorbate peroxidase was localized in the chloroplasts of Chlamydomonas W80 cells; the activity was detected in the stromal fraction but not in the thylakoid membrane. The deduced amino acid sequence of the cDNA showed 54 and 49% homology to chloroplastic and cytosolic ascorbate peroxidase isoenzymes of spinach leaves, respectively. The enzyme from Chlamydomonas W80 cells was purified to electrophoretic homogeneity. The molecular properties of the purified enzyme were similar to those of the other algal ascorbate peroxidases rather than those of ascorbate peroxidases from higher plants. The enzyme was relatively stable in ascorbate-depleted medium compared with the chloroplastic ascorbate peroxidase isoenzymes of higher plants. The presence of NaCl (3%) as well as of beta-d-thiogalactopyranoside was needed for the expression of Chlamydomonas W80 ascorbate peroxidase in Escherichia coli.     

Cloning and sequence analysis of cDNAs encoding the cytosolic precursors of subunits GapA and GapB of chloroplast glyceraldehyde-3-phosphate dehydrogenase from pea and spinach

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is composed of two different subunits, GapA and GapB. cDNA clones containing the entire coding sequences of the cytosolic precursors for GapA from pea and for GapB from pea and spinach have been identified, sequenced and the derived amino acid sequences have been compared to the corresponding sequences from tobacco, maize and mustard. These comparisons show that GapB differs from GapA in about 20% of its amino acid residues and by the presence of a flexible and negatively charged C-terminal extension, possibly responsible for the observed association of the enzyme with chloroplast envelopes in vitro. This C-terminal extension (29 or 30 residues) may be susceptible to proteolytic cleavage thereby leading to a conversion of chloroplast GAPDH isoenzyme I into isoenzyme II. Evolutionary rate comparisons at the amino acid sequence level show that chloroplast GapA and GapB evolve roughly two-fold slower than their cytosolic counterpart GapC. GapA and GapB transit peptides evolve about 10 times faster than the corresponding mature subunits. They are relatively long (68 and 83 residues for pea GapA and spinach GapB respectively) and share a similar amino acid framework with other chloroplast transit peptides.     

Structural Similarities between Spinach Chloroplast and Cytosolic Class I Fructose 1,6-Bisphosphate Aldolases : Immunochemical and Amino-Terminal Amino Acid Sequence Analysis

Immunochemical studies using polyclonal antisera prepared individually against highly purified cytosolic and chloroplast spinach leaf (Spinacia oleracea) fructose bisphosphate aldolases showed significant cross reaction between both forms of spinach aldolase and their heterologous antisera. The individual cross reactions were estimated to be approximately 50% in both cases under conditions of antibody saturation using a highly sensitive enzyme-linked immunosorbent assay. In contrast, the class I procaryotic aldolase from Mycobacterium smegmatis and the class II aldolase from yeast (Saccharomyces cerevisiae) did not cross-react with either type of antiserum. The 29 residue long amino-terminal amino acid sequences of the procaryotic M. smegmatis and the spinach chloroplast aldolases were determined. Comparisons of these sequences with those of other aldolases showed that the amino-terminal primary structure of the chloroplast aldolase is much more similar to the amino-terminal structures of class I cytosolic eucaryotic aldolases than it is to the corresponding region of the M. smegmatis enzyme, especially in that region which forms the first “beta sheet” in the secondary structure of the eucaryotic aldolases. Moreover, results of a systematic comparison of the amino acid compositions of a number of diverse eucaryotic and procaryotic fructose bisphosphate aldolases further suggest that the chloroplast aldolase belongs to the eucaryotic rather than the procaryotic “family” of class I aldolases.     

