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Megadalton Complexes in the Chloroplast Stroma of Arabidopsis thaliana Characterized by Size Exclusion Chromatography, Mass Spectrometry, and Hierarchical Clustering*

  • Paul Dominic B. Olinares
    Affiliations
    ‡Department of Plant Biology, Cornell University, Ithaca, New York 14853

    §Graduate Program of the Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853
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  • Lalit Ponnala
    Affiliations
    ¶Computational Biology Service Unit, and Cornell University, Ithaca, New York 14853
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  • Klaas J. van Wijk
    Correspondence
    To whom correspondence should be addressed:Dept. of Plant Biology, Emerson Hall 332, Cornell University, Ithaca, NY 14853. Tel.: 607-255-3664; Fax: 607-255-3664; E-mail: [email protected]
    Affiliations
    ‡Department of Plant Biology, Cornell University, Ithaca, New York 14853
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  • Author Footnotes
    * This work was supported by National Science Foundation Grant MCB 0343444, United States Department of Agriculture Biochemistry Program Grant 2003-02409, and the New York State Office of Science, Technology and Research (NYSTAR) (to K. J. v. W.).
    This article contains supplemental Fig. 1 and Tables 1 and 2.
    2 B. Zybailov, unpublished data.
    3 H. Rutschow and K. J. van Wijk, unpublished data.
    1 The abbreviations used are:CNcolorless nativeSECsize exclusion chromatographyPDCpyruvate dehydrogenase complexACCaseacetyl-CoA carboxylaseBCCPbiotin carboxyl carrier proteinPRIDEProteomics Identifications databasePEPplastid-encoded RNA polymerasePPDBPlant Proteome DatabaseRubiscoribulose-1,5-bisphosphate carboxylase/oxygenaseSPCspectral countadjSPCadjusted SPCNadjSPCnormalized adjusted SPCNSAFnormalized spectral abundance factorHMhigh massLMlow massRPSribosomal protein small subunitRPLribosomal protein large subunitPSRPplastid-specific ribosomal proteinIFinitiation factorEFelongation factorRFrelease factorRRFribosome recycling factorPDFpeptide deformylaseMAPmethionine aminopeptidasecpSRP54chloroplast signal recognition particleTFtrigger factorCPNchaperoneHSPheat shock proteinPNPasepolynucleotide phosphorylaseTACtranscriptionally active chromosomeCRMchloroplast ribosome maturationcpRNPchloroplast ribonucleoproteinPPRpentatricopeptide repeatRHRNA helicaseTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineDIGdigoxigeninMOWSEmolecular weight searchDALdifferentiation and greening (DAG)-likepTACtranscriptionally active plastid chromosomeCTcarboxyltransferaseACPacyl carrier proteinRAribosome assemblycTPchloroplast transit peptideXMLextensible markup language.
      To characterize MDa-sized macromolecular chloroplast stroma protein assemblies and to extend coverage of the chloroplast stroma proteome, we fractionated soluble chloroplast stroma in the non-denatured state by size exclusion chromatography with a size separation range up to ∼5 MDa. To maximize protein complex stability and resolution of megadalton complexes, ionic strength and composition were optimized. Subsequent high accuracy tandem mass spectrometry analysis (LTQ-Orbitrap) identified 1081 proteins across the complete native mass range. Protein complexes and assembly states above 0.8 MDa were resolved using hierarchical clustering, and protein heat maps were generated from normalized protein spectral counts for each of the size exclusion chromatography fractions; this complemented previous analysis of stromal complexes up to 0.8 MDa (Peltier, J. B., Cai, Y., Sun, Q., Zabrouskov, V., Giacomelli, L., Rudella, A., Ytterberg, A. J., Rutschow, H., and van Wijk, K. J. (2006) The oligomeric stromal proteome of Arabidopsis thaliana chloroplasts. Mol. Cell. Proteomics 5, 114–133). This combined experimental and bioinformatics analyses resolved chloroplast ribosomes in different assembly and functional states (e.g. 30, 50, and 70 S), which enabled the identification of plastid homologues of prokaryotic ribosome assembly factors as well as proteins involved in co-translational modifications, targeting, and folding. The roles of these ribosome-associating proteins will be discussed. Known RNA splice factors (e.g. CAF1/WTF1/RNC1) as well as uncharacterized proteins with RNA-binding domains (pentatricopeptide repeat, RNA recognition motif, and chloroplast ribosome maturation), RNases, and DEAD box helicases were found in various sized complexes. Chloroplast DNA (>3 MDa) was found in association with the complete heteromeric plastid-encoded DNA polymerase complex, and a dozen other DNA-binding proteins, e.g. DNA gyrase, topoisomerase, and various DNA repair enzymes. The heteromeric ≥5-MDa pyruvate dehydrogenase complex and the 0.8–1-MDa acetyl-CoA carboxylase complex associated with uncharacterized biotin carboxyl carrier domain proteins constitute the entry point to fatty acid metabolism in leaves; we suggest that their large size relates to the need for metabolic channeling. Protein annotations and identification data are available through the Plant Proteomics Database, and mass spectrometry data are available through Proteomics Identifications database.
      Chloroplasts are essential plant organelles of prokaryotic origin that perform a variety of metabolic and signaling functions. Best known for their role in photosynthesis, they also carry out the biosynthesis of many primary and secondary metabolites like lipids, amino acids, vitamins, nucleotides, tetrapyrroles, and hormones (
      ). Subcellular localization prediction by TargetP (
      • Emanuelsson O.
      • Nielsen H.
      • Brunak S.
      • von Heijne G.
      Predicting subcellular localization of proteins based on their N-terminal amino acid sequence.
      ) combined with a correction for false positive and false negative rates suggested that all non-green plastid types and chloroplasts together contain some 3500 proteins in Arabidopsis thaliana (
      • van Wijk K.J.
      Plastid proteomics.
      ). More than 95% of the chloroplast proteins are nucleus-encoded and post-translationally imported into the chloroplast (
      • Jarvis P.
      • Robinson C.
      Mechanisms of protein import and routing in chloroplasts.
      ,
      • Soll J.
      • Schleiff E.
      Protein import into chloroplasts.
      ,
      • Kessler F.
      • Schnell D.J.
      The function and diversity of plastid protein import pathways: a multilane GTPase highway into plastids.
      ). Over the last decade, several studies were published that aimed to identify (subfractions of) the Arabidopsis chloroplast proteome (e.g. Refs.
      • Ferro M.
      • Salvi D.
      • Brugiere S.
      • Miras S.
      • Kowalski S.
      • Louwagie M.
      • Garin J.
      • Joyard J.
      • Rolland N.
      Proteomics of the chloroplast envelope membranes from Arabidopsis thaliana.
      ,
      • Kieselbach T.
      • Schröder W.P.
      The proteome of the chloroplast lumen of higher plants.
      ,
      • Zybailov B.
      • Rutschow H.
      • Friso G.
      • Rudella A.
      • Emanuelsson O.
      • Sun Q.
      • van Wijk K.J.
      Sorting signals, N-terminal modifications and abundance of the chloroplast proteome.
      ,
      • Baginsky S.
      • Kleffmann T.
      • von Zychlinski A.
      • Gruissem W.
      Analysis of shotgun proteomics and RNA profiling data from Arabidopsis thaliana chloroplasts.
      ). The precise number of bona fide chloroplast proteins from these proteomics studies is probably somewhere around 1000–1300; comparing this number with the predicted chloroplast proteome indicates that ∼50% of the proteome has still not been observed. Recently, we concluded that when compared with the predicted Arabidopsis chloroplast proteome the chloroplast proteome identified to date is particularly underrepresented (40–70%) for proteins involved in signaling, stress, development, unassigned function, and DNA/RNA metabolism (
      • Zybailov B.
      • Rutschow H.
      • Friso G.
      • Rudella A.
      • Emanuelsson O.
      • Sun Q.
      • van Wijk K.J.
      Sorting signals, N-terminal modifications and abundance of the chloroplast proteome.
      ). To probe deeper into the chloroplast proteome, enrichment for low abundance proteins prior to MS analysis is required.
      Many biochemical functions are executed by protein assemblies. Several studies have catalogued the assembly states of chloroplast proteins in plants. Separation of the oligomeric Arabidopsis stromal proteome by two-dimensional native gel electrophoresis (CN
      The abbreviations used are:
      CN
      colorless native
      SEC
      size exclusion chromatography
      PDC
      pyruvate dehydrogenase complex
      ACCase
      acetyl-CoA carboxylase
      BCCP
      biotin carboxyl carrier protein
      PRIDE
      Proteomics Identifications database
      PEP
      plastid-encoded RNA polymerase
      PPDB
      Plant Proteome Database
      Rubisco
      ribulose-1,5-bisphosphate carboxylase/oxygenase
      SPC
      spectral count
      adjSPC
      adjusted SPC
      NadjSPC
      normalized adjusted SPC
      NSAF
      normalized spectral abundance factor
      HM
      high mass
      LM
      low mass
      RPS
      ribosomal protein small subunit
      RPL
      ribosomal protein large subunit
      PSRP
      plastid-specific ribosomal protein
      IF
      initiation factor
      EF
      elongation factor
      RF
      release factor
      RRF
      ribosome recycling factor
      PDF
      peptide deformylase
      MAP
      methionine aminopeptidase
      cpSRP54
      chloroplast signal recognition particle
      TF
      trigger factor
      CPN
      chaperone
      HSP
      heat shock protein
      PNPase
      polynucleotide phosphorylase
      TAC
      transcriptionally active chromosome
      CRM
      chloroplast ribosome maturation
      cpRNP
      chloroplast ribonucleoprotein
      PPR
      pentatricopeptide repeat
      RH
      RNA helicase
      Tricine
      N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine
      DIG
      digoxigenin
      MOWSE
      molecular weight search
      DAL
      differentiation and greening (DAG)-like
      pTAC
      transcriptionally active plastid chromosome
      CT
      carboxyltransferase
      ACP
      acyl carrier protein
      RA
      ribosome assembly
      cTP
      chloroplast transit peptide
      XML
      extensible markup language.
      -PAGE) profiled 240 non-redundant proteins and captured information for 124 complexes (
      • Peltier J.B.
      • Cai Y.
      • Sun Q.
      • Zabrouskov V.
      • Giacomelli L.
      • Rudella A.
      • Ytterberg A.J.
      • Rutschow H.
      • van Wijk K.J.
      The oligomeric stromal proteome of Arabidopsis thaliana chloroplasts.
      ). However, native gel electrophoresis has a practical size limit, and only protein complexes below ∼1000 kDa can be effectively separated, thereby missing megadalton-sized complexes. Several megadalton-sized complexes in plants have been characterized by targeted purification schemes, including the spinach 30 and 50 S ribosomal particles (
      • Yamaguchi K.
      • von Knoblauch K.
      • Subramanian A.R.
      The plastid ribosomal proteins. Identification of all the proteins in the 30 S subunit of an organelle ribosome (chloroplast).
      ,
      • Yamaguchi K.
      • Subramanian A.R.
      The plastid ribosomal proteins. Identification of all the proteins in the 50 S subunit of an organelle ribosome (chloroplast).
      ,
      • Yamaguchi K.
      • Subramanian A.R.
      Proteomic identification of all plastid-specific ribosomal proteins in higher plant chloroplast 30S ribosomal subunit.
      ), cytosolic ribosomes (
      • Zanetti M.E.
      • Chang I.F.
      • Gong F.
      • Galbraith D.W.
      • Bailey-Serres J.
      Immunopurification of polyribosomal complexes of Arabidopsis for global analysis of gene expression.
      ,
      • Carroll A.J.
      • Heazlewood J.L.
      • Ito J.
      • Millar A.H.
      Analysis of the Arabidopsis cytosolic ribosome proteome provides detailed insights into its components and their post-translational modification.
      ), the tobacco plastid-encoded RNA polymerase (PEP) complex (
      • Suzuki J.Y.
      • Ytterberg A.J.
      • Beardslee T.A.
      • Allison L.A.
      • Wijk K.J.
      • Maliga P.
      Affinity purification of the tobacco plastid RNA polymerase and in vitro reconstitution of the holoenzyme.
      ), maize mitochondrial pyruvate dehydrogenase complex (PDC) (
      • Thelen J.J.
      • Miernyk J.A.
      • Randall D.D.
      Partial purification and characterization of the maize mitochondrial pyruvate dehydrogenase complex.
      ), and pea chloroplast acetyl-CoA carboxylase (ACCase) complex (
      • Sasaki Y.
      • Nagano Y.
      Plant acetyl-CoA carboxylase: structure, biosynthesis, regulation and gene manipulation for plant breeding.
      ). Proteome characterization of a membrane-depleted, Triton-insoluble, and high density pellet from pea plastids was highly enriched for the chloroplast PDC as well as proteins involved in plastid gene expression and carbon fixation (
      • Phinney B.S.
      • Thelen J.J.
      Proteomic characterization of a Triton-insoluble fraction from chloroplasts defines a novel group of proteins associated with macromolecular structures.
      ). However, because no subsequent fractionation was performed, specific protein associations could not be resolved.
      To extend chloroplast proteome coverage and characterize MDa-sized macromolecular assemblies to complement the previous CN-PAGE analysis of complexes up to 0.8 MDa, we fractionated the soluble chloroplast stroma by size exclusion chromatography (SEC) with a particular focus on complexes greater than 0.8 MDa. Proteins were identified by mass spectrometry analysis using an LTQ-Orbitrap, a high accuracy and high sensitivity hybrid instrument (
      • Hu Q.
      • Noll R.J.
      • Li H.
      • Makarov A.
      • Hardman M.
      • Graham Cooks R.
      The Orbitrap: a new mass spectrometer.
      ,
      • Scigelova M.
      • Makarov A.
      Orbitrap mass analyzer—overview and applications in proteomics.
      ). SEC migration profiles for identified proteins were generated from matched spectral counts. Hierarchical clustering and protein heat maps of the SEC migration profiles revealed that the identified protein complexes include 30, 50, and 70 S ribosomal particles; PDC; PEP; and ACCase, indicating successful MDa size fractionation. In addition, many “new” proteins were detected, and they were enriched for functions in plastid gene expression, in particular putative ribosomal biogenesis factors. Finally, protein annotations and identification data are available via the Plant Proteomics Database (PPDB) at http://ppdb.tc.cornell.edu/, and mass spectrometry data with their metadata were deposited in the Proteomics Identifications database (PRIDE) (http://www.ebi.ac.uk/pride/) under accession numbers 11459-11568.
      The concept of using chromatography (or other continuous fractionation techniques) of protein complexes (or other types of cellular protein fractions) with mass spectrometry-based quantification to determine co-localization has been applied using stable isotope labeling (
      • Dunkley T.P.
      • Watson R.
      • Griffin J.L.
      • Dupree P.
      • Lilley K.S.
      Localization of organelle proteins by isotope tagging (LOPIT).
      ,
      • Dong M.
      • Yang L.L.
      • Williams K.
      • Fisher S.J.
      • Hall S.C.
      • Biggin M.D.
      • Jin J.
      • Witkowska H.E.
      A “tagless” strategy for identification of stable protein complexes genome-wide by multidimensional orthogonal chromatographic separation and iTRAQ reagent tracking.
      ) or label-free techniques (
      • Andersen J.S.
      • Wilkinson C.J.
      • Mayor T.
      • Mortensen P.
      • Nigg E.A.
      • Mann M.
      Proteomic characterization of the human centrosome by protein correlation profiling.
      ,
      • Majeran W.
      • Zybailov B.
      • Ytterberg A.J.
      • Dunsmore J.
      • Sun Q.
      • van Wijk K.J.
      Consequences of C4 differentiation for chloroplast membrane proteomes in maize mesophyll and bundle sheath cells.
      ). When combined with cluster analysis (this study and Ref.
      • Dong M.
      • Yang L.L.
      • Williams K.
      • Fisher S.J.
      • Hall S.C.
      • Biggin M.D.
      • Jin J.
      • Witkowska H.E.
      A “tagless” strategy for identification of stable protein complexes genome-wide by multidimensional orthogonal chromatographic separation and iTRAQ reagent tracking.
      ), principle component analysis (
      • Dunkley T.P.
      • Watson R.
      • Griffin J.L.
      • Dupree P.
      • Lilley K.S.
      Localization of organelle proteins by isotope tagging (LOPIT).
      ), or correlation of normalized elution profiles (this study and Refs.
      • Andersen J.S.
      • Wilkinson C.J.
      • Mayor T.
      • Mortensen P.
      • Nigg E.A.
      • Mann M.
      Proteomic characterization of the human centrosome by protein correlation profiling.
      and
      • Majeran W.
      • Zybailov B.
      • Ytterberg A.J.
      • Dunsmore J.
      • Sun Q.
      • van Wijk K.J.
      Consequences of C4 differentiation for chloroplast membrane proteomes in maize mesophyll and bundle sheath cells.
      ), this strategy is clearly a powerful tool and is widely applicable to other subcellular proteomes.

