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J. Biol. Chem., Vol. 275, Issue 37, 28466-28482, September 15, 2000

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The Plastid Ribosomal Proteins

IDENTIFICATION OF ALL THE PROTEINS IN THE 50 S SUBUNIT OF AN ORGANELLE RIBOSOME (CHLOROPLAST)^*

Kenichi Yamaguchi and Alap R. Subramanian

From the Department of Biochemistry, The University of Arizona, Tucson, Arizona 85712 and the Max-Planck-Institut für Molekulare Genetik, Berlin-Dahlem, Germany 14195

Received for publication, June 9, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have completed identification of all the ribosomal proteins (RPs) in spinach plastid (chloroplast) ribosomal 50 S subunit via a proteomic approach using two-dimensional electrophoresis, electroblotting/protein sequencing, high performance liquid chromatography purification, polymerase chain reaction-based screening of cDNA library/nucleotide sequencing, and mass spectrometry (reversed-phase HPLC coupled to electrospray ionization mass spectrometry and electrospray ionization mass spectrometry). Spinach plastid 50 S subunit comprises 33 proteins, of which 31 are orthologues of Escherichia coli RPs and two are plastid-specific RPs (PSRP-5 and PSRP-6) having no homologues in other types of ribosomes. Orthologues of E. coli L25 and L30 are absent in spinach plastid ribosome. 25 of the plastid 50 S RPs are encoded in the nuclear genome and synthesized on cytosolic ribosomes, whereas eight of the plastid RPs are encoded in the plastid organelle genome and synthesized on plastid ribosomes. Sites for transit peptide cleavages in the cytosolic RP precursors and formyl Met processing in the plastid-synthesized RPs were established. Post-translational modifications were observed in several mature plastid RPs, including multiple forms of L10, L18, L31, and PSRP-5 and N-terminal/internal modifications in L2, L11 and L16. Comparison of the RPs in gradient-purified 70 S ribosome with those in the 30 and 50 S subunits revealed an additional protein, in approximately stoichiometric amount, specific to the 70 S ribosome. It was identified to be plastid ribosome recycling factor. Combining with our recent study of the proteins in plastid 30 S subunit (Yamaguchi, K., von Knoblauch, K., and Subramanian, A. R. (2000) J. Biol. Chem. 275, 28455-28465), we show that spinach plastid ribosome comprises 59 proteins (33 in 50 S subunit and 25 in 30 S subunit and ribosome recycling factor in 70 S), of which 53 are E. coli orthologues and 6 are plastid-specific proteins (PSRP-1 to PSRP-6). We propose the hypothesis that PSRPs were evolved to perform functions unique to plastid translation and its regulation, including protein targeting/translocation to thylakoid membrane via plastid 50 S subunit.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The plastid (chloroplast) ribosome is a plant-specific, organelle ribosome that produces proteins encoded by the plastid genome. Plastid ribosomes are responsible for the synthesis of huge amounts of biomass, since the large subunit of ribulose 1,5-bisphophate carboxylase/oxygenase (a most abundant protein in the biosphere) is synthesized in plastids. Plastid ribosomes are very similar to the eubacterial 70 S-type ribosome, in composition and general mode of function (1-4). The rRNAs and most of the characterized ribosomal proteins (RPs)¹ in plastid ribosomes also bear close resemblance to the corresponding components so far identified in cyanobacteria, a correlation supporting the importance of endosymbiotic theory in plastid evolution (5).

The Escherichia coli ribosome, the most well studied of the eubacterial ribosomes (6), is composed of 21 RPs in the 30 S subunit and 33 RPs in the 50 S subunit. Two more possible E. coli RPs have been suggested: protein Y, the product of E. coli yfia gene (7), bearing a distant sequence homology to a chloroplast-specific RP (PSRP-1), and a protein designated S22 (8). Post-translational modifications are found in many E. coli RPs, although a modification in L16 (Arg⁸¹) remains yet to be characterized (see "Results" for plastid L16). We have recently identified all the RPs in spinach plastid 30 S ribosomal subunit, including all its PSRPs and many post-translational modifications (83). The number of RPs in plastid 50 S subunits has so far only been estimated (~35-39; reviewed in Ref. 2) and has not been determined.

Although the constituents of plastid translational machinery in general are similar to those of E. coli, the genes are distributed in two genome compartments: the plastid and the nucleus. The rRNA and tRNA genes are located in the plastid genome, whereas the genes for processing/modification enzymes, aminoacyl tRNA synthetases, and 60% of the RPs are located in the nuclear genome (1-4). The plastid translation system also differs from the eubacterial system in other significant ways, e.g. chloroplast mRNA is often edited (9); about 60% of chloroplast mRNAs lack canonical ribosome-binding sites found in E. coli mRNAs (10); mRNA levels in chloroplasts remain relatively unchanged through dark/light transitions, whereas protein synthesis rates increase dramatically upon illumination (11, 12); nuclear-coded factors mediate light-regulated translation (13); and nuclear mutants occur with defects in chloroplast polysome assembly (14). Gene expression in chloroplasts depends overwhelmingly on nuclear gene products that mediate both transcription/post-transcriptional processing (15, 16) and translation. We speculated (83) that some of the nuclear factors exert their roles through evolutionary alterations in the plastid ribosome. Because few evolutionary changes are observed in the plastid rRNA, but many are observed in plastid RP (e.g. PSRPs), a key to understanding light-dependent translational regulation might involve chloroplast RPs.

We have applied a proteomic approach (two-dimensional PAGE, HPLC separation, protein sequencing, PCR-based screening/DNA sequencing, LC/MS, and ESI MS) to the plastid ribosomal 50 S subunit to establish a complete identification of all its protein components. Proteins in both 50 S subunits and 70 S ribosomes were identified, yielding an unexpected result that plastid RRF is present in the approximate stoichiometry of one in the 70 S ribosome. Transit peptide cleavage sites in all 25 cytosolically synthesized plastid RP precursors, post-translational modifications in many of the mature PRPs, the absence of the orthologues of two E. coli RPs, and possible function of a 70 S ribosome-bound plastid RRF are discussed.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Preparation of Spinach Chloroplast 70 S Ribosome, 50 S Subunit, TP50, and TP70-- Spinach (Spinacia oleracea cv. Alwaro) ribosomes were prepared as described previously (17). Purification of 70 S ribosomes and the 50 S subunits were done by zonal centrifugation (5000 A₂₆₀ units of ribosome/zonal rotor for 70 S purification and 3000 A₂₆₀ units of purified 70 S/zonal rotor for 50 S isolation) as described previously (18). The approximate conversion factor used for the estimation of protein amount was: 1 A₂₆₀ unit of 70 S or 50 S = 20 µg of protein. TP50 and TP70 (total proteins of 50 S subunit and 70 S ribosome, respectively) were extracted basically according to Hardy et al. (19). Sample preparations for two-dimensional PAGE analysis, HPLC resolution (1 mg of TP50/run), and LC/MS analysis were as described earlier (30 S) (83).

Reversed-phase HPLC-- The HPLC system was assembled with a WellChrom Maxi-Star K-1000 HPLC pump (Knauer), an injection valve (number 7125; Rheodyne), and a UV detector, UV-VIS S-3702 (Soma). Separation of TP50 was performed basically according to Kamp and Wittmann-Liebold (20) using Vydac C18 column (4.6 × 250 mm) and a gradient with 0.1% trifluoroacetic acid and 0.1% trifluoroacetic acid in isopropanol at a flow rate of 0.5 ml/min.

Protein Electrophoresis and Electroblotting-- SDS-PAGE was done by the method of Laemmli (21). Tricine SDS-PAGE was performed according to the method of Schägger and von Jagow (22). Two-dimensional PAGE (basic and acidic) were done as described previously (23, 24). Electroblotting was done according to Walsh et al. (25) using a tank blotting chamber and performed in 25 mM Tris-HCl, pH 8.4, 0.5 mM dithioerythritol, 0.02% SDS at 500 mA for 16 h at 4 °C. PVDF membrane (Sequi-Blot, Bio-Rad) was used for transfer membrane. After electroblotting, the resulting blot was rinsed three times in water for 5 s and stained with 0.1% Amido Black 10B (Sigma) in 50% methanol for 5 min, destained with 50% methanol until the background disappeared, rinsed three times in water, then dried on a Whatman 3MM filter paper, and stored in the dark at 20 °C until used.

Protein Sequencing-- Protein sequencing was carried out at the Laboratory for Protein Sequencing and Analysis (University of Arizona) using Applied Biosystem 477A Protein/Peptide sequencer interfaced with 120A HPLC analyzer to determine phenylthiohydantoin (PTH) amino acids. After the conversion step, 50 µl of the PTH-derivative (of 135 µl or 37%) is injected into an ABI PTH-Narrowbore C18 column (2.1 × 250 mm) for detection of PTH-derivatives (remaining sample going to fraction collector).

PCR Screening for cDNAs of PrpL19, PrpL34, Psrp-5, and Psrp-6-- A gt11 spinach cDNA library prepared in our laboratory previously (26) was screened by thermal gradient PCR using a Mastercycler gradient PCR apparatus (Eppendorf Scientific). Thermal gradient PCR was performed for 3 min at 94 °C, 35 cycles of 1 min at 94 °C, 1 min at 43-60 °C, 1 min 30 s at 72 °C with 0.25 unit of Taq DNA polymerase (Life Technologies, Inc.) in a 20-µl reaction volume containing 1 µl of spinach cDNA library (~10⁸ plaque-forming units), 5 µM gene specific primer or 10 µM degenerate primer, 5 µM  arm primer (PF or PR), 20 µM each dNTP, 1.5 mM MgCl₂, and 50 mM KCl in 20 mM Tris-HCl, pH 8.4.