Characterization of cDNAs encoding CuZn-superoxide dismutases in Scots pine

A Scots pine (Pinus sylvestris L.) cDNA library was screened with two heterologous cDNA probes (P31 and T10) encoding cytosolic and chloroplastic superoxide dismutases (SOD) from tomato. Several positive clones for cytosolic and chloroplastic superoxide dismutases were isolated, subcloned, mapped and sequenced. One of the cDNA clones (PS3) had a full-length open reading frame of 465 bp corresponding to 154 amino acid residues and showed approximately 85% homology with the amino acid sequences of angiosperm cytosolic SOD counterparts. Another cDNA clone (PST13) was incomplete, but encoded a putative protein with 93% homology to pea and tomato chloroplastic superoxide dismutase. The derived amino acid sequence from both cDNA clones matched the corresponding N-terminal amino acid sequence of the purified mature SOD isozymes. Northern blot hybridizations showed that, cytosolic and chloroplastic CuZn-SOD are expressed at different levels in Scots pine organs. Sequence data and Southern blot hybridization confirm that CuZn-SODs in Scots pine belong to a multigene family. The results are discussed in relation to earlier observations of CuZn-SODs in plants.     

Isolation and characterization of the cytosolic and chloroplast forms of spinach leaf fructose diphosphate aldolase

Two different isoenzymes of fructose-P2 aldolase can be resolved by chromatography of crude spinach leaf extracts on DEAE-cellulose columns. The acidic isoenzyme comprises about 85% of the total leaf aldolase activity. The two forms differ in primary structure as judged by their distinctive amino acid compositions, tryptic peptide patterns, and immunological properties. Only the acidic isoenzyme was detected in extracts of isolated chloroplasts, suggesting that this molecule represents the chloroplast form of spinach leaf aldolase while the basic isoenzyme is of cytosolic origin. The cytosolic (basic) isoenzyme and chicken aldolase A4 are similar in the following respects. 1) They have similar specific catalytic activity (10-15 units/mg); 2) they are both highly sensitive to inactivation by very limited digestion with bovine pancreatic carboxypeptidase A; 3) they both have subunit molecular weights of 40,000; 4) they both have derivatized (blocked) NH2-terminal structures; 5) they are both resistant to thermal denaturation at 50 degrees C; and 6) they both regain catalytic activity following reversible denaturation at pH 2.3 or in 5.8 M urea. Also, the cytosolic aldolase cross-reacted immunologically with the single aldolases present in spinach seeds and in wheat germ. Further, this isoenzyme readily "hybridized" with chicken aldolase A4 in vitro. These observations demonstrate the close homology between the cytosolic aldolases derived from plant and animal origins. The chloroplast aldolase had a specific catalytic activity of about 8 units/mg and, like its cytosolic counterpart, was severely inactivated by limited digestion with carboxypeptidase A. However, this isoenzyme was distinct from the cytosolic aldolase in the following characteristics: 1) its "small" subunit size (Mr congruent to 38,000); 2) its underivatized NH2-terminal structure; 3) its high sensitivity to thermal denaturation at 50 degrees C; and 4) its inability to refold into an enzymatically active conformation following denaturation at pH 2.3 or in 5.8 M urea. The distinctive properties of the chloroplast aldolase may be expected for an enzyme which is synthesized as a higher molecular weight precursor on cytosolic polysomes and is then proteolytically processed to the "mature" form during its migration into the chloroplast organelle.     

Antigenic relationships between chloroplast and cytosolic fructose-1,6-bisphosphatases.