      EXPERIMENTAL PROCEDURES

       Plant Growth, Chloroplast Stroma Proteome Isolation, and Size Fractionation

      A. thaliana (Col 0) was grown under 10-h light/14-h dark cycles at 25/17 °C in controlled growth chambers (Conviron) for about 55 days, and leaves were collected from mature rosettes about 1 week prior to bolting. Leaves were briefly homogenized in grinding medium (50 mm HEPES-KOH, pH 8.0, 330 mm sorbitol, 2 mm EDTA, 5 mm ascorbic acid, 5 mm cysteine, 0.03% BSA) and filtered through a nylon mesh. The crude plastids were then collected by a 2-min spin at 1100 × g and further purified on 40–85% Percoll cushions (Percoll in 0.6% Ficoll, 1.8% polyethylene glycol) by a 10-min spin at 3750 × g and one additional wash in the grinding medium without ascorbic acid, cysteine, and BSA. Chloroplasts were subsequently lysed in 10 mm HEPES-KOH, pH 8.0, 5 mm MgCl2 with a mixture of protease inhibitors under mild mechanical disruption. The lysate was then subjected to ultracentrifugation (100,000 × g) to pellet the membrane components. The supernatant (stroma) was then collected and concentrated using an Amicon 10-kDa-molecular mass cutoff filter (Millipore). Protein amounts were determined using the Bradford reagent (Bio-Rad) or the BCA protein assay kit (ThermoScientific).
      Stroma (1–3 mg) was loaded in a Superose 6 10/300 GL column (GE Healthcare) using an ÄKTA FPLC system (Amersham Biosciences) with a mercury lamp as a detector. Absorbance was measured at 280 nm. Elution was performed with buffer A (25 mm HEPES, pH 8.0, 10 mm NaCl, 10 mm MgCl2) or buffer B (50 mm HEPES, 50 mm NaCl, pH 8.0, 5 mm MgCl2) at an optimal flow rate of 0.25 ml/min. 300-γl subfractions were initially collected. Subfractions were pooled as follows: three for fractions 1–6, four for fractions 7–12, and six for fraction 13 (see Fig. 2 for the SEC chromatogram and the fraction designations). Proteins from pooled fractions were either concentrated using an Amicon Microcon YM-10 filter (Millipore) or precipitated with 80% acetone and were then separated further using SDS-PAGE on 12% T Laemmli or 12% T Tricine minigels. Protein bands were visualized using fluorescent SYPRO Ruby for fractions 1–5 of SEC-separated sample from buffer A, and the rest of the gels were stained with Coomassie Blue. Each gel lane was excised into four or five bands followed by reduction, alkylation, in-gel digestion with trypsin, and peptide extraction as described (
      • Shevchenko A.
      • Tomas H.
      • Havlis J.
      • Olsen J.V.
      • Mann M.
      In-gel digestion for mass spectrometric characterization of proteins and proteomes.
      ). Peptide extracts were dried down and resuspended in 15–20 γl of 5% formic acid for MS/MS analysis.
      Figure thumbnail gr2
      Fig. 2Size exclusion fractionation of Arabidopsis stroma. A, chromatogram of SEC-fractionated stroma (∼3 mg of proteins) eluted with buffer A. The SEC separation with buffer A is further divided into HM-A (fractions 1–5) and LM-A (fractions 6–13) with their corresponding SDS-PAGE gels. Protein bands in the HM-A gel were visualized with fluorescent SYPRO Ruby, whereas those for LM-A were stained with Coomassie Blue. 100% of fractions 1–5 was loaded on the gel, whereas approximately equal amounts of proteins (estimated from the integrated peak areas for each fraction) were loaded for the LM range (fractions 6–13). The Rubisco large and small subunits are indicated by L and S, respectively. The asterisk indicates the CPN60 complex of 800 kDa. Abs, absorbance; mAU, milliabsorbance units. B, comparison of the HM-A and LM-A data sets based on the functional distribution of the non-overlapping data sets shown as stacked bar graphs. C, distribution of chloroplast rRNA of 30 and 50 S ribosomal particles as well as the plastid chromosome. The upper panels show Northern blots of extracted RNA for 16 and 23 S rRNA using antibodies targeting DIG-labeled rRNAs. The lower panel shows chloroplast DNA detected by PCR amplification of the plastid gene encoding for the chloroplast 16 S rRNA.

       Nano-LC-LTQ-Orbitrap Analysis and Data Processing

      The resuspended peptide extracts were analyzed by data-dependent MS/MS using an on-line LC-LTQ-Orbitrap (Thermo Electron Corp.). Peptide samples were automatically loaded on a guard column (LC Packings MGU-30-C18PM) via an autosampler followed by separation on a PepMap C18 reverse-phase nanocolumn (LC Packings nan75-15-03-C18PM) using 90-min gradients with 95% water, 5% ACN, 0.1% formic acid (solvent A) and 95% ACN, 5% water, 0.1% formic acid (solvent B) at a flow rate of 200 nl/min. Two blanks were run after every sample (see Zybailov et al. (
      • Zybailov B.
      • Rutschow H.
      • Friso G.
      • Rudella A.
      • Emanuelsson O.
      • Sun Q.
      • van Wijk K.J.
      Sorting signals, N-terminal modifications and abundance of the chloroplast proteome.
      ) for the gradient and sample injection scheme). The acquisition cycle consisted of a survey MS scan in the Orbitrap with a set mass range from 350 to 1800 m/z at the highest resolving power (100,000) followed by five data-dependent MS/MS scans acquired in the LTQ. Dynamic exclusion was used with the following parameters: exclusion size, 500; repeat count, 2; repeat duration, 30 s; exclusion time, 180 s; exclusion window, ±6 or ±100 ppm. Target values were set at 5 × 105 and 104 for the survey and tandem MS scans, respectively. Regular scans were used both for the precursor and tandem MS.
      Peak lists (.mgf format) were generated using DTASuperCharge (v1.19) software (SourceForge) and searched with Mascot v2.2 (Matrix Science). For off-line calibration, first a preliminary search was conducted with the precursor tolerance window set at ±30 ppm. Peptides with ion scores above 33 were chosen as benchmarks to determine the offset for each LC-MS/MS run. This particular ion score value (
      • Zybailov B.
      • Coleman M.K.
      • Florens L.
      • Washburn M.P.
      Correlation of relative abundance ratios derived from peptide ion chromatograms and spectrum counting for quantitative proteomic analysis using stable isotope labeling.
      ) was chosen in accordance with the results of the search against the target-decoy database (see further below and Ref.
      • Zybailov B.
      • Sun Q.
      • van Wijk K.J.
      Workflow for large scale detection and validation of peptide modifications by RPLC-LTQ-Orbitrap: application to the Arabidopsis thaliana leaf proteome and an online modified peptide library.
      ). This offset was then applied to adjust precursor masses in the peak lists of the respective .mgf file for recalibration using a Perl script.
      B. Zybailov, unpublished data.
      The recalibrated peak lists were searched against The Arabidopsis Information Resource A. thaliana database v8, including sequences for known contaminants (e.g. keratin and trypsin) (total 33,013 entries) with or without a concatenated decoy database where all the sequences were in reverse orientation. Each of the peak lists were searched using Mascot v2.2 (maximum p value of 0.01) for fully tryptic peptides using a precursor ion tolerance window set at ±6 ppm, variable methionine oxidation, fixed cysteine carbamidomethylation, a minimal ion score threshold of 33, and mass range of 700–3500 Da for precursor ions. To reduce the false identification rate of proteins identified by one peptide, the Mascot search results were further filtered as follows: the ion score threshold was increased to 35, and mass accuracy on the precursor ion was required to be within ±3 ppm. Overall, this yielded a peptide false discovery rate of 1.5% with peptide false positive rate calculated as 2 × (decoy_hits/total_hits) derived from searches against the target-decoy database. The false protein identification rate of protein identified with two or more peptides was zero. All filtered results were uploaded into the PPDB (http://ppdb.tc.cornell.edu/) (
      • Sun Q.
      • Zybailov B.
      • Majeran W.
      • Friso G.
      • Olinares P.D.
      • van Wijk K.J.
      PPDB, the Plant Proteomics Database at Cornell.
      ). All mass spectral data (the .mgf files reformatted as PRIDE XML files) were made available via the PRIDE at http://www.ebi.ac.uk/pride/.

       Determination of Protein SEC Elution Profiles and Relative Protein Abundance

      To determine the relative protein abundance distribution by spectral counting, the number of matched MS/MS spectra or spectral count (SPC) for each protein was obtained. This was further classified as total SPC, unique SPC (uniquely matching to an accession), and adjusted SPC (adjSPC). The latter is the sum of unique SPCs and SPCs from shared peptides across accessions with SPC distributed in proportion to their unique SPCs if applicable. Proteins that shared more than 80% of their matched peptides with other proteins across the complete data set were grouped into families. For many Arabidopsis genes, more than one protein model is predicted. In this study, protein models with the highest total adjSPC across all experiments were used; if the protein models did not differ in total adjSPC, protein model 1 was selected. To increase the robustness and significance of the data set, we removed all proteins that were identified with only one amino acid sequence irrespective of charge state, post-translational modifications, or number of SPCs. Proteins that were quantified with two or fewer adjSPCs were also removed. To generate protein elution heat maps for the “high mass” (HM) data set, the adjSPC for each protein per SEC fraction was normalized to the highest adjSPC across five HM fractions (NadjSPC).
      To calculate the relative abundance for each protein across all HM fractions, the total adjSPC was divided by the number of observable tryptic peptides within the mass range 700–3500 Da (with the predicted transit peptide removed), yielding the spectral abundance factor (SAF). The SAF values were then normalized to the total SAF in the whole data set, yielding normalized spectral abundance factors (NSAFs).