Plaque Screening of cDNA Library/Cloning of Spinach PrpL5 and PrpL34-- The gt11 spinach cDNA library (26) was screened using random-primed, ³²P-labeled Arabidopsis EST clone (E10B7T7) and a 5'-PRPL34 cDNA portion (PF/PL34R1), respectively, as probes for PrpL5 and PrpL34. Clone E10B7T7 was from Arabidopsis Biological Resource Center (Ohio State University). Radiolabeling was carried out as described by the supplier of Random Primed DNA Labeling Kit (Roche Molecular Biochemicals). 150,000 plaque-forming units were plated on four 132-mm plates, and plaques were lifted onto Nylon filters. Prehybridization was performed in 500 mM sodium phosphate, pH 7.0, at 50 °C (for PrpL5) or 65 °C (for PrpL34) for 2 h, and hybridization was performed in 500 mM sodium phosphate, pH 7.0, 7% SDS at 50 °C for 4 h (for PrpL5) or at 65 °C for 16 h (for PrpL34). The filters were washed twice in 100 mM sodium phosphate, pH 7.0, 1% SDS at 37 °C for 10 min followed by a 10-min wash in 40 mM sodium phosphate, pH 7.0, 1% SDS at 37 °C and autoradiographed. Plaques giving positive signals were purified by two further rounds of screening (27).

Oligonucleotide Primers and DNA Sequencing-- The oligonucleotide primers used in this study were: PF, 5'-CGGGATCCGGTGGCGACGACTCCTGGAGCCCG-3'; PR, 5'-CGGGATCCCAACTGGTAATGGTAGCGACCGGC-3'; PL5F1, 5'-TGGCACTGATTACTGGGCAAAGGC-3'; PL5R1, 5'-GTGTTTTGTTACACGGAATGC-3'; PL5R2, 5'-TACCTTCTCTGACCTTAAACCCTG-3'; PL19F1, 5'-AARGARATHAARGTIGTIGCICAYMG-3'; PL19F2, 5'-CAATGACTTGAATTTCCCTG-3'; PL19F3, 5'-GCCATTGAAGAAGCAATTAG-3'; PL19F4, 5'-GGAGACATTGTGCAAATCAG-3'; PL19R1, 5'-CTTAGATAGTATAGCCTTGCC-3'; PL19R2, 5'-CTGATTTGCACAATGTCTCC-3'; PL19R3, 5'-GCTATCCTCCGCTTCCGACC-3'; PL19R4, 5'-CTAATTGCTTCTTCAATGGC-3'; PL34F1, 5'-GGIAARGCIGCIYTIISIYTIACIAARMG-3'; PL34F2, 5'-GTCATTGGCTCGGACACATG-3'; PL34R1, 5'-GCTCATTCGCAGACGAAAACC-3'; PL34R2, 5'-AGAGCAATGGAGTGACCCGG-3'; PPSRP-5F1, 5'-GGAATTCTAGATATCGTCGACGAGAGATGGCACTCCTTTC-3'; PPSRP-5F2, 5'-GAAGCTAACATCTCAGTTCAG-3'; PPSRP-5R1, 5'-GGAATTCGTCGACGCGTTTTTGAGAAAAAGATTTACACTG-3'; PPSRP-5R2, 5'-GCCTGTTCCTTCGGAGTCTG-3'; PPSRP-6F1, 5'-GGAATTCTAGATATCGTCGACCCTTCCAKAGCAAAATAGAAAAAAAAGAGG-3'; PPSRP-6R1, 5'-CATRTGRTGIGCIGTICCYTTYTTYTG-3'; PPSRP-6R2, 5'-TCATCAAACAGTTCATATGC-3'; PTAG1, 5'-GGAATTCTAGATATCGTC-G-3'; PTAG2, 5'-GGAATTCGTCGACGCG-3'; PT7, 5'-TAATACGACTCACTATAGGG-3'; and PT3, 5'-AATTAACCCTCACTAAAGGG-3'.

PF (forward primer) and PR (reverse primer) are complementary to the cloning site of gt11. Degenerate oligonucleotide primers PL19F1, PL34F1, and PPSRP-6R1 were designed from PRPL19 peptide 1 (sequence region, KEIKVVSHR), N-terminal sequence of PRPL34 (sequence region, GKAALXLTKR), and N-terminal sequence of PSRP-6 (sequence region, QKKGTAHHM), respectively (Table I). Gene-specific PCR primers for PrpL19 and PrpL34 (PL19R1 and PL34R1) were designed from the nucleotide sequences of PCR products PL19F1/PR and PL34F1/PR, respectively, which were amplified using primer sets (PL19F1 and PR) and (PL34F1 and PR). Gene-specific PCR primer for Psrp-6 (PPSRP-6F1) was designed from the nucleotide sequence of PF/PPSRP-6R1 and tagged with PTAG1 sequence. Gene-specific PCR primers for Psrp-5 (PPSRP-5F1 and PPSRP-5R1) were designed from the nucleotide sequence of spinach chloroplast L40 (28) and tagged with PTAG1 and PTAG2 sequences, respectively. Other sequencing primers shown above were designed from obtained DNA sequences during primer walking. PCR products were analyzed by agarose gel electrophoresis using 1% agarose and visualized by ethidium bromide staining. PrpL5 insert DNA in the phage clone (L5F2-1) was amplified by PCR using primer sets PF and PR and sequenced using primers PF, PL5F1, PL5R1, and PL5R2. The nucleotide sequence of PrpL19 was obtained by sequencing PCR products PF/PL19R1 and PL19F1/PR using sequencing primers: PL19F2, PL19F3, PL19F3, PL19F4, PL19R2, PL19R3, and PL19R4. For PrpL34, insert DNA in the phage clone (L34D2-1-1) was amplified by PCR using primer sets PF and PR, then cleaved by EcoRI digestion, and subcloned into the plasmid vector pBluescript SK (Stratagene). The insert DNA in PrpL34 plasmid clone was sequenced using primers, PT3, PL34F2, PT7, and PL34R2. The nucleotide sequence of Psrp-5 was obtained by sequencing PPSRP-5F1/PPSRP-5R1 using sequencing primers: PTAG1, PPSRP-5F2, PTAG2, and PPSRP-5R2. The nucleotide sequence of Psrp-6 was obtained by sequencing PPSRP-6F1/PR using sequencing primers: PTAG1 and PPSRP-6R2. Nucleotide sequences were determined at the DNA Sequencing Facility, University of Arizona, using an Applied Biosystems model 377 sequencer.

View this table: [in this window] [in a new window]   Table I N-terminal and internal peptide sequences of spinach plastid 50 S ribosomal proteins and a 70 S ribosome-specific protein X, amino acid not identified.* +, N-terminal alanine of L2 is -N-monomethylated (30). , 10% N-terminal methionine of L16 is -N-monomethylated; 90% is blocked to Edman reaction. Protein sequences determined in the current study are represented in bold type (other data are published from our laboratory). Prefix PRP is omitted.

Mass Spectrometry-- Mass spectrometry was done at the Mass Spectrometry Facility (Chemistry Department, University of Arizona). ESI MS was carried out on a Finnigan LCQ system. LC/MS was done on the same LCQ system interfaced with a Michrom HPLC system (Magic 2002) using a Microbore C18 column (1 × 150 mm). The solvent system was 0.1% trifluoroacetic acid in 2% acetonitrile (solvent #1) and 0.1% trifluoroacetic acid in 90% acetonitrile (solvent #2). 50 pmol of purified PRP in 10 µl of 4% acetic acid was subjected to ESI MS. For LC/MS, 20 µl of TP50 (100 pmol) in solvent A was injected to the Microbore C18 column.

Internal Peptide Preparation-- "In gel" digestion of PRPL19 was performed basically according to Hellman et al. (29). Five spots were excised from Coomassie Blue-stained two-dimensional gels of TP50 (200 pmol/gel) and equilibrated by mixing for 40 min at 30 °C in 2 ml of 50 mM Tris-HCl, pH 8.5, in 50% acetonitrile. The gel pieces were completely dried in a Speed-Vac and then rehydrated with 150 µl of 50 mM Tris-HCl, pH 8.5, 0.02% polyoxyethylenesorbitan monolaurate (Tween 20), 10% acetonitrile containing 0.4 µg (~ enzyme/substrate ratio by weight) endoproteinase Lys-C (Sigma) and incubated at 30 °C for 16 h. The enzyme reaction was stopped by adding reaction volume of 10% trifluoroacetic acid. Gel pieces were transferred into 500 µl of 0.1% trifluoroacetic acid in 60% acetonitrile, and peptides were extracted by shaking at 30 °C for 80 min. The extract was dried in a Speed-Vac and dissolved in 50 µl of 5% acetic acid, and peptides were purified by reversed-phase HPLC using a Vydac C8 (4.6 × 50 mm) column in trifluoroacetic acid-acetonitrile solvent system. The purified peptides (PRPL19 peptides 1 and 2, see Table I) were dried, dissolved in 25 µl of 30% acetic acid, and subjected to protein sequencing.