Cytosolic fructose-1,6-biphosphatases (FBPase, EC 3.1.3.11) from pea (Pisum sativum L. cv Lincoln) and spinach (Spinacia oleracea L. cv Winter Giant) did not cross-react by double immunodiffusion and western blotting with either of the antisera raised against the chloroplast enzyme of both species; similarly, pea and spinach chloroplast FBPases did not react with the spinach cytosolic FBPase antiserum. On the other hand, spinach and pea chloroplast FBPases showed strong cross-reactions against the antisera to chloroplast FBPases, in the same way that the pea and spinach cytosolic enzymes displayed good cross-reactions against the antiserum to spinach cytosolic FBPase. Crude extracts from spinach and pea leaves, as well as the corresponding purified chloroplast enzymes, showed by western blotting only one band (44 and 43 kD, respectively) in reaction with either of the antisera against the chloroplast enzymes. A unique fraction of molecular mass 38 kD appeared when either of the crude extracts or the purified spinach cytosolic FBPase were analyzed against the spinach cytosolic FBPase antiserum. These molecular sizes are in accordance with those reported for the subunits of the photosynthetic and gluconeogenic FBPases. Chloroplast and cytosolic FBPases underwent increasing inactivation when increasing concentrations of chloroplast or cytosolic anti-FBPase immunoglobulin G (IgG), respectively, were added to the reaction mixture. However, inactivations were not observed when the photosynthetic enzyme was incubated with the IgG to cytosolic FBPase, or vice versa. Quantitative results obtained by enzyme-linked immunosorbent assays (ELISA) showed 77% common antigenic determinants between the two chloroplast enzymes when tested against the spinach photosynthetic FBPase antiserum, which shifted to 64% when assayed against the pea antiserum. In contrast, common antigenic determinats between the spinach cytosolic FBPase and the two chloroplast enzymes were less than 10% when the ELISA test was carried out with either of the photosynthetic FBPase antisera, and only 5% when the assay was performed with the antiserum to the spinach cytosolic FBPase. These results were supported by sequencing data: the deduced amino acid sequence of a chloroplast FBPase clone isolated from a pea cDNA library indicated a 39,253 molecular weight protein, with a homology of 85% with the spinach chloroplast FBPase but only 48.5% with the cytosolic enzyme from spinach.     

Molecular mechanisms of the resistance to hydrogen peroxide of enzymes involved in the calvin cycle from halotolerant Chlamydomonas sp. W80

cDNA clones encoding NADP(+)-glyceraldehyde-3-phosphate dehydrogenase (NADP(+)-GAPDH) and sedoheptulose-1,7-bisphosphatase (SBPase) were isolated and characterized from halotolerant Chlamydomonas sp. W80 (C. W80) cells. The cDNA clone for NADP(+)-GAPDH encoded 369 amino acid residues, preceded by the chloroplast transit peptide (37 amino acid residues). The cDNA clone for SBPase encoded 351 amino acids with the chloroplast transit peptide. The activities of NADP(+)-GAPDH and SBPase from C. W80 cells were resistant to H(2)O(2) up to 1 mM, as distinct from spinach chloroplastic thiol-modulated enzymes. The illumination to the dark-adapted cells and dithiothreitol treatment to the crude homogenate had little effect on the activities of NADP(+)-GAPDH and SBPase in C. W80. Modeling of the tertiary structures of NADP(+)-GAPDH and SBPase suggests that resistance of the enzymes to H(2)O(2) in C. W80 is due to the different conformational structures in the vicinity of the Cys residues of the chloroplastic enzymes between higher plant and C. W80 cells.     

Serine acetyltransferase involved in cysteine biosynthesis from spinach: molecular cloning, characterization and expression analysis of cDNA encoding a plastidic isoform.

A cDNA clone that encodes a chloroplast-localizing isoform of serine acetyltransferase (SATase) (EC 2.3.1.30) was isolated from spinach (Spinacia oleracea L.). The cDNA encodes a polypeptide of 347 amino acids containing a putative transit peptide of ca. 60-70 amino acids at the N-terminal. Deduced amino acid sequence of SATase from spinach exhibited homology with other SATases from plants. DNA blot hybridization analysis showed the presence of 2-3 copies of Sat gene in the genome of spinach. RNA blot hybridization analysis indicated the constitutive expression of Sat gene in green and etiolated seedlings of spinach. Bacterial expression of the cDNA could directly rescue the cysteine auxotrophy of Escherchia coli caused by a lack of SATase locus (cysE). Catalytically active SATase protein was produced in E. coli cells. L-Cysteine, an end product of the cysteine biosynthetic pathway, inhibited the activity of recombinant spinach SATase, indicating the regulatory function of SATase in this metabolic pathway. A chloroplastic localization of this spinach SATase was revealed by the analyses of transgenic plant expressing transit peptide of SATase-beta-glucuronidase (GUS) fusion protein, and transient expression using the transit peptide-green fluorescent protein (GFP) fusion protein. The result from in vitro translation analysis suggests that this cDNA may encode both plastidic and cytosolic SATases.     