       Hierarchical Clustering Analysis

      To group proteins with similar elution profiles in the HM data set, hierarchical clustering was utilized using the Statistics toolbox of MATLAB version 7 (Mathworks, Inc.). The linear correlation (ρ) between every pair of proteins with NadjSPC distribution across the SEC HM fractionation range, X1, … Xn and Y1, … Yn where n = 5, was derived.
      ρXY=Σj=1n(XjX¯)(YjY¯)Σj=1n(XjX¯)2Σj=1n(YjY¯)2
      (Eq. 1)


      This was then converted into a distance measure, ΔXY = 1 − ρXY. Protein pairs with similar elution profiles have higher correlations and in turn have smaller distance values. A linkage map based on the average distance among protein pairs was then constructed to yield a hierarchical cluster tree (dendrogram).

       Nucleic Acid Extraction and Subsequent Detection of DNA and Ribosomal RNA from SEC Fractions

      DNA and RNA were isolated from SEC fractions 1–7 by phenol/chloroform extraction followed by ethanol precipitation. Briefly, an aliquot from each fraction was combined with phenol/chloroform/isoamyl alcohol in a 50:50 mixture together with 0.3% SDS, 1.5 mm EDTA, and 20 ng/γl Glycoblue (Ambion). SDS and EDTA were added to dissociate proteins from nucleoprotein complexes. Glycoblue enhances nucleic acid recovery and increases visibility of the sample pellet. The aqueous phase was extracted after phase separation. To increase nucleic acid yield, the organic phase was then back-extracted with TESS buffer (10 mm Tris-HCl, pH 8.0, 1 mm EDTA, 100 mm NaCl, 0.2% SDS). The collected aqueous phase was then combined with the initial extraction. After adjustment of salt concentration with 3 m sodium acetate, nucleic acid was precipitated with 70% ethanol, pelleted, dried, and resuspended in TE buffer (10 mm Tris-HCl, pH 8.0, 1 mm EDTA).
      An aliquot of the extracted DNA/RNA was treated with ribonuclease H (Invitrogen) to degrade RNA from RNA-DNA hybrids, and plastid DNA was probed by PCR amplification of the gene for 16 S rRNA (see Ref.
      • Zybailov B.
      • Friso G.
      • Kim J.
      • Rudella A.
      • Rodríguez V.R.
      • Asakura Y.
      • Sun Q.
      • van Wijk K.J.
      Large scale comparative proteomics of a chloroplast Clp protease mutant reveals folding stress, altered protein homeostasis, and feedback regulation of metabolism.
      for primers used). PCR samples were then separated on agarose gels and visualized by ethidium bromide staining. The presence of 16 and 23 S rRNA in the extracted DNA/RNA was determined by Northern blot analysis through hybridization with digoxigenin (DIG)-11-dUTP-labeled rRNA probes and subsequent detection with anti-DIG antibodies as described previously (
      • Zybailov B.
      • Friso G.
      • Kim J.
      • Rudella A.
      • Rodríguez V.R.
      • Asakura Y.
      • Sun Q.
      • van Wijk K.J.
      Large scale comparative proteomics of a chloroplast Clp protease mutant reveals folding stress, altered protein homeostasis, and feedback regulation of metabolism.
      ).

       Plant Proteomics Database

      Mass spectrometry-based information of all identified proteins was extracted from the Mascot search pages and filtered for significance (e.g. minimum ion scores, etc.), ambiguities, and shared spectra as described previously. This information includes MOWSE scores, number of matching peptides, total SPCs, unique SPCs, adjSPCs, highest peptide score, highest peptide error (in ppm), lowest absolute error (ppm), sequence coverage, and tryptic peptide sequences (
      • Sun Q.
      • Zybailov B.
      • Majeran W.
      • Friso G.
      • Olinares P.D.
      • van Wijk K.J.
      PPDB, the Plant Proteomics Database at Cornell.
      ). All these are available in the PPDB by using the search function “Proteome Experiments” and by selecting the desired output parameters. Alternatively, information for specific accessions (either individually or a group) can be extracted using the search function “Accessions,” and if desired, this search can be limited to specific experiments. Finally information for a particular accession can also be found on each “protein report page.” The MapMan bin system (
      • Thimm O.
      • Bläsing O.
      • Gibon Y.
      • Nagel A.
      • Meyer S.
      • Krüger P.
      • Selbig J.
      • Müller L.A.
      • Rhee S.Y.
      • Stitt M.
      MAPMAN: a user-driven tool to display genomics data sets onto diagrams of metabolic pathways and other biological processes.
      ) was used for functional assignment, and proteins were reassigned to other bins if needed.

      RESULTS

       Isolation, Size Exclusion Fractionation, and MS Analysis of Arabidopsis Stromal Proteome

      This study aimed to extend the chloroplast proteome coverage and examine MDa-sized assemblies. A summary of the complete work flow of the experimental and computational analysis is shown in Fig. 1. Intact chloroplasts were isolated from mature Arabidopsis leaf rosettes and were lysed under non-denaturing conditions. The lysate was subjected to ultracentrifugation to remove chloroplast membrane-bound proteomes (pellet) from the soluble fraction (stroma). Size separation of the non-denatured stromal proteome was then performed by SEC using a column that resolved up to ∼5-MDa complexes followed by SDS-PAGE.
      Figure thumbnail gr1
      Fig. 1Work flow for the experimental and bioinformatics analyses of the Arabidopsis chloroplast stroma proteome.
      Several lysis media and SEC elution conditions with varying MgCl2 concentration, NaCl concentration, and flow rates were tested for optimization of resolution and complex stability. Removal of MgCl2 in the lysis and elution buffers led to the destabilization of high molecular weight complexes such as 70 S ribosomes (data not shown). Chromatograms for runs at various NaCl concentrations (50, 100, and 150 mm NaCl) revealed a slight shift to later elution times with increasing ionic strength, suggesting partial destabilization of protein interactions (not shown). However, similar overall peak profiles were observed, suggesting that the core complexes remained intact but that transient and weak protein-protein or protein-RNA associations were destabilized at higher salt concentrations. Based on these optimizations, SEC fractionation was performed at a flow rate of 0.25 ml/min using either buffer A or buffer B.
      A typical SEC chromatogram of the stromal proteome under native conditions is shown in Fig. 2A. The highest peak at fraction 7 (∼13 ml) corresponds to the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) holocomplex at ∼550 kDa that comprises ∼58% of the total stromal mass (
      • Peltier J.B.
      • Cai Y.
      • Sun Q.
      • Zabrouskov V.
      • Giacomelli L.
      • Rudella A.
      • Ytterberg A.J.
      • Rutschow H.
      • van Wijk K.J.
      The oligomeric stromal proteome of Arabidopsis thaliana chloroplasts.
      ) (Fig. 2A). Five fractions for the SEC run with buffer A covering the mass range of 0.8 to >5 MDa were designated as HM-A (fractions 1–5), and the remainder of the fractions were assigned as LM-A (fractions 6–13), spanning a mass range of up to 0.8 MDa. The LM fraction covers the same native mass range as the CN-PAGE stromal proteome analysis (
      • Peltier J.B.
      • Cai Y.
      • Sun Q.
      • Zabrouskov V.
      • Giacomelli L.
      • Rudella A.
      • Ytterberg A.J.
      • Rutschow H.
      • van Wijk K.J.
      The oligomeric stromal proteome of Arabidopsis thaliana chloroplasts.
      ), whereas the HM fraction is complementary to this CN-PAGE analysis. For the SEC run with buffer B, we only analyzed and discussed the HM fraction (HM-B).
      The proteins in the SEC fractions were separated by SDS-PAGE (Fig. 2A). Each gel lane was excised into slices followed by in-gel trypsin digestion and nano-LC-LTQ-Orbitrap analysis of the extracted peptides using data-dependent acquisition and dynamic exclusion in a work flow previously optimized for identification and quantification by spectral counting (
      • Zybailov B.
      • Rutschow H.
      • Friso G.
      • Rudella A.
      • Emanuelsson O.
      • Sun Q.
      • van Wijk K.J.
      Sorting signals, N-terminal modifications and abundance of the chloroplast proteome.
      ). 110 MS runs were analyzed, and the search results and relevant associated information (e.g. ion scores, mass errors, matched sequences, etc.) were uploaded in the PPDB. The PPDB provides an integrated platform for comparing our protein identifications with other in-house MS-based proteomics experiments, annotated properties, and published information (
      • Sun Q.
      • Zybailov B.
      • Majeran W.
      • Friso G.
      • Olinares P.D.
      • van Wijk K.J.
      PPDB, the Plant Proteomics Database at Cornell.
      ). All mass spectral data (the .mgf files reformatted as PRIDE Extensible Markup Language files) were made available via PRIDE at http://www.ebi.ac.uk/pride/.
      It has been shown for LC-MS-based analyses of proteomes that the number of matching MS/MS spectra, here assigned as SPCs, correlates well with protein abundance if there is a sufficient number of SPCs obtained per protein (
      • Liu H.
      • Sadygov R.G.
      • Yates 3rd., J.R.
      A model for random sampling and estimation of relative protein abundance in shotgun proteomics.
      ,
      • Zybailov B.
      • Coleman M.K.
      • Florens L.
      • Washburn M.P.
      Correlation of relative abundance ratios derived from peptide ion chromatograms and spectrum counting for quantitative proteomic analysis using stable isotope labeling.
      ,
      • Old W.M.
      • Meyer-Arendt K.
      • Aveline-Wolf L.
      • Pierce K.G.
      • Mendoza A.
      • Sevinsky J.R.
      • Resing K.A.
      • Ahn N.G.
      Comparison of label-free methods for quantifying human proteins by shotgun proteomics.
      ,
      • Lu P.
      • Vogel C.
      • Wang R.
      • Yao X.
      • Marcotte E.M.
      Absolute protein expression profiling estimates the relative contributions of transcriptional and translational regulation.
      ). To correct for SPCs derived from peptides shared between proteins, adjSPCs were calculated as the sum of unique SPCs and a proportional distribution of shared SPCs (see “Experimental Procedures”). In addition, the relative concentration for each identified protein in the HM range was calculated as the NSAF derived from adjSPC weighted for the number of theoretical tryptic peptides with a relevant length (“observable peptides”) (
      • Zybailov B.
      • Mosley A.L.
      • Sardiu M.E.
      • Coleman M.K.
      • Florens L.
      • Washburn M.P.
      Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae.
      ). To increase the confidence and significance of the data sets in this study, proteins that were matched to only one amino acid sequence (irrespective of charge state, post-translational modification, or number of SPCs) were excluded from further analysis (with the exception of three small ribosomal proteins; see MS/MS spectra in supplemental Fig. 1).

       Comparison with Other Chloroplast Proteomics Studies Demonstrates Enrichment of Plastid Gene Expression Components

      In total, we identified 1081 proteins in the HM-A, LM-A, and HM-B fractions (not shown, but see PPDB) of which 347 were not identified in our comprehensive total chloroplast proteome analysis (membranes and stroma) in which 1325 proteins were identified (
      • Zybailov B.
      • Rutschow H.
      • Friso G.
      • Rudella A.
      • Emanuelsson O.
      • Sun Q.
      • van Wijk K.J.
      Sorting signals, N-terminal modifications and abundance of the chloroplast proteome.
      ). For the purpose of the current study, we imposed an additional stringency demanding at least two different matched amino acid sequences per protein and at least two adjSPCs, reducing the new data set to 664 proteins. The reason for this extra stringency is that low abundance proteins were the focus in this study (up to more than 100,000-fold lower in abundance than Rubisco), and this extra filter reduced identification of non-chloroplast proteins and false positives even if several “novel” chloroplast proteins were removed (including pentatricopeptide repeat (PPR) proteins). Following the grouping of closely related homologues and removal of 54 non-chloroplast proteins and 47 chloroplast membrane(-associated) proteins, we identified 542 stromal proteins (and protein groups) of which 86% had a TargetP predicted chloroplast transit peptide (cTP) (supplemental Table 1). 60 of these 542 stromal proteins were not observed in our previous analysis (
      • Zybailov B.
      • Rutschow H.
      • Friso G.
      • Rudella A.
      • Emanuelsson O.
      • Sun Q.
      • van Wijk K.J.
      Sorting signals, N-terminal modifications and abundance of the chloroplast proteome.
      ); 37 of those 60 were in the HM set and were dominated by chloroplast ribosomal proteins, various splice factors, and proteins with unknown function, such as PPR domain proteins and DNA-binding proteins. In contrast, the new proteins in the LM fractions had a variety of functions (supplemental Table 1). Thus, the analysis of MDa-sized assemblies revealed low abundance proteins mostly involved in plastid gene expression.