To obtain internal peptides of RRF (the 70 S ribosome-specific protein, identified later as plastid ribosome recycling factor), it was purified from pool 37 from a previous work (30). Pool 37 showed two components, identified ~60% RRF and ~40% PSRP-2, by two-dimensional PAGE analysis. RRF was purified using a Vydac C4 (4.6 × 150 mm) column in trifluoroacetic acid-acetonitrile solvent system. The purified protein (~20 µg) was digested using 0.2 µg of endoproteinase Asp-N (Sigma) in 50 mM Tris-HCl, pH 8.0, 2 M urea, at 37 °C for 16 h. The digest was dried in a Speed-Vac and then subjected to Tricine SDS-PAGE. Peptides separated on the gel were electroblotted onto a PVDF membrane and stained as described. A 3.5-kDa peptide band was excised from the blot and sequenced (RRF peptide 1 shown in Table I).

Small Subunit of RuBisCo-- Spinach SSU (small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase) was present in a pool from previous work, pool 40 (30). It was purified by reversed-phase HPLC using a Vydac C8 (4.6 × 50 mm) column in trifluoroacetic acid-acetonitrile solvent system, and also by two-dimensional PAGE/electroblotting. The N-terminal sequence of the purified protein (12 cycles), methyl-MKVWPTQNMKRY, confirmed its identity as spinach SSU.

Computer Analyses-- The program BLAST from the National Center for Biotechnology Information was used for sequence searches. Protein sequence searches were performed using Blastp program versus nr (nonredundant data base of GenBank^TM CDS translations: PDB, Swiss-Prot, PIR, and PRF). EST searches were done using Tblastn program versus dbEST (nonredundant data base of GenBank^TM, EMBL, DDBJ EST Divisions). ORFs from cDNA sequences were analyzed using the Map program from GCG software (31). Isoelectric points and sequence masses were calculated using Peptidesort program (31). Sequence alignments and comparisons were performed using Pileup and Gap programs (31). LC/MS data were analyzed using the ThermoQuest Finnigan Xcalibur data system. Masses were deconvoluted from resulted m/z using the BIOMASS Deconvolution program, and charge states were convoluted using the BIOMASS Calculation program.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Proteins of Chloroplast Ribosomal 50 S Subunit-- To identify all the PRPs in pure spinach chloroplast 50 S ribosomal subunit, spinach chloroplast 70 S ribosomes were first purified on a zonal gradient and then run through a second dissociating zonal gradient to obtain 30 and 50 S subunits. Efficient dissociation of chloroplast ribosomes required the development of a phosphate-containing dissociation buffer (see Ref. 18 for details and gradient profiles). TP50 was extracted from 50 S subunits, and the proteins were separated by two-dimensional PAGE and transferred onto PVDF membrane. Fig. 1A shows such a membrane, stained with Amido Black. The individual spots have a slightly different staining pattern with Amido Black as compared with staining with Coomassie Blue, e.g. acidic proteins like L12 are stained poorly by Amido Black (compare Fig. 1A with Fig. 8A). Each of the spots was excised and subjected to N-terminal sequencing. Because several of the protein spots were composite-looking, indicating overlapping separations (Fig. 1A), the experiment was repeated, with the spots excised into two or three segments for N-terminal analysis. The sequence data from all these experiments are summarized in Table I.

View larger version (68K): [in this window] [in a new window]   Fig. 1.   Two-dimensional PAGE pattern of spinach plastid 50 S ribosomal subunit proteins and protein spot identification. A, Amido Black-stained electroblot (PVDF membrane) of TP50 (200 pmol) separated by two-dimensional PAGE, as described in Ref. 23. The first dimension was at pH 5.0 in 8 M urea, and the second dimension was at pH 6.7 in 0.2% SDS. Amido Black stains basic proteins well but acidic proteins like L12 poorly (compare with Fig. 8). B, schematic diagram of the spots in A with protein identification (see "Discussion" for RP nomenclature); prefix PRP is omitted in B. Some of the minor spots in the high molecular mass portion of the electropherogram were identified as: L2 dimer (a), L5 dimer (b), L20 dimer (c), L16 dimer (d), L1 fragment (e; see "Discussion"), minor form of L5 (f; seen only in some gels), L28 dimer (g), L34 dimer (h), and not identified (i; yielded no N-terminal sequence and no HPLC/MS data). Spot of PSRP-5 was unusually elongated; L36 and PSRP-5 / forms were not visible on the two-dimensional PAGE but were resolved by HPLC (Fig. 2) and identified. See "Results" for the details of identification strategy.

Most of the N-terminal sequences were sufficient to allow protein identification from similarity using BLAST search and, in several cases, from the identity to reported spinach PRP sequences in data bases. In a few instances, however, the N-terminal data were insufficient to provide a positive identification. In those cases, additional experiments were done to obtain internal peptide sequences and/or to isolate cDNAs from a spinach cDNA library for nucleotide sequencing. PRPs are designated in Table I as L1, L2,  ... L36, as per their sequence similarity to the corresponding E. coli ribosomal proteins. The two-dimensional PAGE pattern is diagramatically represented in Fig. 1B, depicting the positions of all the identified proteins. The designations , ,  ... , PSRP-5, PSRP-6, etc., are discussed later.

To confirm whether all of spinach chloroplast 50 S subunit PRPs appeared in the two-dimensional PAGE pattern shown in Fig. 1, two additional experiments were performed. The two-dimensional system used for Fig. 1 resolves mainly proteins of pI 4.5 or greater (23), as acidic proteins of lower pI do not migrate into the first dimension gel. We therefore ran TP50 in another two-dimensional system suitable for the resolution of acidic proteins (pI 5.5 or lower; Ref. 24). A few spots appeared in this two-dimensional PAGE, but N-terminal sequencing did not reveal any new protein sequence (data not shown). A second problem arises if an N-blocked protein comigrates with an unblocked PRP. A single spot would then appear, giving a single N-terminal sequence under Edman degradation, suggesting that the spot contained one protein. To overcome this problem, we resolved the 50 S subunit PRPs (TP50) on a reversed-phase HPLC column, where hydrophobic interaction (rather than the net charge or peptide chain length) is the key to resolution. HPLC could thus separate proteins that are often unresolved by two-dimensional PAGE. Fig. 2 shows the separation of plastid TP50 obtained by this procedure. Proteins in the individual peaks were identified by two-dimensional PAGE (co-electrophoresis) analysis. The HPLC experiment allowed clean separations of several proteins that were not well resolved by two-dimensional PAGE, e.g. L4/L21, L3/L13, and L20/PSRP-5 form (compare Figs. 1 and 2). Indeed, plastid L36 and the / forms of PSRP-5 did not at all show up on two-dimensional PAGE but gave distinct peaks in the HPLC run, permitting both N-terminal sequence and mass (by LC/MS) determination. Because these three small proteins are very basic, they might have migrated out of the two-dimensional gel. Interestingly, a few proteins on the other hand, L35, L33, L32, L27, and PSRP-5 (-), were each eluted in two different peaks (Fig. 2). Their identities were inferred from two-dimensional PAGE analysis, N-terminal sequencing, as well as mass determinations (ESI MS). This observation could suggest that some plastid RPs may possibly exist as two distinct conformers on the plastid ribosome.

View larger version (40K): [in this window] [in a new window]   Fig. 2.   Resolution of spinach plastid TP50 by reversed-phase HPLC and identification of each eluted PRP by two-dimensional PAGE. 1 mg of TP50 was resolved on a Vydac C18 column (4.6 × 250 mm) using a step linear gradient of solvent 1 (0.1% trifluoroacetic acid) and solvent 2 (0.1% trifluoroacetic acid in isopropanol). The program was 90% solvent 1/10% solvent 2 from 0-10 min, 75% solvent 1/25% solvent 2 at 70 min, 54% solvent 1/46% solvent 2 at 250 min, followed by a washing step, 20% solvent 1/80% solvent 2 at 270 min, at constant flow rate of 0.5 ml/min (fraction size, 375 µl). Two-dimensional gel patterns for three pools (L15/L17, L3, and L12) and their co-electrophoresis (left panels, PRP pool; right panels, PRP pool + TP50) are shown beneath the HPLC profile. Note that PRPs L35, L33, L32, L27, and PSRP-5 - forms were each eluted in two different peaks. ESI MS analysis of the pools of the distinct peaks showed the same protein mass (see "Results").

The average yield of PTH-derivative recovered in the first three cycles of Edman degradation was calculated for each of the sequence runs and is summarized in Fig. 3. Most proteins showed in yields ranging from 20 to 50 pmol, indicating that this amount approximates the average stoichiometry (1 copy/50 S subunit). Plastid L12 gave a yield of almost 200 pmol, four times the highest yield for the other proteins recovered in good yield. The result supports the existence of four copies of L12/plastid 50 S ribosomal subunit, as has been previously deduced (17). Several of the plastid 50 S subunit proteins, e.g. L10, L18, L31, and PSRP-5 exist in multiple forms (named  to , Figs. 1 and 2 and Table I). The summed N-terminal yield of the multiple forms in each of these cases corresponded to the approximate stoichiometric amount for the other PRPs (Fig. 3). Very low yields of N-terminal amino acids were observed in three instances: L16, L22, and L34. As discussed later, 90% of PRPL16 is likely blocked by trimethylation; most of PRPL22 may be N-blocked by an unknown group; and PRPL34, one of the most basic proteins of the 50 S subunit (like L36), partially runs out of the two-dimensional gel.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Yields of N-terminal amino acids from 50 S PRP sequencer runs. 200 pmol of TP50 or TP70 were subjected to two-dimensional PAGE and electroblotted onto PVDF membrane, and individual spots were subjected to N-terminal protein sequencing. Yield was calculated from the average PTH-derivative recovery for the first three sequence cycles for each spot. The average apparent yield for all the 50 S PRPs (L12 was counted as four copies; see text) is 27.5 pmol/protein The actual recovery of PTH-derivative is 74 pmol (37%), because only 50 µl of 135 µl is injected for PTH-derivative identification. PRPs L10, L18, L31, and PSRP-5 exist in multiple forms and are indicated in the inset. Plastid RRF is found to be a 70 S ribosome-specific protein (this work; see text).