Molecular evolution and structure--function relationships of the superoxide dismutase gene families in angiosperms and their relationship to other eukaryotic and prokaryotic superoxide dismutases

This study assesses whether the phylogenetic relationships between SODs from different organisms could assist in elucidating the functional relationships among these enzymes from evolutionarily distinct species. Phylogenetic trees and intron positions were compared to determine the relationships among these enzymes. Alignment of Cu/ZnSOD amino acid sequences indicates high homology among plant sequences, with some features that distinguish chloroplastic from cytosolic Cu/ZnSODs. Among eukaryotes, the plant SODs group together. Alignment of the Mn and FeSOD amino acid sequences indicates a higher degree of homology within the group of MnSODs (>70%) than within FeSODs (approximately 60%). Tree topologies are similar and reflect the taxonomic classification of the corresponding species. Intron number and position in the Cu/Zn Sod genes are highly conserved in plants. Genes encoding cytosolic SODs have seven introns and genes encoding chloroplastic SODs have eight introns, except the chloroplastic maize Sod1, which has seven. In Mn Sod genes the number and position of introns are highly conserved among plant species, but not among nonplant species. The link between the phylogenetic relationships and SOD functions remains unclear. Our findings suggest that the 5' region of these genes played a pivotal role in the evolution of function of these enzymes. Nevertheless, the system of SODs is highly structured and it is critical to understand the physiological differences between the SODs in response to different stresses in order to compare their functions and evolutionary history.     

Isolation of two cDNA clones from tomato containing two different superoxide dismutase sequences

A cDNA library was derived from the poly(A)⁺ RNA of young tomato leaves. The library was cloned in a gt11 system and screened by synthetic oligonucleotide probes having sequences that match the codes of conserved regions of amino acid sequences of Cu,Zn superoxide dismutase (SOD) proteins from a wide range of eukaryotic organisms. Two cDNAs were isolated, cloned and sequenced. One of the cDNAs, P31, had a full-size open reading frame of 456 bp with a deduced amino acid sequence having an 80% homology with the deduced amino acid sequence of the cytosolic SOD-2 cDNA of maize. The other cDNA, T10 (extended by T1), had a 651 bp open reading frame that revealed, upon computer translation, 90% homology to the amino acid sequence of mature spinach chloroplast SOD. The 5 end of the reading frame seems to code for a putative transit peptide. This work thus suggests for the first time an amino acid sequence for the transit peptide of chloroplast SOD. Northern hybridizations indicated that each of the P31 and T10 clones hybridized to a blotted poly(A)⁺ RNA species. These two species are differentially expressed in the plant organs: e.g., the species having the T10 sequence was detected in the leaves but not in roots, while the one with the P31 sequence was expressed in both leaves and roots. The cDNA clones P31 and T10 were also hybridized to Southern blots of endonuclease fragmented tomato DNA. The clones hybridized to specific fragments and no cross hybridization between the two clones was revealed under stringent hybridization conditions; the hybridization pattern indicated that, most probably, only one locus is coding for each of the two mRNA species.     