       Comparison of HM and LM Data Sets Reveals Effective SEC Fractionation and Enrichment for Plastid Gene Expression Components

      Fig. 2B compares the identified stromal proteins in HM-A and LM-A fractions. Importantly, there was only an 11% overlap (76 proteins) between these two data sets, showing that SEC effectively preserved and separated specific protein complexes above and below 0.8 MDa. This overlap consisted predominantly of highly abundant proteins involved in primary carbon metabolism, such as Rubisco components. Comparison of the distribution of functions of the non-overlapping protein fractions showed a >5-fold enrichment in plastid gene expression in the HM fractions (47% all proteins) at the expense of all other functions except for protein homeostasis components (e.g. chaperones).
      Table I and supplemental Table 2 list annotated functions and relative abundance of proteins (NSAF) in the HM sets. Moreover, the heat maps (from NadjSPC) show the distribution for each protein across the SEC mass range. Excluding the 77 proteins that were also found in the LM data set, 27 of the 30 most abundant proteins are ribosomal subunits, further confirming that the HM fractions are highly enriched with proteins involved in plastid gene expression. This enrichment is consistent with the presence of plastid rRNAs and plastid DNA in the HM fraction (Fig. 2C). The average relative abundance of ribosomal proteins across the HM fractions is 0.01 (Table I), and this will be used to assess the accumulation levels of ribosome-associated proteins (see further below).
      Table IElution profiles of stromal proteins identified in megadalton-sized SEC fractions
      Table thumbnail fx2

       Profiling of Macromolecular Assemblies in Stroma

      The SEC elution profiles of proteins, derived from NadjSPC values across the chromatogram, reflect the size range(s) of complexes in which they participate as well as their quantitative distribution over these complexes. To obtain a global view of SEC migration protein profiles and to facilitate the identification of putative interacting proteins, hierarchical clustering was used to group proteins that exhibit similar SEC elution trends (Fig. 1). Several dendrograms from clustering with different minimum thresholds for total adjSPCs were analyzed. Based on these tests, we chose a minimum threshold of 20 total adjSPCs per protein; this threshold minimized noise that might skew the linkages among correlated proteins. The resulting dendrogram (Fig. 3) displayed three main clusters, namely cluster I (essentially proteins confined to complexes >2 MDa), cluster II (1–3 MDa), and cluster III (<1 MDa). The first two clusters are linked, and they are both dominated by ribosomal proteins and associated factors as will be discussed in the following sections. Cluster III is subdivided into cluster III-1 containing several ribosomal proteins and cluster III-2 consisting mainly of proteins that are starting to elute below 1 MDa. Cluster III-2 included components of the Rubisco complex (∼550 kDa), chaperone 60 (∼800 kDa), ferredoxin-glutamine:2-oxoglutarate amidotransferase (∼700 kDa), and Glyceraldehyde 3-phosphate dehydrogenase (600 kDa), which were well resolved by CN-PAGE (
      • Peltier J.B.
      • Cai Y.
      • Sun Q.
      • Zabrouskov V.
      • Giacomelli L.
      • Rudella A.
      • Ytterberg A.J.
      • Rutschow H.
      • van Wijk K.J.
      The oligomeric stromal proteome of Arabidopsis thaliana chloroplasts.
      ).
      Figure thumbnail gr3
      Fig. 3Overview of SEC migration profiles of MDa-sized complexes. A, dendrogram of SEC migration profiles obtained by hierarchical clustering of HM-A proteins (total adjSPC cutoff of 20). The color code representing the relative abundance of the protein per SEC fraction is shown. The three main clusters (I, II, and III) and subclusters of III (III-1 and III-2) are also noted. B, the subcluster (marked as an orange star in A) of the pyruvate dehydrogenase complex (composed of E1, two E2 proteins, and E3 subunits) with SEC elution profile peaking at fraction 1 (mass range >5 MDa).
      Many of the proteins that were grouped by hierarchical clustering coincided with known assemblies, such as plastid PDC; 30, 50, and 70 S ribosomal particles; and ACCase (Fig. 3). For instance, the maize mitochondrial PDC was found to have an estimated mass of about 8–9 MDa (
      • Thelen J.J.
      • Miernyk J.A.
      • Randall D.D.
      Partial purification and characterization of the maize mitochondrial pyruvate dehydrogenase complex.
      ,
      • Thelen J.J.
      • Muszynski M.G.
      • David N.R.
      • Luethy M.H.
      • Elthon T.E.
      • Miernyk J.A.
      • Randall D.D.
      The dihydrolipoamide S-acetyltransferase subunit of the mitochondrial pyruvate dehydrogenase complex from maize contains a single lipoyl domain.
      ). Similarly to the mitochondria PDC, the Arabidopsis plastid-localized PDC components clustered in the mass range greater than 3 MDa (Fig. 3B). As we will show in more detail below, the clustering further confirmed that SEC effectively separated stromal complexes. The remainder of the “Results” will discuss the composition, organization, and function of the observed MDa-sized macromolecular assemblies.

       70 S Ribosomes

      The spinach plastid 70 S ribosome consists of 58 ribosomal proteins with 25 and 33 components comprising the 30 and 50 S complexes, respectively (
      • Yamaguchi K.
      • von Knoblauch K.
      • Subramanian A.R.
      The plastid ribosomal proteins. Identification of all the proteins in the 30 S subunit of an organelle ribosome (chloroplast).
      ,
      • Yamaguchi K.
      • Subramanian A.R.
      The plastid ribosomal proteins. Identification of all the proteins in the 50 S subunit of an organelle ribosome (chloroplast).
      ,
      • Yamaguchi K.
      • Subramanian A.R.
      Proteomic identification of all plastid-specific ribosomal proteins in higher plant chloroplast 30S ribosomal subunit.
      ). The majority of these proteins are homologous to bacterial 70 S ribosome components, but several are unique to the chloroplast (plastid-specific ribosomal proteins (PSRPs)), and they are proposed to perform plastid-specialized ribosomal functions (
      • Yamaguchi K.
      • von Knoblauch K.
      • Subramanian A.R.
      The plastid ribosomal proteins. Identification of all the proteins in the 30 S subunit of an organelle ribosome (chloroplast).
      ,
      • Yamaguchi K.
      • Subramanian A.R.
      Proteomic identification of all plastid-specific ribosomal proteins in higher plant chloroplast 30S ribosomal subunit.
      ,
      • Sharma M.R.
      • Wilson D.N.
      • Datta P.P.
      • Barat C.
      • Schluenzen F.
      • Fucini P.
      • Agrawal R.K.
      Cryo-EM study of the spinach chloroplast ribosome reveals the structural and functional roles of plastid-specific ribosomal proteins.
      ). PSRP-2, -3, and -4 associate with the 30 S subunit, whereas PSRP-5 and PSRP-6 associate with the 50 S subunit (
      • Yamaguchi K.
      • von Knoblauch K.
      • Subramanian A.R.
      The plastid ribosomal proteins. Identification of all the proteins in the 30 S subunit of an organelle ribosome (chloroplast).
      ,
      • Yamaguchi K.
      • Subramanian A.R.
      The plastid ribosomal proteins. Identification of all the proteins in the 50 S subunit of an organelle ribosome (chloroplast).
      ,
      • Yamaguchi K.
      • Subramanian A.R.
      Proteomic identification of all plastid-specific ribosomal proteins in higher plant chloroplast 30S ribosomal subunit.
      ). PSRP-7 interacts with the 30 S particle in Chlamydomonas reinhardtii, Arabidopsis, and rice but is missing in spinach (
      • Beligni M.V.
      • Yamaguchi K.
      • Mayfield S.P.
      Chloroplast elongation factor ts pro-protein is an evolutionarily conserved fusion with the s1 domain-containing plastid-specific ribosomal protein-7.
      ). PSRP-7 is synthesized as a polyprotein consisting of the mature PSRP-7 and the elongation factor (EF)-Ts (PETs), which might be post-translationally processed to render various fused or independent proteins (
      • Beligni M.V.
      • Yamaguchi K.
      • Mayfield S.P.
      Chloroplast elongation factor ts pro-protein is an evolutionarily conserved fusion with the s1 domain-containing plastid-specific ribosomal protein-7.
      ). Arabidopsis orthologues for all but three ribosomal proteins were identified in the HM fractions, accounting for 57 ribosomal proteins (Table I). About 50 and 75% of the 30 and 50 S ribosomal components in Arabidopsis are nucleus-encoded, respectively, consistent with that observed in spinach (
      • Yamaguchi K.
      • von Knoblauch K.
      • Subramanian A.R.
      The plastid ribosomal proteins. Identification of all the proteins in the 30 S subunit of an organelle ribosome (chloroplast).
      ,
      • Yamaguchi K.
      • Subramanian A.R.
      The plastid ribosomal proteins. Identification of all the proteins in the 50 S subunit of an organelle ribosome (chloroplast).
      ,
      • Yamaguchi K.
      • Subramanian A.R.
      Proteomic identification of all plastid-specific ribosomal proteins in higher plant chloroplast 30S ribosomal subunit.
      ). Interestingly, RPL23 is nucleus-encoded in spinach but has two chloroplast-encoded genes in Arabidopsis (Atcg00840.1 and Atcg01300.1), suggesting evolutionary divergence.
      Several Arabidopsis chloroplast ribosomal proteins that were identified are encoded by multiple genes. Five of these, namely RPS7, RPS12, RPL2, RPL12, and RPL23, comprise two or three identical gene products. Four of these protein groups are chloroplast-encoded, suggesting a possible regulatory role or adaptation to specific conditions in plastid gene expression. In addition, two paralogues each were assigned to RPL18 (23% identity) and to RPL19 (74% identity, variable N-terminal regions), and these related proteins were distinguishable by MS analysis. Interestingly, RPL18A was found to be 6 times more abundant than RPL18B. In addition, one chloroplast and two nuclear genes encode for RPS16 in Arabidopsis (
      • Asakura Y.
      • Barkan A.
      Arabidopsis orthologs of maize chloroplast Splicing factors promote splicing of orthologous and species-specific group II introns.
      ,
      • Ueda T.
      • Matsuda N.
      • Anai T.
      • Tsukaya H.
      • Uchimiya H.
      • Nakano A.
      An Arabidopsis gene isolated by a novel method for detecting genetic interaction in yeast encodes the GDP dissociation inhibitor of Ara4 GTPase.
      ), but only one paralogue (At4g34620.1) was detected in this study.
      The ribosomal components RPS21, RPL34, and PSRP-6 were not detected in this study or in previous published chloroplast proteomics studies on Arabidopsis. One reason might be that they are short, lysine- and arginine-rich proteins, yielding small tryptic peptides that were not amenable for LC-MS analysis. Another possibility is the location and function of these proteins in the ribosome where they could easily be detached. RPS21 is found comprising the top of the head of the 30 S region in prokaryotic ribosomes (
      • Liljas A.
      ). PSRP-6 has been found to be loosely associated with the ribosome (
      • Sharma M.R.
      • Wilson D.N.
      • Datta P.P.
      • Barat C.
      • Schluenzen F.
      • Fucini P.
      • Agrawal R.K.
      Cryo-EM study of the spinach chloroplast ribosome reveals the structural and functional roles of plastid-specific ribosomal proteins.
      ) and is detected at lower amounts compared with other spinach ribosomal proteins (
      • Yamaguchi K.
      • von Knoblauch K.
      • Subramanian A.R.
      The plastid ribosomal proteins. Identification of all the proteins in the 30 S subunit of an organelle ribosome (chloroplast).
      ).
      To explore the assembly state of the ribosome and to co-localize ribosome-associated factors, we carried out a separate hierarchical clustering of all 53 assigned ribosomal proteins, (co-)translational factors, and ribosome biogenesis factors with 20 or more adjSPCs (Fig. 4A). The left-hand panel shows the complete dendrogram in which three clusters (A, B, and C) were distinguished with cluster A representing the 30 S particle, cluster B representing the 50 S particle, and cluster C representing the translating 70 S ribosome. Close-ups of these three clusters with protein names are shown in the three other panels of Fig. 4A. Protein components and factors known/expected to specifically associate with the 30 S particle are in blue, and those part of the 50 S particle are in black. Proteins that have a function in ribosome biogenesis or translation but that are not an integral part of the ribosome are listed in red and italics. The 30 S particle peaked in the 1–2-MDa range (fraction 4), and the 50 S particle peaked in the 2–3-MDa range (fraction 3), whereas the 70 S ribosomes peaked in fraction 2 (Fig. 4A). Northern blot analyses of rRNAs for 30 and 50 S particles extracted from these fractions were consistent with these profiles (Fig. 2C). Overall, the protein and RNA profiles of these ribonucleoparticles correspond to 30 and 50 S subunits (possibly in different stages of maturation), 70 S ribosomes, and polysomes.
      Figure thumbnail gr4
      Fig. 4Characterization of 30 and 50 S ribosomal particles and associated proteins by hierarchical clustering, annotation, and heat maps. A, hierarchical clustering of all 53 assigned ribosomal proteins, (co-)translational factors, and ribosome biogenesis factors with 20 or more adjSPCs. The left-hand panel shows the complete dendrogram; three clusters were distinguished with cluster A representing the 30 S particle, cluster B representing the 50 S particle, and cluster C representing the translating 70 S ribosome. Close-ups of these three clusters with protein names are shown in the three other panels. Protein components and factors specifically associated with the 30 S particle are in blue, and those part of the 50 S particle are in black. Proteins that have a function in ribosome biogenesis or translation but that are not an integral part of the ribosome are listed in red and in italics. B, schematic summarizing the overview of the ribosome assembly process (upper panel) and protein translation process (lower panel) with identified factors indicated. C, heat map of annotated proteins (based on NadjSPC) identified with two or more adjSPCs and known or expected to associate with ribosomes and preribosomal particles. The proteins are grouped according to function as indicated (translation and co-translational modifications, ribosome biogenesis, and RNA maturation (mat.)). The scale is indicated with NadjSPC values for each fraction normalized to the maximum value (see ).
      We note that the ribosome-associated factors are substoichiometric to the ribosomal proteins, and many were quantified with fewer than 20 total adjSPCs, and they were therefore excluded from the clustering in Fig. 4A. However, to show the distribution of these proteins across the HM fractions, we show a heat map of all 30 proteins in Fig. 4B. In the next sections, we will discuss these ribosome-associated factors in more detail.