The N-terminal sequence data in Table I allowed the identification of the orthologues of 28 E. coli RPs in spinach plastid ribosome, beside two plastid specific proteins, PSRP-5 and PSRP-6. E. coli ribosomal 50 S subunit contains (making three subtractions for L7, -N-acetylated form of L12; L8, a complex of L7/L12 and L10, and L26 = S20) 33 canonical RPs: L1 to L36 (32). Hence our data so far have not allowed positive identifications for five possible E. coli orthologues. These are: L5, L19, L25, L30, and L34, of which three (L5, L19, and L34) were identified with additional sequence data obtained (using the protein sequence information) by screening a spinach cDNA library.

The N-terminal sequence of the protein, later identified as PRPL5 (25 amino acids), did not show significant homology to any data base proteins (BLAST search using the Blastp program), but the same data when used to screen the EST data bank with the Tblastn program gave two matches with Arabidopsis EST clones. One of these, E10B7T7, was obtained from the Arabidopsis Biological Resource Center (Ohio State University) and used as probe to screen our spinach gt11 cDNA library (26). Several positive clones were isolated and the longest cDNA clone (L5F2-1) was sequenced (data deposited in GenBank^TM, accession number AF250923). Clone L5F2-1 encodes 207 amino acid residues, which is 13 residues short of the complete sequence of mature PRPL5. The 25-residue N-terminal sequence (Table I), however, provided this missing sequence as well as a 12-residue overlap with the cDNA-derived sequence. The sequence comparisons of spinach PRPL5 with its homologues in a higher plant (A. thaliana), an alga (Porphyra purpurea), a photosynthetic bacterium (Synechocystis PCC6803), and E. coli are shown in Fig. 4.

View larger version (77K): [in this window] [in a new window]   Fig. 4.   Alignments of five spinach plastid 50 S subunit protein sequences with homologous sequences from eubacteria, algae, and land plants. The spinach plastid sequence (derived from cDNA, see "Results") is at top. Black arrows indicate cleavage sites for removing the transit peptide; gray arrows indicate sites that are either alternative transit peptide cleavage site or processing points after PRP is imported into the plastid. Tilde and dot indicate blanks and gaps, respectively. Underlined text shows protein sequences determined experimentally (Table I). The chain length of mature protein (that of precursor in parentheses), and percentages of identity (I) and similarity (S) are shown after C terminus. PSRP-5 data: Sol1, PSRP-5 sequenced in this work; Sol2, sequence reported as spinach L40 (28): Psa, pea sequence reported as PsCL18 (33). PSRP-6 data: Sol, PSRP-6 sequenced in this work; Psa, pea sequence reported as PsCL25 (33). Sol, S. oleracea; Ath, Arabidopsis thaliana; Ppu, Porphyra purpurea (alga); Syn, Synechocystis PCC6803 (cyanobacterium); Eco, E. coli; Psa, Pisum sativum (pea). New accession numbers from this work are: Sol L5, AF250923; Sol L19, AF250384; Sol L34, AF238221; Sol PSRP-5, AF261940; and Sol PSRP-6, AF245292.

Spinach PRPL5 contains a 16-amino acid-long NTE and a positively charged 26-amino acid long CTE, as compared with E. coli L5. The plastid protein has amino acid identity of 72.5% to the A. thaliana L5 and 56.4%, 60.0%, and 48.6%, respectively to the L5 proteins of P. purpurea, Synechocystis PCC6803, and E. coli. The amino acid sequences of the NTE and CTE of plastid L5 yielded no significant matches when searched against protein data bases.

The N-terminal and internal sequences of plastid L19 and the N-terminal sequence of plastid L34 did not show significant similarity to any proteins in data bases. We therefore screened the spinach gt11 cDNA library (26) using inosine-containing degenerate oligonucleotide primers designed from PRPL19 peptide 1 and the N-terminal sequence of PRPL34. Thermal gradient PCR allowed us to find the optimal amplification conditions using primer sets of degenerate primers and  arm primers (PF or PR). Amplified PCR products (PL19F1/PR and PL34F1/PR) were sequenced, and further PCR amplifications were done using sets of PF and gene specific primers (based on the obtained DNA sequence). The nucleotide sequences of PRPL19 cDNA was thus obtained entirely by sequencing of PCR products (submitted to GenBank^TM, accession number AF 250384).

As compared with E. coli L19, spinach plastid L19 contains a negatively charged, 47-amino acid-long NTE. However, the NTE showed a remarkably low similarity to the corresponding region in the two L19-like sequences in the Arabidopsis data base (Ath1 and Ath2; Fig. 4) and showed no significant matches against the sequences in protein data bases. Overall, spinach PRPL19 showed amino acid identities of only 50.4 and 47.7% to the two Arabidopsis sequences (genes F3I6.17 and AT4 g11630) and 38.5, 42.7, and 37.3%, respectively, to the L19 protein sequences from P. purpurea, Synechocystis PCC6803, and E. coli.

For PRPL34, a PCR product encoding the 5'-region of its cDNA (PF/PL34R) was first obtained and was used as probe to screen the spinach cDNA library. Several clones were isolated, and the one containing the longest cDNA was subcloned into a plasmid vector and was sequenced. The nucleotide sequence is submitted to GenBank^TM (accession number AF238221). As compared with E. coli L34, plastid L34 contains a 7-residue-long NTE and a 10-residue-long CTE. These extensions are absent in both cyanobacterial and algal L34 proteins. Core sequences between PRPL34 and the L34 proteins from P. purpurea, Synechocystis PCC6803, and E. coli are relatively well conserved (Fig. 4), the percentage identities being, 45.5, 41.9, and 50.0%, respectively.

Including the three proteins identified via DNA work, a total of 31 orthologues of E. coli RPs are present in spinach plastid ribosome. There are thus two E. coli RPs (L25 and L30; see "Discussion"), for which we could obtain no evidence of occurrence in spinach plastid ribosome.

Plastid-specific Ribosomal Proteins in Chloroplast 50 S Subunit-- One of the additional N-terminal sequences in Table I (PSRP-5) corresponded to the reported sequence of spinach plastid L40 (28), which is the homologue of an earlier reported protein from pea, named PsCL18 (33). Another N-terminal sequence we determined (PSRP-6, Table I) showed similarity to pea PsCL25 (33); spinach homologue of PsCL25 has not been reported. We screened our spinach cDNA library (26) for the cDNAs corresponding to these two proteins, and the clones obtained were sequenced. The proteins were designated PSRP-5 (previous L40) and PSRP-6 (homologue of pea PsCL25), in accordance with our proposed nomenclature (see "Discussion"). Nucleotide sequences of PSRP-5 and PSRP-6 cDNA are deposited in GenBank^TM (accession numbers AF261940 and AF245292, respectively). Our PSRP-5 data showed 6 amino acids differences, all at the C-terminal portion, from the reported L40 data (28), leaving it uncertain at this point whether the differences reflect spinach cultivar differences (cv. Alwaro versus Géant d'hiver) or sequencing errors. Three post-translationally modified forms of PSRP-5 were identified from our protein sequencing results (Table I), and the modifications were in the N-terminal portion of the protein (see "Discussion"). Both PSRP-5 and PSRP-6 are unique to the plastid ribosome, homologous sequences being not found in the RPs of E. coli, archaebacteria, yeast (cytosolic or mitochondrial), or mammals.