Non-chloroplast α-1,4-glucan phosphorylase from pea leaves: characterization and in situ localization by indirect immunofluorescence

Leaf extracts of Pisum sativum L. contain three forms of α-1,4-glucan phosphorylase (EC 2.4.1.1) activity. One of these (form I) is located outside the chloroplast; the other two reside inside this organelle (Steup, M. and Latzko, E. (1979) Planta 145, 69–75). The extra-chloroplastic enzyme form, which represents the major proportion of the total extractable phosphorylase activity, was purified and characterized. Its in situ location was determined by indirect immunofluorescence performed with cryostat sections of formaldehyde-fixed leaf. By this technique the enzyme was localized in the cytoplasm of mesophyll and guard cells, whereas the other epidermal cells lacked the enzyme. In its kinetic properties, especially glucan specificity, the enzyme was very similar to the cytosolic phosphorylase from spinach leaves; it has a low affinity towards low-molecular-weight glucans but a very high affinity towards branched polysaccharides such as strach and glycogen. The immunological properties of the enzyme and its peptide pattern were determined and compared with those of other plant phosphorylase. The pea phosphorylase form I was immunologically different from the two chloroplastic phosphorylase forms, and it reacted more strongly with antibodies raised against the spinach cytosolic phosphorylase than with those directed against the spinach chloroplastic counterpart. Peptide patterns obtained after cleavage with N-chlorosuccinimide were very similar for the cytosolic spinach and pea leaf phosphorylase forms, suggesting a high degree of homology between both proteins.     

Chloroplast and cytosolic triosephosphate isomerases from spinach: purification,microsequencing and cDNA cloning of the chloroplast enzyme

Chloroplast and cytosolic triosephosphate isomerases from spinach were separated and purified to homogeneity. Both enzymes were partially sequenced by Edman degradation. Using degenerate primers designed against the amino acid sequences, a homologous probe for the chloroplast enzyme was amplified and used to isolate several full-size cDNA clones. Chloroplast triosephosphate isomerase is encoded by a single gene in spinach. Analysis of the chloroplast cDNA sequence in the context of its homologues from eukaryotes and eubacteria reveals that the gene arose through duplication of its pre-existing nuclear counterpart for the cytosolic enzyme during plant evolution.Abbreviations TPI triosephosphate isomerase - PEG polyethylene glycol - cp plastid - c cytosolic - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - PVP polyvinylpyrrolidone - PCR polymerase chain reaction - PGK 3-phosphoglycerate kinase     

The Arabidopsis thaliana genome encodes at least four thioredoxins m and a new prokaryotic-like thioredoxin

Screening of cDNA libraries at low stringency and complete sequencing of EST clones with homology to thioredoxins allowed us to characterize five new prokaryotic type Arabidopsis thaliana thioredoxins. All present N-terminal extensions with characteristics of transit peptides. Four are clustered in a phylogenetic tree with the chloroplastic thioredoxin m from red and green algae and higher plants, and their transit peptides have typical characteristics of chloroplastic transit peptides. One is clearly divergent and defines a new prokaryotic thioredoxin type that we have named thioredoxin x. Its transit peptide sequence presents characteristics of both chloroplastic and mitochondrial transit peptides. The five corresponding genes are expressed at different levels, but mostly in green tissues and in in-vitro cultivated cells.     

Nucleotide sequence and characterization of a cDNA encoding the acetohydroxy acid isomeroreductase from Arabidopsis thaliana

The primary structure of acetohydroxy acid isomeroreductase from Arabidopsis thaliana was deduced from two overlapping cDNA. The full-length cDNA sequence predicts an amino acid sequence for the protein precursor of 591 residues including a putative transit peptide of 67 amino acids. Comparison of the A. thaliana and spinach acetohydroxy acid isomeroreductases reveals that the sequences are conserved in the mature protein regions, but divergent in the transit peptides and around their putative processing site.     

Extended amino acid sequences around the active-site lysine residue of class-I fructose 1,6-bisphosphate aldolases from rabbit muscle, sturgeon muscle, trout muscle and ox liver.