       Protein Translation

      The translation machinery requires the participation of ribosome-associating initiation, elongation, and termination factors as well as proteins aiding in ribosome recycling (
      • Liljas A.
      ,
      • Ramakrishnan V.
      Ribosome structure and the mechanism of translation.
      ). Translation initiation proceeds with the binding of the mRNA transcript to a free 30 S subunit followed by association of the initiator tRNA (fMet-tRNA) and the initiation factors IF1, IF2 and IF3, together forming a preinitiation complex (
      • Liljas A.
      ,
      • Ramakrishnan V.
      Ribosome structure and the mechanism of translation.
      ) (Fig. 4C). We observed all three IFs with IF1 and IF2 being ∼10-fold more abundant than IF3.
      Elongation ensues after binding of the mature 50 S subunit to the preinitiation complex and subsequent release of the three IFs (Fig. 4C). Several rounds of elongation of the nascent peptide proceed by alternating actions of EF-Tu, which introduces new aminoacyl-tRNAs into the peptidyl transfer center in the 50 S ribosome, and EF-G, which translocates the peptidyl-tRNA after spontaneous peptidyl transfer occurs. During each round, EF-Tu dissociates from the peptidyl transfer center upon GTP hydrolysis with the help of EF-Ts, its nucleotide exchange factor (
      • Liljas A.
      ,
      • Ramakrishnan V.
      Ribosome structure and the mechanism of translation.
      ). A point mutation in the chloroplast-localized EF-G protein leads to impairment of chloroplast development within cotyledons (which become white) but not in true leaves (
      • Albrecht V.
      • Ingenfeld A.
      • Apel K.
      Characterization of the snowy cotyledon 1 mutant of Arabidopsis thaliana: the impact of chloroplast elongation factor G on chloroplast development and plant vitality.
      ). Aside from its function in translation, the EF-Tu orthologue in maize plastids was suggested to also serve as a chaperone during heat stress (
      • Bhadula S.K.
      • Elthon T.E.
      • Habben J.E.
      • Helentjaris T.G.
      • Jiao S.
      • Ristic Z.
      Heat-stress induced synthesis of chloroplast protein synthesis elongation factor (EF-Tu) in a heat-tolerant maize line.
      ,
      • Rao D.
      • Momcilovic I.
      • Kobayashi S.
      • Callegari E.
      • Ristic Z.
      Chaperone activity of recombinant maize chloroplast protein synthesis elongation factor, EF-Tu.
      ). Additional translation elongation factors include the GTPases LepA and TypA/BipA proteins; in bacteria, both have been shown to bind to the 70 S ribosomes at the same site as EF-G (
      • Caldon C.E.
      • Yoong P.
      • March P.E.
      Evolution of a molecular switch: universal bacterial GTPases regulate ribosome function.
      ,
      • Caldon C.E.
      • March P.E.
      Function of the universally conserved bacterial GTPases.
      ,
      • Owens R.M.
      • Pritchard G.
      • Skipp P.
      • Hodey M.
      • Connell S.R.
      • Nierhaus K.H.
      • O'Connor C.D.
      A dedicated translation factor controls the synthesis of the global regulator Fis.
      ). In Escherichia coli, LepA is proposed to recognize ribosomes with mistranslocated tRNAs and induce back-translocation for corrective retranslocation (
      • Qin Y.
      • Polacek N.
      • Vesper O.
      • Staub E.
      • Einfeldt E.
      • Wilson D.N.
      • Nierhaus K.H.
      The highly conserved LepA is a ribosomal elongation factor that back-translocates the ribosome.
      ). The bacterial and plant TypA/BipA appear to be particularly important under stress conditions (
      • Pfennig P.L.
      • Flower A.M.
      BipA is required for growth of Escherichia coli K12 at low temperature.
      ,
      • Beckering C.L.
      • Steil L.
      • Weber M.H.
      • Völker U.
      • Marahiel M.A.
      Genomewide transcriptional analysis of the cold shock response in Bacillus subtilis.
      ,
      • Eymann C.
      • Homuth G.
      • Scharf C.
      • Hecker M.
      Bacillus subtilis functional genomics: global characterization of the stringent response by proteome and transcriptome analysis.
      ,
      • Krishnan K.
      • Flower A.M.
      Suppression of Delta bipA phenotypes in Escherichia coli by abolishment of pseudouridylation at specific sites on the 23S rRNA.
      ,
      • Wang F.
      • Zhong N.Q.
      • Gao P.
      • Wang G.L.
      • Wang H.Y.
      • Xia G.X.
      SsTypA1, a chloroplast-specific TypA/BipA-type GTPase from the halophytic plant Suaeda salsa, plays a role in oxidative stress tolerance.
      ).
      The elongation factors detected in the HM fractions are EF-Tu, EF-G, EF-Ts, LepA, and TypA/BipA (Fig. 4B). These elongation factors have different SEC elution profiles (Fig. 4, A and B), but they were mostly found in the 2–5-MDa range and were most likely associated with 70 S ribosomes or polysomes. The primary elongation factor EF-Tu was an abundant protein (same abundance range as the ribosomes) and was seen as a broad peak across all five fractions (Fig. 4, B and C). Similarly, CN-PAGE analysis of stroma showed EF-Tu migrating at multiple native masses (
      • Peltier J.B.
      • Cai Y.
      • Sun Q.
      • Zabrouskov V.
      • Giacomelli L.
      • Rudella A.
      • Ytterberg A.J.
      • Rutschow H.
      • van Wijk K.J.
      The oligomeric stromal proteome of Arabidopsis thaliana chloroplasts.
      ). TypA/BipA, LepA, and PSRP-1 exhibited similar accumulation levels at about 20% of the ribosome. The PETs, which harbors the EF-Ts domain fused to PSRP-7, peaked in the 0.8–1-MDa range (Table I). Cryoelectron microscopy structure analysis and genetic and biochemical studies have shown that PSRP-1 is a translation factor rather than an integral ribosomal protein (
      • Sharma M.R.
      • Wilson D.N.
      • Datta P.P.
      • Barat C.
      • Schluenzen F.
      • Fucini P.
      • Agrawal R.K.
      Cryo-EM study of the spinach chloroplast ribosome reveals the structural and functional roles of plastid-specific ribosomal proteins.
      ,
      • Sharma M.R.
      • Dönhöfer A.
      • Barat C.
      • Marquez V.
      • Datta P.P.
      • Fucini P.
      • Wilson D.N.
      • Agrawal R.K.
      PSRP1 is not a ribosomal protein, but a ribosome binding factor that is recycled by RRF and EF-G.
      ). PSRP-1 contacts the space between the 30 and 50 S subunits, thereby stabilizing the 70 S ribosome, and is proposed to be involved in translation regulation during stress (
      • Sharma M.R.
      • Dönhöfer A.
      • Barat C.
      • Marquez V.
      • Datta P.P.
      • Fucini P.
      • Wilson D.N.
      • Agrawal R.K.
      PSRP1 is not a ribosomal protein, but a ribosome binding factor that is recycled by RRF and EF-G.
      ). PSRP-1 clustered with the 50 S particle and 70 S ribosomes, supporting its role as a translation factor.
      Translation termination occurs after release factors (RFs), which recognize the stop codon, bind to the ribosome. Ribosome recycling then occurs wherein a ribosome recycling factor (RRF) binds to post-termination complexes and, in coordination with EF-G, splits the 70 S ribosomes for the next round of translation (
      • Liljas A.
      ,
      • Ramakrishnan V.
      Ribosome structure and the mechanism of translation.
      ). No RFs and RRFs were observed in the HM fraction, but Arabidopsis orthologues of RF1 (At3g62910.1) and RRF (At3g63190.1) were found in LM-A fractions (see supplemental Table 1).

       Co-translational Protein Processing and Folding

      During translation, the nascent polypeptide chain that extends out of the peptidyl exit tunnel of the 70 S ribosome is subjected to N-terminal modifications as well as protein folding (
      • Kramer G.
      • Boehringer D.
      • Ban N.
      • Bukau B.
      The ribosome as a platform for co-translational processing, folding and targeting of newly synthesized proteins.
      ). Proteins involved in such co-translational activities were indeed found in fractions with 70 S ribosomes (Table I and Fig. 4). These include the enzymes peptide deformylase (PDF) and methionine aminopeptidase (MAP), the chloroplast signal recognition particle (cpSRP54), and trigger factor (TF).
      PDF and MAP are hydrolytic enzymes that together perform co-translational N-terminal methionine removal. PDF removes all N-formyl groups, exposing the amino group of the first methionine, a prerequisite for the subsequent action of MAP (
      • Kramer G.
      • Boehringer D.
      • Ban N.
      • Bukau B.
      The ribosome as a platform for co-translational processing, folding and targeting of newly synthesized proteins.
      ). Two PDFs (PDF1A and PDF1B) have been found in Arabidopsis, but only PDF1B (At5g14660.1), shown to be dually targeted to the chloroplast and mitochondria in Arabidopsis (
      • Serero A.
      • Giglione C.
      • Meinnel T.
      Distinctive features of the two classes of eukaryotic peptide deformylases.
      ,
      • Giglione C.
      • Serero A.
      • Pierre M.
      • Boisson B.
      • Meinnel T.
      Identification of eukaryotic peptide deformylases reveals universality of N-terminal protein processing mechanisms.
      ,
      • Dirk L.M.
      • Williams M.A.
      • Houtz R.L.
      Eukaryotic peptide deformylases. Nuclear-encoded and chloroplast-targeted enzymes in Arabidopsis.
      ), was observed in this study. Several MAP proteins (MAP1B, MAP1C, and MAP1D) exist in plants, and the chloroplast-localized MAP1B (At1g13270.1) (
      • Giglione C.
      • Serero A.
      • Pierre M.
      • Boisson B.
      • Meinnel T.
      Identification of eukaryotic peptide deformylases reveals universality of N-terminal protein processing mechanisms.
      ) was found in this study.
      In co-translational protein targeting, nascent polypeptides with hydrophobic domains are recognized by the signal recognition particle, and they are then targeted as a ribosome-nascent chain complex to membranes (
      • Kramer G.
      • Boehringer D.
      • Ban N.
      • Bukau B.
      The ribosome as a platform for co-translational processing, folding and targeting of newly synthesized proteins.
      ). The chloroplast sorting component cpSRP54 has a role in post-translational targeting of nucleus-encoded thylakoid proteins and has also been implicated in co-translational targeting (
      • Franklin A.E.
      • Hoffman N.E.
      Characterization of a chloroplast homologue of the 54-kDa subunit of the signal recognition particle.
      ,
      • Li X.
      • Henry R.
      • Yuan J.
      • Cline K.
      • Hoffman N.E.
      A chloroplast homologue of the signal recognition particle subunit SRP54 is involved in the posttranslational integration of a protein into thylakoid membranes.
      ,
      • Nilsson R.
      • Brunner J.
      • Hoffman N.E.
      • van Wijk K.J.
      Interactions of ribosome nascent chain complexes of the chloroplast-encoded D1 thylakoid membrane protein with cpSRP54.
      ,
      • Rutschow H.
      • Ytterberg A.J.
      • Friso G.
      • Nilsson R.
      • van Wijk K.J.
      Quantitative proteomics of a chloroplast SRP54 sorting mutant and its genetic interactions with CLPC1 in Arabidopsis.
      ) and, consistently, was detected in this study peaking with ribosomes at the 2–5-MDa range (Fig. 4). In addition, polypeptides are being folded as they emerge from the peptide exit tunnel. The E. coli TF binds to the 70 S exit tunnel and prevents misfolding and aggregation of emerging nascent proteins (
      • Ferbitz L.
      • Maier T.
      • Patzelt H.
      • Bukau B.
      • Deuerling E.
      • Ban N.
      Trigger factor in complex with the ribosome forms a molecular cradle for nascent proteins.
      ). A 55-kDa TF (At5g55220.1) with conserved domains and a 19-kDa truncated form of TF (At2g30695.1) were identified with the 70 S ribosomal subunits. The 55-kDa full-length TF protein was 3-fold more abundant than the truncated form (Table I).
      Chloroplasts contain two dominant protein chaperones systems, namely the CPN60/CPN21/CPN10 (
      • Nishio K.
      • Hirohashi T.
      • Nakai M.
      Chloroplast chaperonins: evidence for heterogeneous assembly of alpha and beta cpn60 polypeptides into a chaperonin oligomer.
      ,
      • Sharkia R.
      • Bonshtien A.L.
      • Mizrahi I.
      • Weiss C.
      • Niv A.
      • Lustig A.
      • Viitanen P.V.
      • Azem A.
      On the oligomeric state of chloroplast chaperonin 10 and chaperonin 20.
      ) and the HSP70/GrpE system (
      • Schroda M.
      • Vallon O.
      • Whitelegge J.P.
      • Beck C.F.
      • Wollman F.A.
      The chloroplastic GrpE homolog of Chlamydomonas: two isoforms generated by differential splicing.
      ). The Arabidopsis HSP70-1 (At4g24280.1) and HSP70-2 (At5g49910.1) are abundant proteins with a broad substrate pool, and their abundance was constant across all the HM-A fractions, consistent with their wide range of substrates. The nucleotide exchange factor of HSP-70, GrpE (At5g17710.1), showed a similar distribution. Based on information about E. coli homologues, it is quite likely that HSP70 also functions in co-translational folding, thereby assisting the TF protein (
      • Kramer G.
      • Boehringer D.
      • Ban N.
      • Bukau B.
      The ribosome as a platform for co-translational processing, folding and targeting of newly synthesized proteins.
      ).