Mass Spectrometry of 50 S PRPs-- Spinach TP50 was analyzed by LC/MS. Fig. 5A shows the relative abundance of summed mass/charge ratio (m/z) of the PRPs in the m/z range of 400-2000 versus HPLC elution time. An example of a mass spectrum at the elution interval of 41.5-42.0 min (summed scans for 30 s) is shown in Fig. 5B. Protein mass was calculated by deconvolution of the m/z series. For example, from deconvolution of the m/z series in Fig. 5B, a major mass of 13,811.0 Da and a minor mass 13850.2 Da were derived (Fig. 5C). Because the observed mass 13,811.0 Da is very close to the sequence mass of PRPL12, i.e. 13,814.54 Da, this peak was identified as of plastid L12. In cases where sequence mass were not available, ESI MS of HPLC pools, containing proteins identified by two-dimensional PAGE (shown in Fig. 2), allowed mass identification. Every 30-s interval was analyzed as stated above, and the resultant mass values are summarized in Table II. The combined LC/MS analysis of TP50 and ESI MS analysis of HPLC pools allowed the identification of most individual 50 S PRP masses, except for PRPs L2, L20, the multiple forms of L10, L18, L31, and PSRP-6. An extreme case was PRPL2, which was not detected either by LC/MS of TP50 or ESI MS of HPLC pools; it probably represents a very poorly ionizing polypeptide.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   LC/MS analysis of spinach chloroplast 50 S subunit proteins. A, relative abundance (m/z) of PRPs from the range of m/z 400-2000 versus HPLC elution time. TP50 (100 pmol) was loaded on a Microbore C18 (1 × 150 mm) column and resolved by a step linear gradient of solvent 1 (0.1% trifluoroacetic acid in 2% acetonitrile) and solvent 2 (0.1% trifluoroacetic acid in 90% acetonitrile): 95% solvent 1/5% solvent 2 at 0 min, 65% solvent 1/35% solvent 2 at 22 min, 64% solvent 1/36% solvent 2 at 23 min, 35% solvent 1/65% solvent 2 at 55 min, followed by a washing step, 5% solvent 1/95% solvent 2 at 56 min, at constant flow rate of 50 µl/min. The ion series of a protein discussed in B is indicated by arrow. B, an example of mass spectrum for elution interval 41.5-42.0 min. Peaks represent individual charged ions. Convoluted charge and m/z values are indicated above each peak. C, deconvoluted mass spectrum of the m/z series in B. It indicates a major protein of mass 13811.0 Da (PRPL12) and a minor form, 13850.2 Da (see text).

Edman Degradation of PRPL16-- The plastid located prpL16 gene codes for the N-terminal sequence, formyl Met-Leu-Ser- (34), whereas our N-terminal sequence analysis of PRPL16 yielded Xaa-Leu-Ser- (where Xaa is a modified, unidentified amino acid). The N terminus of E. coli L16 is -N-monomethylated (35), and so we suspected the same modification in plastid L16. To test this hypothesis, we purified spinach SSU (small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase), which has monomethylated methionine at the N terminus (36). About 200 pmol each of SSU HPLC (SSU purified by HPLC), SSU two-dimensional/blot (SSU purified using two-dimensional PAGE/electroblotting), and PRPL16 two-dimensional/blot were subjected to N-terminal analysis (Fig. 6). The SSU HPLC yielded PTH-monomethyl methionine (retention time 26.37 min, appearing between PTH-tryptophan and PTH-phenylalanine), whereas SSU two-dimensional/blot showed an unusual PTH-derivative with a retention time of 10.30 min, just after the N'N-dimethyl-N'-phenylthiourea peak (peak a; retention time, 10.25 min). Alkylation of monomethyl methionine with acrylamide under mildly alkaline conditions (during two-dimensional PAGE and/or electroblotting), just as the similar modification of cysteine during the same procedure (37), could produce the latter (uncharacterized) compound. PRPL16 two-dimensional/blot showed the same unusual PTH-derivative with the same retention time in cycle 1. In cycle 2, PRPL16 two-dimensional/blot gave the expected residue, PTH-Leu, but the yield (2.7 pmol) was only about one-tenth the expected amount (the average yield of N-terminal PTH-derivative of 50 S PRPs was 27.5 pmol; Fig. 3), whereas the two SSU preparations gave the normal yield of PTH-Lys (Fig. 6). We therefore infer that post-translationally about 10% of PRPL16 is -N-monomethylated, whereas 90% of it is blocked to Edman degradation, probably by trimethylation (see "Discussion").

View larger version (30K): [in this window] [in a new window]   Fig. 6.   Evidence for -N-monomethyl methionine in plastid L16. Samples of L16 and SSU (small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase), 200 pmol each, were subjected to N-terminal analysis. Results from the first two cycles are shown. NMM, PTH--N-monomethyl methionine; NMM-deriv., uncharacterized PTH--N-monomethyl methionine derivative; a, N',N-dimethyl-N'-phenylthiourea; b, N',N-diphenylthiourea. Minor amounts of PTH-Ala (L27) in cycle 1 and PTH-His (L27) in cycle 2 of PRPL16 sequencer run were from L27 contamination (see Fig. 1). SSU HPLC and SSU two-dimensional/blot and L16 two-dimensional/blot stand for SSU purified by HPLC, SSU purified by two-dimensional PAGE/blotting, and L16 purified by two-dimensional PAGE/blotting, respectively (L16 could not be purified by HPLC, see Fig. 2).

Identification of a 70 S Ribosome-specific Protein-- During the course of this work we realized that a prominent protein spot, always present in TP70 gels, was absent in either TP30 or TP50 gels (Fig. 7). To investigate further, a TP70 blot was prepared, and this spot and several other spots (from both 30 and 50 S proteins) were excised and subjected to N-terminal sequence analysis. The analysis yielded a single N-terminal sequence, specific to a protein found only on plastid 70 S ribosome, the spot indicated by an arrow in Fig. 7C. The N-terminal sequence of 21 residues (Table I), however, did not show homology to any of the reported proteins in data bases. Therefore, the 70 S ribosome-specific protein was purified from a previously fractionated spinach chloroplast ribosomal protein pool, number 37 (30), and an internal peptide was obtained by endoproteinase Asp-N digestion. At the time we sequenced the internal peptide, the nucleotide sequence of spinach chloroplast RRF was reported (38). Our N-terminal and internal sequences matched 100% with the corresponding sequences of RRF (N-terminal positions 1-21 and internal positions 116-139). Thus, the 70 S ribosome-specific protein we identified in this work is plastid RRF. In contrast to the plastid situation, E. coli RRF is mostly found in post-ribosomal supernatant (see "Discussion").

View larger version (86K): [in this window] [in a new window]   Fig. 7.   A protein is identified as present in plastid 70 S ribosome but absent in either 30 or 50 S subunits. A portion of Coomassie Blue-stained two-dimensional PAGE patterns of TP30, TP50 and TP70 are shown. The arrow indicates a protein spot (marked RRF) in TP70, and its absence in the two-dimensional gels of TP30 and TP50. PRPs S1 and L1 are shown for orientation. The N-terminal and an internal peptide sequences of the 70 S-specific protein were determined that permitted its identification.

We have previously identified all the proteins in spinach chloroplast 30 S subunit, a total of 25 proteins: 21 E. coli orthologues and 4 PSRPs (83). Here we have identified all the proteins in spinach chloroplast 50 S subunit, a total of 33 proteins: 31 E. coli orthologues and 2 PSRPs. In addition, a protein found only on plastid 70 S ribosome was characterized, and this 70 S-specific protein was identified as plastid RRF. To round off the picture, a two-dimensional PAGE of TP70 was done (Fig. 8A), and all the spots were cut out and analyzed. The protein identifications confirmed the results from the 30 and 50 S subunit experiments and, as diagramatically represented in Fig. 8B, revealed no additional proteins. It should be mentioned that PRPL36 and two of the forms of PSRP-5 ( and ) did not show up in the two-dimensional PAGE patterns but were identified with the help of HPLC (Fig. 2). As we noted in the 30 S identification paper, the diffusely staining minor spots visible in the upper (high molecular) part of the two-dimensional PAGE (Figs. 1A and 8A) represent mainly minor aggregates of a few PRPs exhibiting tendency to polymerize (see legend to Fig. 1) and small amounts of nonribosomal proteins that probably have functional associations with ribosomes.

View larger version (50K): [in this window] [in a new window]   Fig. 8.   Plastid ribosomal proteins of spinach chloroplast 70 S ribosome separated by two-dimensional PAGE. A, Coomassie Blue-stained electropherogram of spinach chloroplast 70 S RPs. TP70 (200 pmol) was subjected to two-dimensional PAGE as described (Ref. 23; see legend to Fig. 1 for details). B, schematic diagram of the spots in A with protein identification. 30 S PRPs are shown in light gray, and 50 S PRPs are in dark gray. The 70 S ribosome-specific protein is shown in black. Plastid-specific ribosomal proteins (PSRPs) are written in bold type. Proteins L36 and the / forms of PSRP-5 were not seen in the two-dimensional PAGE; they were isolated and identified by HPLC resolution (Fig. 2).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this paper we present sufficient protein and nucleic acid sequence data to establish the identification of all the protein components in the 50 S subunit of a land plant chloroplast ribosome. Similar results for chloroplast 30 S subunit are in the accompanying article (83). Taken together these two publications present the first complete identification of the protein components of an organelle (plastid) ribosome, complementing the previously reported such complete identifications for E. coli (6), and the cytosolic ribosomes of yeast (39) and mammals (40). Recent reports indicate that a complete protein identification for a mitochondrial ribosome may soon be forthcoming (41-43).

In the companion 30 S subunit paper we presented a nomenclature for plastid RPs and their genes that accords with both the current usage for mitochondrial RPs and the Commission on Plant Gene Nomenclature rules for plant gene names. In brief, chloroplast RPs are designated PRPs, with gene names written in italics, but having the first letter in capital for the RP genes that are located in the nuclear genome and the first letter in lowercase for the RP genes that are located in the plastid genome. Thus the plastid homologue of E. coli L1 is designated PRPL1, and its nucleus-located gene is designated PrpL1, whereas the plastid homologue of E. coli L2 is designated PRPL2, and its plastid-located gene is designated prpL2. As described in the companion paper, chloroplast ribosomes contain proteins that do not have homologues in E. coli, archaebacteria, or in yeast/mammalian cytosolic ribosomes. These proteins are designated PSRP-1 etc (plastid-specific RP), and their genes, which are all located in the nuclear genome, are designated Psrp-1, etc.