1. Amino acid sequences covering the region between residues 173 and 248 [adopting the numbering system proposed by Lai, Nakai & Chang (1974) Science 183, 1204-1206] were derived for trout (Salmo trutta) muscle aldolase and for ox liver aldolase. A comparable sequence was derived for residues 180-248 of sturgeon (Acipenser transmontanus) muscle aldolase. The close homology with the rabbit muscle enzyme was used to align the peptides of the other aldolases from which the sequences were derived. The results also allowed a partial sequence for the N-terminal 39 residues for the ox liver enzyme to be deduced. 2. In the light of the strong homology evinced for these enzymes, a re-investigation of the amino acid sequence of rabbit muscle aldolase between residues 181 and 185 was undertaken. This indicated the presence of a hitherto unsuspected -Ile-Val-sequence between residues 181 and 182 and the need to invert the sequence -Glu-Val- to -Val-Glx- at positions 184 and 185. 3. Comparison of the available amino acid sequences of these enzymes suggested an early evolutionary divergence of the genes for muscle and liver aldolases. It was also consistent with other evidence that the central region of the primary structure of these enzymes (which includes the active-site lysine-227) forms part of a conserved folding domain in the protein subunit. 4. Detailed evidence for the amino acid sequences proposed has been deposited as Suy Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.     

Cloning, nucleotide sequence and mutational analysis of the gene encoding the Photosystem II manganese-stabilizing polypeptide of Synechocystis 6803

Summary Affinity purified, polyclonal antibodies raised against the Photosystem II 33 kDa manganese-stabilizing polypeptide of the spinach oxygen-evolving complex were used to isolate the gene encoding the homologous protein from Synechocystis 6803. Comparison of the amino acid sequence deduced from the Synechocystis psb1 nucleotide sequence with recently published sequences of spinach and pea confirms the homology indicated by antigenic crossreactivity and shows that the cyanobacterial and higher plant sequences are 43% identical and 63% conserved. Regions of identity, varying in length from 1 to 10 consecutive residues, are distributed throughout the protein. The 28 residues at the amino terminus of the psb1 gene product, characteristic of prokaryotic signal peptides, show homology with the carboxyl-terminal third of the transit sequences of pea and spinach and are most likely needed for the transport of the manganese-stabilizing protein across the thylakoid membrane to its destination of the lumen. Synechocystis mutants which contain a kanamycin resistance gene cassette inserted into the coding region for the 32 kDa polypeptide were constructed. These mutants contain no detectable 32 kDa polypeptide, do not evolve oxygen, and are incapable of photoautotrophic growth.     

Cloning and DNA sequencing of cytosolic Cu/Zn superoxide dismutase gene from chinese cabbage

A cDNA clone for the cytosolic Cu/Zn superoxide dismutase (Cu/Zn SOD) from Chinese cabbage (Brassica campestris ssp.pekinensis) was isolated and its DNA sequence was determined. The cDNA clone contains a complete coding sequence which encodes a protein of 152 amino acids and a 3-untranslated region including a poly A signal. The deduced amino acid sequence shows that it is highly homologous to the Cu/Zn SODs from other plants (60–90%). The lack of a putative chloroplast targeting transit peptide indicates that the clone represents a cytosolic form of Cu/Zn SOD. Genomic Southern hybridization suggests that cytosolic Cu/Zn SOD genes are present in 1 or 2 copies per genome.     

Presence in the stroma of chloroplasts of a large pool of a ribosomal protein not structurally related to any Escherichia coli ribosomal protein

Summary A search was made for the presence of a pool of free ribosomal proteins in the stroma of the spinach chloroplast. The results showed that a relatively large amount of one protein, CS-S5, is present in the stroma. Immunoprecipitation experiments showed that this protein is encoded by the nuclear genome. Clones were isolated from a cDNA library constructed in the expression vector lambda gtll, using specific antibodies raised against the CS-S5 protein. A full-length cDNA was sequenced which contains an open reading frame (ORF) for the precursor of the CS-S5 protein, as shown by immunoprecipitation. This precursor contains a putative transit peptide of 66 amino acids and the mature product has no significant homology with any of the Escherichia coli ribosomal proteins, in contrast to the other ribosomal protein gene products so far identified in spinach chloroplasts.