       Ribosome Biogenesis

      The formation of a functional ribosome from more than 50 proteins and four rRNA molecules entails a complex series of coordinated processes, including processing and modification of ribosomal components, assembly, and maturation (
      • Kaczanowska M.
      • Rydén-Aulin M.
      Ribosome biogenesis and the translation process in Escherichia coli.
      ,
      • Connolly K.
      • Culver G.
      Deconstructing ribosome construction.
      ). Several Arabidopsis orthologues of bacterial ribosome assembly factors were identified in the HM-A fractions (Fig. 4B and Table I). Most of these proteins were initially annotated with unknown function, but careful domain analysis, observed homology to bacterial ribosome biogenesis factors (from PSI-BLAST searches (
      • Altschul S.F.
      • Madden T.L.
      • Schäffer A.A.
      • Zhang J.
      • Zhang Z.
      • Miller W.
      • Lipman D.J.
      Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.
      )), and their detection in the ribosomal fractions provided support for their involvement in ribosome biogenesis.
      Several GTPases have been implicated in ribosome assembly in bacteria (
      • Caldon C.E.
      • Yoong P.
      • March P.E.
      Evolution of a molecular switch: universal bacterial GTPases regulate ribosome function.
      ,
      • Caldon C.E.
      • March P.E.
      Function of the universally conserved bacterial GTPases.
      ,
      • Britton R.A.
      Role of GTPases in bacterial ribosome assembly.
      ). The bacterial Obg (ObgE) co-fractionates with the 50 S subunit (
      • Scott J.M.
      • Haldenwang W.G.
      Obg, an essential GTP binding protein of Bacillus subtilis, is necessary for stress activation of transcription factor sigm▵B).
      ,
      • Lin B.
      • Thayer D.A.
      • Maddock J.R.
      The Caulobacter crescentus CgtA(C) protein cosediments with the free 50S ribosomal subunit.
      ,
      • Sato A.
      • Kobayashi G.
      • Hayashi H.
      • Yoshida H.
      • Wada A.
      • Maeda M.
      • Hiraga S.
      • Takeyasu K.
      • Wada C.
      The GTP binding protein Obg homolog ObgE is involved in ribosome maturation.
      ) and the 30 S subunit but not with the 70 S ribosomes (
      • Sato A.
      • Kobayashi G.
      • Hayashi H.
      • Yoshida H.
      • Wada A.
      • Maeda M.
      • Hiraga S.
      • Takeyasu K.
      • Wada C.
      The GTP binding protein Obg homolog ObgE is involved in ribosome maturation.
      ). It associates with 16 and 23 S rRNAs in vitro and with an RNA helicase (
      • Sato A.
      • Kobayashi G.
      • Hayashi H.
      • Yoshida H.
      • Wada A.
      • Maeda M.
      • Hiraga S.
      • Takeyasu K.
      • Wada C.
      The GTP binding protein Obg homolog ObgE is involved in ribosome maturation.
      ). A chloroplast-localized Arabidopsis Obg homologue, AtObgC (At5g18570.1), has been identified, and its GTPase activity has been demonstrated (
      • Bang W.Y.
      • Hata A.
      • Jeong I.S.
      • Umeda T.
      • Masuda T.
      • Chen J.
      • Yoko I.
      • Suwastika I.N.
      • Kim D.W.
      • Im C.H.
      • Lee B.H.
      • Lee Y.
      • Lee K.W.
      • Shiina T.
      • Bahk J.D.
      AtObgC, a plant ortholog of bacterial Obg, is a chloroplast-targeting GTPase essential for early embryogenesis.
      ). Knock-out mutants of AtObgC are embryo-lethal, showing its crucial role in chloroplast development (
      • Bang W.Y.
      • Hata A.
      • Jeong I.S.
      • Umeda T.
      • Masuda T.
      • Chen J.
      • Yoko I.
      • Suwastika I.N.
      • Kim D.W.
      • Im C.H.
      • Lee B.H.
      • Lee Y.
      • Lee K.W.
      • Shiina T.
      • Bahk J.D.
      AtObgC, a plant ortholog of bacterial Obg, is a chloroplast-targeting GTPase essential for early embryogenesis.
      ,
      • Chigri F.
      • Sippel C.
      • Kolb M.
      • Vothknecht U.C.
      Arabidopsis OBG-like GTPase (AtOBGL) is localized in chloroplasts and has an essential function in embryo development.
      ), and studies on knockdown mutants have shown that AtObgC is essential for plastid rRNA processing (
      • Bang W.Y.
      • Hata A.
      • Jeong I.S.
      • Umeda T.
      • Masuda T.
      • Chen J.
      • Yoko I.
      • Suwastika I.N.
      • Kim D.W.
      • Im C.H.
      • Lee B.H.
      • Lee Y.
      • Lee K.W.
      • Shiina T.
      • Bahk J.D.
      AtObgC, a plant ortholog of bacterial Obg, is a chloroplast-targeting GTPase essential for early embryogenesis.
      ). Hflx, a GTPase that is related to ObgE, associates with the 50 S particle (
      • Jain N.
      • Dhimole N.
      • Khan A.R.
      • De D.
      • Tomar S.K.
      • Sajish M.
      • Dutta D.
      • Parrack P.
      • Prakash B.
      E. coli HflX interacts with 50S ribosomal subunits in presence of nucleotides.
      ,
      • Shields M.J.
      • Fischer J.J.
      • Wieden H.J.
      Toward understanding the function of the universally conserved GTPase HflX from Escherichia coli: a kinetic approach.
      ), the 70 S ribosomes (
      • Shields M.J.
      • Fischer J.J.
      • Wieden H.J.
      Toward understanding the function of the universally conserved GTPase HflX from Escherichia coli: a kinetic approach.
      ), and both 16 and 23 S rRNAs (
      • Jain N.
      • Dhimole N.
      • Khan A.R.
      • De D.
      • Tomar S.K.
      • Sajish M.
      • Dutta D.
      • Parrack P.
      • Prakash B.
      E. coli HflX interacts with 50S ribosomal subunits in presence of nucleotides.
      ). In this study, both AtObgC and the Hflx homologues (At5g7960.1) were observed mainly at ∼2 MDa (the 50 S ribosomal fraction), consistent with the results from bacterial studies. YchF/OLA1 is another subclass of the Obg family and exhibits altered nucleotide specificity by hydrolyzing ATP more efficiently than GTP (
      • Koller-Eichhorn R.
      • Marquardt T.
      • Gail R.
      • Wittinghofer A.
      • Kostrewa D.
      • Kutay U.
      • Kambach C.
      Human OLA1 defines an ATPase subfamily in the Obg family of GTP-binding proteins.
      ). The Arabidopsis YchF/OLA1 protein (At1g56050.1) migrated in the 3–5-MDa range (Fig. 4B).
      YqeH, a circularly permuted GTPase, also co-sediments with the 30 S particle and is essential for 16 S rRNA maturation (
      • Anand B.
      • Surana P.
      • Bhogaraju S.
      • Pahari S.
      • Prakash B.
      Circularly permuted GTPase YqeH binds 30S ribosomal subunit: implications for its role in ribosome assembly.
      ). Two YqeH orthologues in Arabidopsis were identified in the HM fractions, and they behave similarly (found in the 30 and 70 S fractions). One of these orthologues, the AtNOA1 protein (At3g47450.1), exhibits GTPase activity in vitro (
      • Moreau M.
      • Lee G.I.
      • Wang Y.
      • Crane B.R.
      • Klessig D.F.
      AtNOS/AtNOA1 is a functional Arabidopsis thaliana cGTPase and not a nitric-oxide synthase.
      ). Moreover, the bacterial YqeH complements AtNOA1-deficient mutants, providing further evidence that AtNOA1 functions as a chloroplast-localized YqeH in plants (
      • Flores-Pérez U.
      • Sauret-Güeto S.
      • Gas E.
      • Jarvis P.
      • Rodríguez-Concepción M.
      A mutant impaired in the production of plastome-encoded proteins uncovers a mechanism for the homeostasis of isoprenoid biosynthetic enzymes in Arabidopsis plastids.
      ,
      • Sudhamsu J.
      • Lee G.I.
      • Klessig D.F.
      • Crane B.R.
      The structure of YqeH. An AtNOS1/AtNOA1 ortholog that couples GTP hydrolysis to molecular recognition.
      ).
      Era is another E. coli GTPase that binds 16 S rRNA and is involved in 30 S subunit maturation (
      • Tu C.
      • Zhou X.
      • Tropea J.E.
      • Austin B.P.
      • Waugh D.S.
      • Court D.L.
      • Ji X.
      Structure of ERA in complex with the 3′ end of 16S rRNA: Implications for ribosome biogenesis.
      ). We observed that the Era orthologue in Arabidopsis (At5g66470.1) was present in the 70 S fractions rather than the 30 S fraction. The E. coli EngA (YphC in Bacillus subtilis) has two contiguous GTPase domains whose nucleotide occupancy modulates its binding to either 30 S alone or to 30, 50, and 70 S ribosomes (
      • Tomar S.K.
      • Dhimole N.
      • Chatterjee M.
      • Prakash B.
      Distinct GDP/GTP bound states of the tandem G-domains of EngA regulate ribosome binding.
      ). It co-sediments with 16 and 23 S rRNAs (
      • Tomar S.K.
      • Dhimole N.
      • Chatterjee M.
      • Prakash B.
      Distinct GDP/GTP bound states of the tandem G-domains of EngA regulate ribosome binding.
      ) and is essential for bacterial growth (
      • Hwang J.
      • Inouye M.
      The tandem GTPase, Der, is essential for the biogenesis of 50S ribosomal subunits in Escherichia coli.
      ). The Arabidopsis orthologue of EngA (At5g6050.1) migrates at >2-MDa range (50 S, 70 S, and polysome fractions). Two mutant alleles of the Arabidopsis EngA are arrested in embryogenesis at the globular stage (
      • Meinke D.
      • Sweeney C.
      • Muralla R.
      Integrating the genetic and physical maps of Arabidopsis thaliana: identification of mapped alleles of cloned essential (EMB) genes.
      ), consistent with an essential function in chloroplast development.
      Additional ribosomal biogenesis factors involved in 16 S rRNA processing and 30 S ribosome maturation include RimM and the ribosome binding factor A protein (RbfA) (
      • Bylund G.O.
      • Wipemo L.C.
      • Lundberg L.A.
      • Wikström P.M.
      RimM and RbfA are essential for efficient processing of 16S rRNA in Escherichia coli.
      ,
      • Lövgren J.M.
      • Bylund G.O.
      • Srivastava M.K.
      • Lundberg L.A.
      • Persson O.P.
      • Wingsle G.
      • Wikström P.M.
      The PRC-barrel domain of the ribosome maturation protein RimM mediates binding to ribosomal protein S19 in the 30S ribosomal subunits.
      ,
      • Datta P.P.
      • Wilson D.N.
      • Kawazoe M.
      • Swami N.K.
      • Kaminishi T.
      • Sharma M.R.
      • Booth T.M.
      • Takemoto C.
      • Fucini P.
      • Yokoyama S.
      • Agrawal R.K.
      Structural aspects of RbfA action during small ribosomal subunit.
      ,
      • Xia B.
      • Ke H.
      • Shinde U.
      • Inouye M.
      The role of RbfA in 16S rRNA processing and cell growth at low temperature in Escherichia coli.
      ). The Arabidopsis homologues of RimM (At5g46420.1) peaked at the 30 S fraction (0.8–2 MDa), and RbfA peaked with the 70 S ribosome (Fig. 4B and Table I).
      rRNA modifications, particularly base methylations, are common to all organisms. Among the bacterial rRNA/tRNA methyltransferases are RsmD, YrdC, and SpoU (
      • Lesnyak D.V.
      • Osipiuk J.
      • Skarina T.
      • Sergiev P.V.
      • Bogdanov A.A.
      • Edwards A.
      • Savchenko A.
      • Joachimiak A.
      • Dontsova O.A.
      Methyltransferase that modifies guanine 966 of the 16 S rRNA. Functional identification and tertiary structure.
      ,
      • Kaczanowska M.
      • Rydén-Aulin M.
      The YrdC protein—a putative ribosome maturation factor.
      ,
      • El Yacoubi B.
      • Lyons B.
      • Cruz Y.
      • Reddy R.
      • Nordin B.
      • Agnelli F.
      • Williamson J.R.
      • Schimmel P.
      • Swairjo M.A.
      • de Crécy-Lagard V.
      The universal YrdC/Sua5 family is required for the formation of threonylcarbamoyladenosine in tRNA.
      ,
      • Tkaczuk K.L.
      • Dunin-Horkawicz S.
      • Purta E.
      • Bujnicki J.M.
      Structural and evolutionary bioinformatics of the SPOUT superfamily of methyltransferases.
      ). The Arabidopsis orthologues of these rRNA-modifying enzymes migrated at different masses with RsmD (At3g28460.1) at 0.8–2 MDa, YrdC (at3g01920.1) at 2–3 MDa, and SpoU (At2g19870.1) at >5 MDa (Fig. 4B and Table I). Other maturation factors include the DAG-like protein DAL and IOJAP. DAL (At2g33430.1) eluted at the 0.8–1-MDa range and is involved in the maturation of rRNAs and ribosome assembly (
      • Bisanz C.
      • Bégot L.
      • Carol P.
      • Perez P.
      • Bligny M.
      • Pesey H.
      • Gallois J.L.
      • Lerbs-Mache S.
      • Mache R.
      The Arabidopsis nuclear DAL gene encodes a chloroplast protein which is required for the maturation of the plastid ribosomal RNAs and is essential for chloroplast differentiation.
      ). The IOJAP protein (At3g12930.1) eluted at the 2–3-MDa fraction. In maize, IOJAP-deficient leaf tissues are ribosomeless, and they lack 16 and 23 S rRNAs and protein synthesis activity (
      • Walbot V.
      • Coe E.H.
      Nuclear gene Iojap conditions a programmed change to ribosome-less plastids in Zea mays.
      ).