Proteins of Plastid Ribosomal 50 S Subunit-- Spinach chloroplast 50 S ribosomal subunit contains 33 proteins, the same number as in E. coli 50 S ribosomal subunit. However, only 31 of these proteins are orthologues of corresponding E. coli RPs, whereas the remaining two are plastid specific ribosomal proteins (PSRPs: Figs. 1B and 3 and Table I). The 31 orthologues of E. coli 50 S RPs are designated (PRP): L1-L6, L9-L24, L27-L29, and L31-L36 (E. coli designations L7, L8, and L26 do not stand for distinct RPs). Thus two E. coli 50 S RPs, L25 and L30, do not have orthologues (or homologues) in the plastid 50 S subunit. Because plastid 30 S subunit contains the orthologues of all of the 21 E. coli 30 S RPs (83), it follows that spinach plastid ribosome has maintained all E. coli RPs except L25 and L30.

The two plastid-specific RPs that are identified in spinach plastid 50 S subunit were first reported in pea, derived from cDNA sequences and named PsCL18 and PsCL25 (33). Based on the present comprehensive protein study, we can state that no additional PSRPs are present in spinach chloroplast 50 S subunit (Figs. 1-3 and Table I). The companion comprehensive study on plastid 30 S subunit (83), has revealed four PSRPs, (designated PSRP-1 to PSRP-4) in the 30 S subunit. The plastid ribosome thus maintains six plastid-specific proteins: four of them in the 30 S subunit and two in the 50 S subunit. The two PSRPs in the 50 S subunit are designated PSRP-5 and PSRP-6.

Sequence similarity between a barley plastid ribosomal protein (BPRL28) and PsCL25 (PSRP-6) has been recently noted (44). Interestingly, despite being specific to plastids, the sequence identities among PSRP-5 and PSRP-6 homologues are relatively low, e.g. only 53.8 and 54.0%, respectively, between the spinach and pea proteins (Fig. 4). There was no significant sequence similarity between PSRP-5 or PSRP-6 and any of the bacterial proteins in data bases, suggesting the appearance of these two proteins in plants after the endosymbiotic event in plastid evolution. In contrast, all four 30 S subunit PSRPs show some sequence homology to eubacterial proteins: PSRP-1, PSRP-2, and PSRP-3 to cyanobacterial proteins and PSRP-4 to a protein only reported from Thermus thermophilus,² suggesting their evolution from ancestral eubacterial genes.

Post-translational Processing: Plastid 50 S RPs Encoded in the Organelle Genome-- The genes encoding eight of the spinach plastid 50 S subunit PRPs are maintained in the plastid genome. They are synthesized on the plastid ribosome with initiating formyl-Met tRNA. The post-translational, N-terminal formyl-Met processing undergone by these PRPs are shown in Table III. Five of the proteins (L2, L20, L22, L32, and L33) have the entire formyl-Met group excised, whereas three (L14, L16, and L36) have only the formyl group removed, leaving methionine at the N terminus. The N-terminal alanine of L2 is monomethylated, as reported earlier (Ref. 30; first description of post-translational modification in a plastid RP). The modifications in plastid L16 are discussed later (see below). We had previously suspected N-terminal modification in plastid L36 (45), but the present study revealed unmodified N-terminal methionine. The MS mass results for the plastid encoded L14, L32, and L33 accorded with their calculated sequence molar masses (Table II), indicating no further post-translational modifications in these PRPs.

Post-translational Processing: Plastid 50 S RPs Encoded in the Nuclear Genome-- Twenty-five 50 S subunit PRPs, orthologues of 23 E. coli RPs and the two PSRPs, are encoded in the nuclear genome and are thus synthesized on the cytosolic ribosomes as precursors. All plastid RP precursor molecules contain an extra, routing peptide sequence (transit peptide), which is cleaved off upon entry of the mature PRP into the plastid. Table III lists the immediate post-cleavage N-terminal sequences for all of these 25 PRPs. Their pre-cleavage peptide sequences are also listed in Table III, where they are known (for 13 PRPs). Remarkably, a consensus flanking peptide sequence that could specify the cleavage site for the transit peptidase enzyme was not discernible in these data.

Only a minority of 50 S subunit RP genes are located in the plastid DNA (8 of 33, or 24%), the majority being in the nuclear genome. For the 30 S subunit, the RP genes are about equally distributed between the plastid genome and the nuclear genome. As pointed out in a previous review (3), this may reflect a co-evolutionary linkage, arising from the main function of the 30 S subunit, i.e. formation of 30 S initiation complex with mRNA transcribed in the plastid. A correlation between the PRPs that are synthesized on the plastid ribosome and the early steps of ribosome assembly (plastid rRNA is transcribed in the plastid) has been suggested, but it is applicable for the 30 S subunit and not for the 50 S subunit.

Post-translational Modifications in Mature 50 S PRPs-- The post-translational modifications of individual plastid 50 S PRPs, as derived from this study, are discussed below.

PRPL1 showed a minor fragment (spot e in Fig. 1) with N-terminal truncation of 23-amino acids, i.e. starting with the N-terminal sequence, TLPSPTKPKKGKAAL, positions 24-38 of PRPL1 sequence (GenBank^TM accession number X76932; Ref. 46).³ Because this fragment was identified as present only in TP50 preparations but not in TP70 (compare Figs. 1 and 8), its significance is at present unclear.

Spinach PRPL2 gene sequence has been reported (accession number X00797; Ref. 47), but the C-terminal portion of the deduced amino acid sequence lacked homology to bacterial and reported chloroplast L2 sequences in data bases. We have therefore resequenced the rpL2 gene from spinach chloroplast DNA, and our data (GenBank^TM accession number AJ244023) confirm a suspected frameshift from a sequencing mistake in the earlier submission. The corrected spinach PRPL2 amino acid sequence shows a conserved C-terminal portion, with the expected homology. Mature spinach plastid L2 is post-translationally modified, with -N-monomethyl alanine at the N terminus (30).

In previous work using a different cultivar of spinach, the spot of PRPL4 had appeared in a different, slightly more basic two-dimensional PAGE position (Fig. 1 in Ref. 2), whereas all the other 50 S and 30 S PRPs showed the same positions as in this study. Thus PRPL4 may be one of the few plastid RPs that harbor detectable strain-specific differences. A mass difference of 58-71 is seen between the sequence mass of PRPL4 (GenBank^TM accession number X93160, cv. Melody; Ref. 48) and the observed MS mass in this study (cv. Alwaro; Table II). Possibly, it may reflect a cultivar difference, or a post-translational modification.

PRPL10 exists in three forms of differing chain length (designated , , and ). The relative amounts were 80% for the  form, 12% for the  form, and about 8% for the minor  form, as estimated from the yields of N-terminal PTH-derivatives. The major form, PRPL10  (mass 20,305 Da) was identified by the LC/MS analysis (Table II), but the other two forms (both approximately 16.5 kDa, as estimated by SDS-PAGE) were not detected in the MS analysis, probably because of low ionization and smaller amounts. Because all three forms gave the same N-terminal sequence, their differences arise from post-translational modifications in the internal and/or C-terminal regions of the molecule.

PRPL11 has been previously shown to be epsilon-N-trimethylated at positions Lys-9 and Lys-45 (49).⁴ The E. coli L11 protein is trimethylated at the corresponding positions in its sequence context (50). LC/MS data showed that plastid L11 has a mass increment of 81.5 Da over its sequence mass (Table II). This increment is close to the 84.2-Da mass for two trimethyl modifications. In the case of E. coli L11, the N-terminal amino acid Ala is -N-trimethylated (50). The N-terminal Ala of PRPL11 is mainly unmodified, but there is indirect evidence for a partial modification of this residue, resulting in the presence of a minor form (this paper and Ref. 49).

PRPL12 was obtained in a significantly higher yield (Fig. 3), consistent with the presence of four copies/50 S subunit, as has been previously deduced for plastid 70 S ribosome (17). In E. coli, this protein exists in two forms: L12, with free N terminus and L7, -N-acetylated form, the sum of the two forms constituting four copies/50 S ribosomal subunit (51), and the ratio of the two forms altering during the bacterial growth cycle (52). The N terminus of plastid L12 is essentially unmodified (Fig. 3), and thus the roles played by N-acetylation (and its variation with growth) in E. coli are apparently abolished in plastid metabolism. Interestingly, a minor mass of 13,850.2 Da was observed in the LC/MS of TP50, at the same elution interval as PRPL12 (mass of 13,811 Da), as shown in Fig. 5C. The mass increment of 39.2 Da might indicate the presence of a minor modification (including a minor acetylated form, +42.04 Da).

PRPL16 is partially -N-monomethylated (10%), whereas the bulk of it is N-blocked by an unknown modification (surmised as trimethylation; see "Results"). In E. coli the L16 protein is -N-monomethylated (35), and its Arg⁸¹ is modified by an as yet uncharacterized group (35). The observed mass of E. coli L16 by a recent matrix-assisted laser desorption/ionization time-of-flight MS analysis (53) is 44.9 Da heavier than the sequence mass. Subtracting the mass of -N-monomethylation (14 Da) from 44.9 Da, the uncharacterized group in Arg⁸¹ of E. coli L16 would be of mass 30.9 Da. Our LC/MS analysis of 50 S PRPs (Table II) indicated that plastid L16 is modified with a mass increment of 74.1 Da. Plastid L16 has a conserved Arg residue (Arg⁸²), in a relatively conserved sequence context, that would correspond to E. coli Arg⁸¹. It is conceivable that Arg⁸² of PRPL16 maintains the same modification as in E. coli Arg⁸¹ (+30.9). Hypothetically, if most of plastid L16 N terminus is blocked by trimethylation (+42.08 Da), the total mass increment would be 72.98 (30.9+42.08), close to the observed 74.1 Da increment in the mass of PRPL16 (Table II).