       RNA Maturation and Degradation

      Three ribonucleases were observed in the HM fractions (Fig. 4B and Table I), and various lines of evidence suggest their involvement in rRNA maturation in various ribosomal assembly stages. CSP41A (At3g63140.1) and CSP41B (At1g09340.1) are two related endoribonucleases with multiple roles in chloroplast gene expression (
      • Beligni M.V.
      • Mayfield S.P.
      Arabidopsis thaliana mutants reveal a role for CSP41a and CSP41b, two ribosome-associated endonucleases, in chloroplast ribosomal RNA metabolism.
      ,
      • Bollenbach T.J.
      • Sharwood R.E.
      • Gutierrez R.
      • Lerbs-Mache S.
      • Stern D.B.
      The RNA-binding proteins CSP41a and CSP41b may regulate transcription and translation of chloroplast-encoded RNAs in Arabidopsis.
      ). In Arabidopsis, CSP41B co-purified with preribosomal particles but not with mature ribosomes or polysomes (
      • Beligni M.V.
      • Mayfield S.P.
      Arabidopsis thaliana mutants reveal a role for CSP41a and CSP41b, two ribosome-associated endonucleases, in chloroplast ribosomal RNA metabolism.
      ,
      • Bollenbach T.J.
      • Sharwood R.E.
      • Gutierrez R.
      • Lerbs-Mache S.
      • Stern D.B.
      The RNA-binding proteins CSP41a and CSP41b may regulate transcription and translation of chloroplast-encoded RNAs in Arabidopsis.
      ). CN-PAGE analysis of stroma showed that both CSP41A and CSP41B migrated at the >1-MDa range (in the stacking gel) (
      • Peltier J.B.
      • Cai Y.
      • Sun Q.
      • Zabrouskov V.
      • Giacomelli L.
      • Rudella A.
      • Ytterberg A.J.
      • Rutschow H.
      • van Wijk K.J.
      The oligomeric stromal proteome of Arabidopsis thaliana chloroplasts.
      ). Consistently, we observed only CSP41B mostly in fractions with 30 S particles. In contrast, CSP41A was identified in LM-A fractions together with the remainder of CSP41B in agreement with previous observations (
      • Peltier J.B.
      • Cai Y.
      • Sun Q.
      • Zabrouskov V.
      • Giacomelli L.
      • Rudella A.
      • Ytterberg A.J.
      • Rutschow H.
      • van Wijk K.J.
      The oligomeric stromal proteome of Arabidopsis thaliana chloroplasts.
      ,
      • Bollenbach T.J.
      • Sharwood R.E.
      • Gutierrez R.
      • Lerbs-Mache S.
      • Stern D.B.
      The RNA-binding proteins CSP41a and CSP41b may regulate transcription and translation of chloroplast-encoded RNAs in Arabidopsis.
      ).
      Polynucleotide phosphorylase (PNPase) (At3g03710.1) is an exoribonuclease that is indispensable for 3′-end maturation of 23 S rRNA transcripts and the efficiency of the 3′-end processing and polyadenylation of mRNAs as well as RNA degradation (
      • Walter M.
      • Kilian J.
      • Kudla J.
      PNPase activity determines the efficiency of mRNA 3′-end processing, the degradation of tRNA and the extent of polyadenylation in chloroplasts.
      ,
      • Yehudai-Resheff S.
      • Hirsh M.
      • Schuster G.
      Polynucleotide phosphorylase functions as both an exonuclease and a poly(A) polymerase in spinach chloroplasts.
      ,
      • Yehudai-Resheff S.
      • Portnoy V.
      • Yogev S.
      • Adir N.
      • Schuster G.
      Domain analysis of the chloroplast polynucleotide phosphorylase reveals discrete functions in RNA degradation, polyadenylation, and sequence homology with exosome proteins.
      ,
      • Slomovic S.
      • Portnoy V.
      • Yehudai-Resheff S.
      • Bronshtein E.
      • Schuster G.
      Polynucleotide phosphorylase and the archaeal exosome as poly(A)-polymerases.
      ). In E. coli, PNPase is part of a “degradosome” complex along with the endoribonuclease RNase E, a DEAD box RNA helicase, and the glycolytic enzyme enolase (
      • Carpousis A.J.
      The Escherichia coli RNA degradosome: structure, function and relationship in other ribonucleolytic multienzyme complexes.
      ). However, the chloroplast PNPase has been observed as a 600-kDa homo-oligomer in spinach (
      • Baginsky S.
      • Shteiman-Kotler A.
      • Liveanu V.
      • Yehudai-Resheff S.
      • Bellaoui M.
      • Settlage R.E.
      • Shabanowitz J.
      • Hunt D.F.
      • Schuster G.
      • Gruissem W.
      Chloroplast PNPase exists as a homo-multimer enzyme complex that is distinct from the Escherichia coli degradosome.
      ) and as a 410-kDa tetramer in Arabidopsis (
      • Peltier J.B.
      • Cai Y.
      • Sun Q.
      • Zabrouskov V.
      • Giacomelli L.
      • Rudella A.
      • Ytterberg A.J.
      • Rutschow H.
      • van Wijk K.J.
      The oligomeric stromal proteome of Arabidopsis thaliana chloroplasts.
      ). Interestingly, PNPase in the SEC fraction was observed to peak in ∼1–3 MDa (50 S fractions) (Fig. 4B), suggesting interactions with RNA-containing complexes.
      RNase J is another endonuclease that is implicated in 16 S rRNA maturation and associates with both assembled 70 S ribosomes and 30 S particles, suggesting a role in ribosome assembly (
      • Britton R.A.
      • Wen T.
      • Schaefer L.
      • Pellegrini O.
      • Uicker W.C.
      • Mathy N.
      • Tobin C.
      • Daou R.
      • Szyk J.
      • Condon C.
      Maturation of the 5′ end of Bacillus subtilis 16S rRNA by the essential ribonuclease YkqC/RNase J1.
      ). The Arabidopsis orthologue (At5g63420.1) was found to be essential for embryogenesis (EMB2746) (
      • Meinke D.
      • Sweeney C.
      • Muralla R.
      Integrating the genetic and physical maps of Arabidopsis thaliana: identification of mapped alleles of cloned essential (EMB) genes.
      ). In this study, it was found to elute at a wide range of masses (>1 MDa) at relatively abundant levels, suggesting interactions with RNA bound to various ribonucleoprotein complexes (polysomes and 70 and 30 S particles).

       RNA Helicases

      DEAD box proteins possess the characteristic Asp-Glu-Ala-Asp sequence, an RNA-binding motif, and an ATP-hydrolyzing domain and are mainly involved in ATP-dependent rearrangement of inter- and intramolecular RNA structures or remodeling of ribonucleoprotein complexes (
      • Rocak S.
      • Linder P.
      Dead-box proteins: The driving forces behind RNA metabolism.
      ,
      • Linder P.
      Dead-box proteins: a family affair–active and passive players in RNP-remodeling.
      ). There are 58 predicted genes for DEAD box proteins in Arabidopsis (
      • Boudet N.
      • Aubourg S.
      • Toffano-Nioche C.
      • Kreis M.
      • Lecharny A.
      Evolution of intron/exon structure of DEAD helicase family genes in Arabidopsis, Caenorhabditis, and Drosophila.
      ,
      • Mingam A.
      • Toffano-Nioche C.
      • Brunaud V.
      • Boudet N.
      • Kreis M.
      • Lecharny A.
      DEAD-box RNA helicases in Arabidopsis thaliana: establishing a link between quantitative expression, gene structure and evolution of a family of genes.
      ); nine of these are predicted by TargetP (
      • Emanuelsson O.
      • Nielsen H.
      • Brunak S.
      • von Heijne G.
      Predicting subcellular localization of proteins based on their N-terminal amino acid sequence.
      ) to be plastid-localized.
      Several DEAD box RNA helicases (RHs) were identified in this study and appeared in almost all fractions >1 MDa, suggesting associations with a variety of RNA-containing protein complexes. RH3 (At5g26742.1), which belongs to a subgroup of DEAD box proteins containing a Gly/Arg/Ser-rich C-terminal extension (
      • Aubourg S.
      • Kreis M.
      • Lecharny A.
      The DEAD box RNA helicase family in Arabidopsis thaliana.
      ), was found to be the most abundant (10× higher) of these RHs. RH3 was also identified in fractions above 1 MDa in the CN-PAGE fractionation of stroma (
      • Peltier J.B.
      • Cai Y.
      • Sun Q.
      • Zabrouskov V.
      • Giacomelli L.
      • Rudella A.
      • Ytterberg A.J.
      • Rutschow H.
      • van Wijk K.J.
      The oligomeric stromal proteome of Arabidopsis thaliana chloroplasts.
      ). An Arabidopsis RH3 knock-out mutant is embryo-lethal (EMB1138) (
      • Meinke D.
      • Sweeney C.
      • Muralla R.
      Integrating the genetic and physical maps of Arabidopsis thaliana: identification of mapped alleles of cloned essential (EMB) genes.
      ), indicating a crucial role in plant development. RH26 (At5g0860.1) has a long N-terminal extension containing seven internal Arg-Ser-Asp repeats (
      • Aubourg S.
      • Kreis M.
      • Lecharny A.
      The DEAD box RNA helicase family in Arabidopsis thaliana.
      ). In addition, RH26-deficient plants display a pale green leaf phenotype, suggesting impairment in chloroplast development (
      • Myouga F.
      • Akiyama K.
      • Motohashi R.
      • Kuromori T.
      • Ito T.
      • Iizumi H.
      • Ryusui R.
      • Sakurai T.
      • Shinozaki K.
      The Chloroplast Function Database: a large-scale collection of Arabidopsis Ds/Spm- or T-DNA-tagged homozygous lines for nuclear-encoded chloroplast proteins, and their systematic phenotype analysis.
      ). RH50 (At3g06980.1) is the Arabidopsis orthologue of the rice OsBIRH1, which exhibits RNA helicase activities in vitro and is involved in conferring plant resistance against various stresses (
      • Li D.
      • Liu H.
      • Zhang H.
      • Wang X.
      • Song F.
      OsBIRH1, a DEAD-box RNA helicase with functions in modulating defence responses against pathogen infection and oxidative stress.
      ).

       Transcription and DNA Binding

      Proteins involved in transcription and other functions involving DNA association were observed in the HM fractions although at relatively low concentrations ( 110 of the ribosomal abundance) (Table I). The PEP complex is composed of four subunits, namely RpoA, RpoB, RpoC1, and RpoC2 (
      • Little M.C.
      • Hallick R.B.
      Chloroplast rpoA, rpoB, and rpoC genes specify at least three components of a chloroplast DNA-dependent RNA polymerase active in tRNA and mRNA transcription.
      ), and is the predominant transcription complex in mature chloroplasts (
      • Liere K.
      • Börner T.
      Transcription and transcriptional regulation in plastids.
      ). The tobacco PEP complex was affinity-purified and was observed in a native gradient gel migrating at >900 kDa (
      • Suzuki J.Y.
      • Ytterberg A.J.
      • Beardslee T.A.
      • Allison L.A.
      • Wijk K.J.
      • Maliga P.
      Affinity purification of the tobacco plastid RNA polymerase and in vitro reconstitution of the holoenzyme.
      ). In the current study, the PEP complex was observed to migrate at masses >2 MDa, suggesting association with HM complexes, most likely plastid DNA as part of the nucleoid (DNA-protein assembly). Indeed, PCR analysis confirmed the presence of plastid DNA, particularly in fractions with masses >3 MDa (Fig. 2C).
      Analysis of purified transcriptionally active chromosomes (TACs) from Arabidopsis chloroplasts revealed 35 proteins involved in plastid gene expression, including the PEP complex and 18 proteins (pTACs 1 to 18) that contain RNA/DNA-binding domains (
      • Pfalz J.
      • Liere K.
      • Kandlbinder A.
      • Dietz K.J.
      • Oelmüller R.
      pTAC2, -6, and -12 are components of the transcriptionally active plastid chromosome that are required for plastid gene expression.
      ). In the current analysis, we found pTACs 2, 16, and 17 eluting with the PEP fraction. Moreover, a DNA gyrase (At3g10690.1), a thioredoxin protein (At3g06730.1), and a pfkB-type carbohydrate kinase (At1g69200.1), which were found in the TAC preparations, were also seen in the current study. In addition, several DNA-binding proteins that were not observed in the TAC analysis (
      • Pfalz J.
      • Liere K.
      • Kandlbinder A.
      • Dietz K.J.
      • Oelmüller R.
      pTAC2, -6, and -12 are components of the transcriptionally active plastid chromosome that are required for plastid gene expression.
      ) clearly co-eluted with the PEP complex and pTAC proteins in the current study. These include two proteins involved in DNA damage repair, namely DNA mismatch repair MutS (At1g65070.1) and DNA repair protein RecA (At1g79050.1). A DNA topoisomerase (At4g31210.1) and a relatively high abundance DNA helicase (At45g35790.1) were observed peaking at 1–2 MDa. Finally, several proteins with DnaJ domains (At2g22360.1 and At4g39960.1) were found in fractions >2 MDa, and the protein heat map suggested association with the nucleoid (Table I).