PRPL18 exists in two forms ( and ) of different size but with the same N-terminal sequence ( form, 35% and  form, 65%, as estimated from the yield of PTH-derivative). The modification in PRPL18 was not characterized in this study.

PRPL19 is a highly diverged protein with a negatively charged, 46-amino acid-long NTE compared with E. coli L19 (Fig. 4). The presence of a phosphorylated protein in plastid ribosomal 50 S subunit has been reported (54, 55); the protein spot in those reports corresponds to PRPL19 (Figs. 1 and 8), as correlated in Ref. 2. Because the observed protein mass of PRPL19 is very close to the sequence mass (Table II), MS data would suggest no post-translational modification. However, phosphorylation is a reversible process, and the plastid ribosomes used in the present study could be dephophorylated. The sequence (RLSSLRASTSKS) in the C-terminal portion of Xenopus 40 S subunit ribosomal protein S6 (56) is phosphorylated (S in bold). The consensus recognition motif for S6 kinase II is reported to be -RXXS- (57), and for casein kinase II is (S/T)XX(E/D) (58). The motif -RXXS- is present in both spinach (⁸³RRLS⁸⁶) and Arabidopsis (RRVS) plastid L19 sequences, whereas (¹SEAE⁴ and ²⁶SEAE³⁰) are present only in spinach L19. Thus, this study cannot rule out (or rule in) plastid L19 phosphorylation.

Bubunenko et al. (59) have reported that plastid L23 protein in the chloroplast ribosomes of a certain group of plants (Caryophyllidae, spinach and relatives) is replaced by a homologue of the cytosolic L23 protein, an unusual evolutionary event (59). Table II shows the observed mass of plastid L23 by ESI MS analysis (13,553.5 Da), a value close to the sequence mass (13,553.7 Da), calculated from the nucleotide of sequence of plastid L23 cDNA (GenBank^TM accession number X90414).⁵ The mature plastid L23 thus appears post-translationally unmodified. The N-terminal sequence of PRPL23 shown in Table I corresponds to the reported cDNA; it does not correspond to the deduced N-terminal sequence of a prokaryotic-type rpL23 gene occurring in spinach plastid DNA. Moreover, an N-terminal sequence corresponding to the plastid rpL23 gene was not observed in this comprehensive study. These results thus confirm the previous conclusion (59) that the plastid-encoded rpL23 gene in spinach is a pseudogene. cDNAs derived from two distinct but closely related genes for spinach cytosolic L23, the presumed progenitor of the nuclear gene for spinach plastid L23, have been isolated and sequenced (GenBank^TM accession numbers X92367 and X92350).⁴

PRPL31 exists in three forms ( to ) with the same N-terminal sequence but differing in charge:  form 55%,  form 31%, and  14% by estimation from PTH-derivative yields. E. coli L31 may apparently exist in two forms, a truncated form missing the C-terminal sequence, RFNIPGSK, and the full-length form as deduced from the rpL31 (rpmE) gene sequence (60, 61). The modifications in plastid L31 forms are uncharacterized.

PRPL34 (61 amino acids) is the smallest and the most basic (pI = 12.99) of the 25 nucleus-coded RPs of spinach plastid 50 S subunit. Its transit peptide is 91 amino acids long (Fig. 4) and thus is the longest of all spinach chloroplast RP transit peptides. PRPL35 (73 amino acids, pI = 12.15) also has a relatively long transit peptide of 86 amino acids (62). An average size plastid ribosomal protein, PRPL19 (156 amino acids, pI = 10.51) has a 77-amino acid-long transit peptide. The largest plastid ribosomal protein PRPS1 (370 amino acids, pI = 4.83) has only a 41-amino acid-long transit peptide (63). In general, a correlation can be made that shorter the mature protein, the longer the transit peptide in the RP precursor. Longer transit peptides might thus be a requirement for the proper import and processing of small basic ribosomal proteins.

Among the two plastid-specific proteins of the 50 S subunit, PSRP-5 exists in three forms ( to ), differing apparently in the cleavage points in the precursor form. The reported spinach L40 sequence (28) corresponds to PSRP-5  form in protein sequence length. The cDNA-derived protein sequences of L40 and PSRP-5 differ in 6 amino acids at the C-terminal portion (Fig. 4). These differences could be due to cultivar difference (Alwaro versus Géant d'hiver) or possibly sequencing errors. The protein mass of all three forms of PSRP-5 were observed upon LC/MS analysis of TP50 (Table II), suggesting that all the forms exist on plastid 50 S subunit. Mass data also indicate that two of the forms ( and ) are unlikely to be post-translationally modified. In contrast, Edman degradation of PSRP-5  gave only 6% of the expected average yield of PTH-derivative, as compared with the yields of 28 and 30%, respectively, for PSRP-5  and PSRP-5  (Fig. 3). We surmise that the major portion of PSRP-5  is blocked at the N terminus. Because the LC/MS mass data for PSRP-5  showed a 41-Da increment over the calculated sequence mass (Table II), the blocking group could be acetyl (42.04 Da). The relative occurrence of the three forms of PSRP-5 appear to be  42%,  28%, and  30%. The second plastid specific protein on the 50 S subunit, i.e. PSRP-6, appears N-terminally unmodified (Table I), but because it was not observed in the MS analysis, we have no data on modifications elsewhere in the molecule.

The Case of Ribosomal Proteins L25 and L30-- Plastid 50 S subunit is missing the orthologues of E. coli L25 and L30 (the PSRP-5 and PSRP-6 proteins have no sequence similarity to L25/L30). In E. coli, L25, L18, and L5 are 5 S rRNA-binding proteins, the binding of L18 stimulating the specific binding of L5 (64) and the association of 5 S rRNA with 23 S rRNA requiring all three proteins (65). Is the function of E. coli L25 performed by another plastid 50 S protein? It has been reported that there are striking similarities in the properties of E. coli L25 and spinach plastid L22, in protecting a domain of E. coli 5 S rRNA comprising nucleotides 70-109 (66). Spinach plastid L22 consists of a long NTE and a CTE, and a central core homologous to E. coli L22 (34); but apparently it is this core of plastid L22 that binds to 5 S rRNA (67), even though it is known that E. coli L22 does not bind 5 S rRNA. A homologue of E. coli L25 has been identified in Anabaena (photosynthetic cyanobacterium) RPs (68), and evidence for the occurrence of L25 in Synechocystis PCC 6803 genome sequence has been presented (68). Thus, it appears that L25 protein might have been lost during chloroplast evolution, with plastid L22 protein taking over some of its functions.

E. coli L30 is a 23 S rRNA-binding, late assembly protein that can be mutated out without loss of cell viability (69). Binding of L30 protein to nucleotides 931-938 of 23 S rRNA has been established by cross-linking (70). Interestingly, the three nucleotides (931) on the E. coli 23 S rRNA are replaced by a variable loop of 5-20 nucleotides in chloroplast 23 S rRNA (Refs. 71 and 72; see Fig. 10). Protein sequences showing some similarity to E. coli L30 could be found in several plant ESTs, but the deduced sequences lacked chloroplast transit peptide sequence. Those ESTs thus probably represent cytosolic RPs, mitochondrial RPs, which do not require a separate transit peptide sequence (some mitochondrial RPs are imported into mitochondria without transit peptide), or nonribosomal proteins.

Sequences having significant homology to E. coli L25 and L30 are not always present in the many reported eubacterial genomes. Thus, although both L25 and L30 genes are identified in Haemophilus influenzae, Chlamydia trachomatis, Rickettsia prowazekii, Neisseria meningitidis strains MC58 and Z2491, the L25 gene is reported missing in Aquifex aeolicus, Bacillus subtilis, Borrelia burgdorferi, and Thermotoga maritima, and the L30 gene is missing in Synechocystis PCC6803. Both L25 and L30 genes are missing in Helicobacter pylori strains 26695 and J99, Mycoplasma pneumoniae, Mycobacterium tuberculosis, Mycoplasma genitalium, and Treponema pallidum. L30 gene homologues are present in the reported complete genome sequences from archaebacteria, but L25 gene orthologues are absent. Thus, ribosomal proteins L25 and L30 (and their genes) appear to be evolutionary mavericks.

Comparison of the 50 S Ribosomal Subunits of Chloroplast and E. coli-- Almost all of the plastid 50 S RPs are larger than their E. coli counterparts (Fig. 9, histogram). The mass increases are essentially due to NTEs and/or CTEs added to the E. coli homologous core portions of plastid ribosomal proteins. Significant mass increases are present in PRPs L1, L4, L5, L13, L15, L19, L21, L22, L24, L27, L29, and L31. Interestingly, all these proteins except L22 are encoded in the nucleus. The summed mass of plastid specific NTEs, CTEs, and the two PSRPs is equal to 92.5 kDa. The protein mass of chloroplast 50 S subunit is 529.6 kDa (as compared with the 437.1-kDa protein mass in E. coli 50 S subunit); thus the increase corresponds to 21.2%. For chloroplast 30 S subunit also there is a similar increase in protein mass: addition of 81.5 kDa, corresponding to a 19% increase over E. coli 30 S protein mass (83).

View larger version (66K): [in this window] [in a new window]   Fig. 9.   Changes in molecular mass of individual chloroplast 50 S subunit proteins as compared with that of corresponding RPs of E. coli. Chloroplast (plastid) ribosomal protein masses are taken from Table II. The E. coli 50 S RP masses, as determined by mass spectrometry (matrix-assisted laser desorption/ionization time-of-flight), are from Ref. 53.

The rRNA of land plant plastid 50 S subunit consists of 23, 4.5, and 5 S rRNAs. The 4.5 S rRNA essentially represents the corresponding 3' end portion of bacterial 23 S rRNA (73). The sum of tobacco 23 S rRNA (2804 nucleotides) and 4.5 S rRNA (103 nucleotides) is just three nucleotides larger than E. coli 23 S rRNA (2904 nucleotides). The 5 S rRNA is a conserved molecule (E. coli and tobacco 5 S rRNAs are 120 and 121 nucleotides, respectively), and spinach chloroplast 5 S rRNA was shown to incorporate in vitro with RPs and 23 S rRNA from Bacillus stearothermophilus to form functionally active 50 S subunits (74).

Interestingly, there are several small but distinctly variable regions of nucleotide sequence, identifiable in both tobacco (a dicot plant) and maize (a monocot plant) 23 S rRNA molecules (72). One is the variable sequence discussed earlier regarding L30-binding site. The variable regions are mostly localized in domains I, II, III, and VI in 23 S rRNA structure (Fig. 10). Although these variable regions seem to be randomly distributed in the secondary structure of 23 S rRNA, they have a unique localization in the three-dimensional arrangement of the rRNA in 50 S subunit (75). As seen in Fig. 10, except for the added loop at helix 38.2, most of the other changes are clustered around the bottom of the 50 S subunit structure, near the exit site for the nascent polypeptide.

View larger version (49K): [in this window] [in a new window]   Fig. 10.   Secondary structure differences between E. coli 23 S rRNA and tobacco chloroplast 23 S rRNA: locations in the three-dimensional arrangement of rRNA in the 50 S subunit. Helix numbers shown in the secondary structure schematics and three-dimensional structure are taken from Mueller et al. (75). Only those regions in domains I-III and VI are shown where there are some significant differences (domains IV and V are highly conserved). Domain VI includes 4.5S rRNA. The three-dimensional arrangement of rRNA in the 50 S subunit is also from Ref. 75. Cp, central protuberance; St, L7/12 stalk; Exit, exit site of nascent peptide, indicated by dotted circle (view from solvent side) or arrow (view from L7/L12 stalk side); De9 and De98, positions of deleted helices 9 and 98; Ad38.2, Ad58.2, and Ad100, added loops in chloroplast 23 S rRNA. The Ad 38.2 loop (nucleotides 931-933) interrupts the L30-binding site in 23 S rRNA (see text).

The functions of the large ribosomal subunit include peptide bond formation at the peptidyl transferase center and co-translational protein targeting for membrane and lumenal proteins via interactions with signal recognition particle (SRP) and its receptor. SRPs in E. coli and eukaryotic cytosol are ribonucleoprotein complexes that co-translationally target proteins to the ER and the bacterial inner membrane, respectively. Plants have evolved an additional specific membrane in chloroplasts, namely the thylakoid where photosynthesis occurs. Interestingly, a very large number of the protein components of the two photosystems, cytochrome b/f complex and ATP synthase are synthesized on the plastid ribosome and inserted in the thylakoid. Recently a chloroplast SRP has been reported as a novel SRP, because it lacks the RNA moiety found in bacterial and eukaryotic SRPs (76). It is conceivable that the plastid 23 S rRNA variations (Fig. 10) and the two PSRPs in the 50 S subunit have been evolved, in combination with an RNA-less chloroplast SRP, for protein targeting/translocation functions at the 50 S subunit-thylakoid membrane interface.

Plastid RRF-- We identified plastid RRF as a protein strongly associated with the plastid 70 S ribosome (Fig. 8). Its stoichiometry was approximately one, similar to that of most PRPs in the zonal sucrose gradient-purified 70 S ribosome preparations (Table I). The RRF protein was absent in either 30 or 50 S subunits obtained from the 70 S ribosome preparation (Fig. 7). In contrast, the bulk of the RRF content in E. coli is present in post-ribosomal supernatant, and only a small amount (0.08-0.2 mol/ribosome) is present in the ribosomal pellet; ribosomal pellet from midlog cells contained the lower amount and that from stationary cells contained the higher amount; the amount in gradient-purified ribosomes was negligible (experimental results with radiolabeled E. coli).⁶ Thus, there is a remarkable difference in the apparent ribosomal affinity of RRF between the plastid and E. coli translation systems.

In E. coli, RRF is suggested to catalyze the fourth step of protein synthesis, i.e. the disassembly of the post-termination complex of ribosome, mRNA, and deacylated tRNA (77). In a more recent report, the dissociation of 50 S subunit from the 70 S post-termination complex was proposed to be the step that is catalyzed by RRF, requiring elongation factor-G (EF-G)-dependent GTP hydrolysis (78). The finding that plastid RRF is associated, in approximately stoichiometric amount, with 70 S ribosomes is apparently inconsistent with the catalytic role suggested for E. coli RRF. In an experiment where plastid 70 S ribosomes were treated with 500 mM ammonium chloride in 50% ethanol (at 0 °C for 10 min), half the RRF amount was still found on the ribosome, whereas most of plastid L12 was released (data not shown). This observation further supports a strong binding between RRF and 70 S ribosomes in plastids.

In E. coli, IF-3 is reported to be a ribosome dissociation factor that dissociates run-off 70 S ribosomes to 30 and 50 S subunits, the free 30 S subunit with bound IF-3 initiating a new round of translation (79, 80). In chloroplasts, the IF-3 activity of the plastid IF-3 molecule is100-fold affected by the presence of its plastid-specific N/C-terminal extensions; it has been suggested that plastid IF-3 is capable of being activated (through its NTE/CTE) by a nuclear regulatory factor under appropriate environmental signals, e.g. light (81). We speculate that in chloroplasts the plastid RRF might have a role in holding the 70 S ribosome together (as an inactive form), prior to the activation of IF-3 by a light-dependent regulatory factor and the start of a new round of translation. Plastid RRF might thus function as a true ribosome anti-dissociation factor.

In conclusion, among the eight spinach plastid 50 S subunit RP genes located in the organelle genome, only rpL16 gene contains an intron (rpL2 gene is intron-containing in most plants, except spinach and relatives). To obtain a preliminary idea of the intron-exon structure in the nuclear genes that encode for plastid 50 S RPs, we cloned four PRP genes in our laboratory. One of them, PrpL12 occurs as a cluster of three intron-less genes in A. thaliana (82), whereas the other three, PrpL1, PrpL13, and PrpL35, are present as single copy genes, containing six, three, and two introns, respectively (46).² The complete picture of plastid ribosomal protein genes from a plant should soon be available (e.g. with the completion of the Arabidopsis genome sequencing project), with our protein work facilitating PRP gene identifications.

	ACKNOWLEDGEMENTS

We thank Drs. K. Giese, B. R. Srinivasa, U. Markmann, Y. Ogihara, G. Timmler, S. H. Phua, P.M. Smooker, C. H. Johnson, M. Bubunenko, C. Jayabaskaran, and M. Kavousi (postdoctoral fellows/visiting scientists in A. R. Subramanian's Berlin laboratory) for early contributions, Dr. W. Schröder (Free University, Berlin) for work on PRPL11 methylation, and K. von Knoblauch for devoted technical/computing assistance.

	FOOTNOTES

^* This work was supported by Max-Planck-Gesellschaft through a Sponsored Project Grant (protein synthesis and regulation).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF238221, AF245292, AF250384, AF250923, and AF261940.

To whom correspondence should be addressed: 5110 East Woodgate Ln., Tucson, AZ 85712. Tel./Fax: 520-325-7957; E-mail: alapsubraman@cs.com.

Published, JBC Papers in Press, June 28, 2000, DOI 10.1074/jbc.M005012200

³ M. Kavousi and A. R. Subramanian, unpublished results.

⁴ J. Schmidt and A. R. Subramanian, unpublished results.

⁵ C. Jayabaskaran and A. R. Subramanian, unpublished results.

⁶ A. R. Subramanian, unpublished results.

² K. Yamaguchi and A. R. Subramanian, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: RP, ribosomal protein; PRP, plastid ribosomal protein; PSRP, plastid-specific ribosomal protein; RRF, ribosome recycling factor; TP50, total protein from 50 S subunit; TP70, total protein from 70 S ribosome; two-dimensional PAGE, two-dimensional polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; MS, mass spectrometry; LC/MS, reversed-phase HPLC coupled to electrospray ionization mass spectrometry; ESI, electrospray ionization; EST, expressed sequence tag; AA, amino acid(s); NTE, N-terminal extension; CTE, C-terminal extension; PCR, polymerase chain reaction; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PVDF, polyvinylidene difluoride; PTH, phenylthiohydantoin; SRP, signal recognition particle; SSU, small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase.

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