       Post-transcriptional Events: RNA Intron Splicing, Processing, and Editing

      Proteins harboring single or multiple CRS1-YhbY domains, also called the chloroplast ribosome maturation (CRM) domain, participate in the assembly of catalytic ribonucleoprotein complexes, namely group II intron particles and the 50 S ribosomal subunit (
      • Barkan A.
      • Klipcan L.
      • Ostersetzer O.
      • Kawamura T.
      • Asakura Y.
      • Watkins K.P.
      The CRM domain: an RNA binding module derived from an ancient ribosome-associated protein.
      ). In Arabidopsis, 16 proteins are predicted to have single or multiple CRM domains (
      • Barkan A.
      • Klipcan L.
      • Ostersetzer O.
      • Kawamura T.
      • Asakura Y.
      • Watkins K.P.
      The CRM domain: an RNA binding module derived from an ancient ribosome-associated protein.
      ). CAF1 (At2g20020.1) has two CRM domains and is involved in group II intron RNA splicing (
      • Asakura Y.
      • Barkan A.
      Arabidopsis orthologs of maize chloroplast Splicing factors promote splicing of orthologous and species-specific group II introns.
      ). Aside from CAF1, two other CRM domain proteins were identified in this study. These include At4g39040.1 and At3g18390.1, which have one and three CRM domains, respectively. Two mutant alleles for the latter protein are embryo-lethal (EMB1865) with embryo development arrested at the globular stage, suggesting its importance in plastid development (
      • Meinke D.
      • Sweeney C.
      • Muralla R.
      Integrating the genetic and physical maps of Arabidopsis thaliana: identification of mapped alleles of cloned essential (EMB) genes.
      ).
      In maize chloroplasts, WTF1 is found in group II intron ribonucleoprotein complexes (600–800 kDa) and cooperates with RNC1 in promoting group II intron splicing (
      • Kroeger T.S.
      • Watkins K.P.
      • Friso G.
      • van Wijk K.J.
      • Barkan A.
      A plant-specific RNA-binding domain revealed through analysis of chloroplast group II intron splicing.
      ). At4g01037.1 and At4g37510.1, the respective Arabidopsis orthologues of maize WTF1 and RNC1, did not exhibit identical elution profiles in our SEC analysis but still indicated interactions with ribonucleoprotein complexes with WTF1 eluting at 1–2 MDa and RNC1 eluting at 3–5 MDa (Table I). In addition, a chloroplast-encoded maturase K (Atcg00040.1) was observed at the 1–2-MDa region in this study (Table I). The RNA binding properties and function of maturase K in other plants have been studied (
      • Liere K.
      • Link G.
      RNA-binding activity of the matK protein encoded by the chloroplast trnK intron from mustard (Sinapis alba L.).
      ,
      • Vogel J.
      • Hübschmann T.
      • Börner T.
      • Hess W.R.
      Splicing and intron-internal RNA editing of trnK-matK transcripts in barley plastids: support for MatK as an essential splice factor.
      ).
      About 450 PPR proteins are predicted in Arabidopsis, and they are recognized as major mediators of organelle gene expression, although their specific functions remain poorly characterized (
      • Lurin C.
      • Andrés C.
      • Aubourg S.
      • Bellaoui M.
      • Bitton F.
      • Bruyère C.
      • Caboche M.
      • Debast C.
      • Gualberto J.
      • Hoffmann B.
      • Lecharny A.
      • Le Ret M.
      • Martin-Magniette M.L.
      • Mireau H.
      • Peeters N.
      • Renou J.P.
      • Szurek B.
      • Taconnat L.
      • Small I.
      Genome-wide analysis of Arabidopsis pentatricopeptide repeat proteins reveals their essential role in organelle biogenesis.
      ). Eight PPRs were observed in the HM fractions (Table I). In maize, the PPR proteins PPR4 and CRP1 are required for the translation of several chloroplast-encoded transcripts (
      • Schmitz-Linneweber C.
      • Williams-Carrier R.E.
      • Williams-Voelker P.M.
      • Kroeger T.S.
      • Vichas A.
      • Barkan A.
      A pentatricopeptide repeat protein facilitates the trans-splicing of the maize chloroplast rps12 pre-mRNA.
      ,
      • Schmitz-Linneweber C.
      • Williams-Carrier R.
      • Barkan A.
      RNA immunoprecipitation and microarray analysis show a chloroplast Pentatricopeptide repeat protein to be associated with the 5′ region of mRNAs whose translation it activates.
      ,
      • Barkan A.
      • Walker M.
      • Nolasco M.
      • Johnson D.
      A nuclear mutation in maize blocks the processing and translation of several chloroplast mRNAs and provides evidence for the differential translation of alternative mRNA forms.
      ,
      • Fisk D.G.
      • Walker M.B.
      • Barkan A.
      Molecular cloning of the maize gene crp1 reveals similarity between regulators of mitochondrial and chloroplast gene expression.
      ). The Arabidopsis orthologues of PPR4 (At5g04810.1) and CRP1 (At5g432310.1) eluted at 3–5 and 1–3 MDa, respectively. In addition, the RNA-binding P67 (At4g16390.1) (
      • Lahmy S.
      • Barnèche F.
      • Derancourt J.
      • Filipowicz W.
      • Delseny M.
      • Echeverria M.
      A chloroplastic RNA-binding protein is a new member of the PPR family.
      ) was found at fractions >2 MDa (Table I).
      Three proteins with S1 RNA-binding domains (At1g17200.1, At3g23700.1, and At1g1280.1.) were found to elute at fractions >1MDa with At3g23700.1 being 10-fold more abundant than the other two. Plants deficient in At1g7200.1 expression exhibit pale green cotyledons and leaves (
      • Myouga F.
      • Akiyama K.
      • Motohashi R.
      • Kuromori T.
      • Ito T.
      • Iizumi H.
      • Ryusui R.
      • Sakurai T.
      • Shinozaki K.
      The Chloroplast Function Database: a large-scale collection of Arabidopsis Ds/Spm- or T-DNA-tagged homozygous lines for nuclear-encoded chloroplast proteins, and their systematic phenotype analysis.
      ), indicating a role in chloroplast development. Another RNA-binding protein, HCF173 (At1g16720.1), was found in the 1–3-MDa fractions, consistent with its detection in mRNA-containing HM complexes (
      • Schult K.
      • Meierhoff K.
      • Paradies S.
      • Töller T.
      • Wolff P.
      • Westhoff P.
      The nuclear-encoded factor HCF173 is involved in the initiation of translation of the psbA mRNA in Arabidopsis thaliana.
      ).
      Chloroplast ribonucleoproteins (cpRNPs) are highly abundant proteins that associate with various RNA species for RNA processing and/or stabilization (
      • Nakamura T.
      • Ohta M.
      • Sugiura M.
      • Sugita M.
      Chloroplast ribonucleoproteins function as a stabilizing factor of ribosome-free mRNAs in the stroma.
      ). In tobacco, cp29A, cp29B, and cp33 were mostly found as non-ribosome-bound ribonucleoprotein complexes but were also detected in fractions >600 kDa (
      • Nakamura T.
      • Ohta M.
      • Sugiura M.
      • Sugita M.
      Chloroplast ribonucleoproteins function as a stabilizing factor of ribosome-free mRNAs in the stroma.
      ). In this study, cp29B (At2g37220.1) and two cp33 proteins (At1g01080.1 and At2g35410.1) were observed in the 0.8–2-MDa range with similar elution profiles (Table I). Several cpRNPs are involved in RNA editing (
      • Hirose T.
      • Sugiura M.
      Involvement of a site-specific trans-acting factor and a common RNA-binding protein in the editing of chloroplast mRNAs: development of a chloroplast in vitro RNA editing system.
      ,
      • Tillich M.
      • Hardel S.L.
      • Kupsch C.
      • Armbruster U.
      • Delannoy E.
      • Gualberto J.M.
      • Lehwark P.
      • Leister D.
      • Small I.D.
      • Schmitz-Linneweber C.
      Chloroplast ribonucleoprotein CP31A is required for editing and stability of specific chloroplast mRNAs.
      ), although this has not yet been demonstrated for the cpRNPs identified in this study.

       Lipid Metabolism

      Aside from ribosomes and ribosome-associated proteins, the other function that is enriched in the HM data set is fatty acid synthesis. Two hetero-oligomeric complexes, namely PDC and ACCase, were observed (see Fig. 3 and Table I).
      The PDC is composed of multiple copies of three enzymes. The E1 component is a pyruvate dehydrogenase (consisting of α- and β-subunits), E2 is a dihydrolipoamide acetyltransferase, and E3 is a dihydrolipoamide dehydrogenase. PDCs form large complexes composed of a core complex of eight trimers (cube) or 20 trimers (pentagonal dodecahedron) of E2 with E1 with E3 promoting substrate channeling across the three enzyme components (
      • Mooney B.P.
      • Miernyk J.A.
      • Randall D.D.
      The complex fate of alpha-ketoacids.
      ). The maize mitochondrial PDC was found to have an estimated mass of about 8–9 MDa (
      • Thelen J.J.
      • Miernyk J.A.
      • Randall D.D.
      Partial purification and characterization of the maize mitochondrial pyruvate dehydrogenase complex.
      ) due in part to the 2.7-MDa E2 core (
      • Thelen J.J.
      • Muszynski M.G.
      • David N.R.
      • Luethy M.H.
      • Elthon T.E.
      • Miernyk J.A.
      • Randall D.D.
      The dihydrolipoamide S-acetyltransferase subunit of the mitochondrial pyruvate dehydrogenase complex from maize contains a single lipoyl domain.
      ). The plastid-localized PDC from pea chloroplasts dissociates rapidly in vitro, making the estimation of its organization and composition difficult (
      • Camp P.J.
      • Randall D.D.
      Purification and characterization of the pea chloroplast pyruvate dehydrogenase complex: a source of acetyl-CoA and NADH for fatty acid biosynthesis.
      ).
      The PDC components eluted at a mass range >5 MDa (Fig. 3 and Table I). All of the subunits of the plastid-localized PDC are nucleus-encoded, and all, except the E1 α component (At1g01090.1), are encoded by more than one gene. An E1 β-subunit (At1g3020.1) has been characterized previously (
      • Johnston M.L.
      • Luethy M.H.
      • Miernyk J.A.
      • Randall D.D.
      Cloning and molecular analyses of the Arabidopsis thaliana plastid pyruvate dehydrogenase subunits.
      ) and was observed in this experiment with unique peptides. LTA2 (At3g25860.1) is an E2 component and has been shown to exhibit dihydrolipoamide acetyltransferase activity (
      • Mooney B.P.
      • Miernyk J.A.
      • Randall D.D.
      Cloning and characterization of the dihydrolipoamide S-acetyltransferase subunit of the plastid pyruvate dehydrogenase complex (E2) from Arabidopsis.
      ). The T-DNA insertion mutant for LTA2 is embryo-lethal (
      • Lin M.
      • Behal R.
      • Oliver D.J.
      Disruption of plE2, the gene for the E2 subunit of the plastid pyruvate dehydrogenase complex, in Arabidopsis causes an early embryo lethal phenotype.
      ). Another E2 subunit (At1g34330.1) was found in the genome database (
      • Mooney B.P.
      • Miernyk J.A.
      • Randall D.D.
      The complex fate of alpha-ketoacids.
      ), and a T-DNA insertion mutant (EMB3003) for this gene is also embryo-lethal (
      • Meinke D.
      • Sweeney C.
      • Muralla R.
      Integrating the genetic and physical maps of Arabidopsis thaliana: identification of mapped alleles of cloned essential (EMB) genes.
      ). Interestingly, two elution peaks were observed for E2, one at >5 MDa and another at 1–2 MDa (see Table I), suggesting different oligomeric states for the E2 core with or without bound E1 and E3. The two plastidic E3 isoforms (At3g16950.1 or ptlpd1 and At4g16155.1 or ptlpd2) are 85% identical and were characterized previously (
      • Lutziger I.
      • Oliver D.J.
      Molecular evidence of a unique lipoamide dehydrogenase in plastids: analysis of plastidic lipoamide dehydrogenase from Arabidopsis thaliana.
      ,
      • Drea S.C.
      • Mould R.M.
      • Hibberd J.M.
      • Gray J.C.
      • Kavanagh T.A.
      Tissue-specific and developmental-specific expression of an Arabidopsis thaliana gene encoding the lipoamide dehydrogenase component of the plastid pyruvate dehydrogenase complex.
      ). The presence of different paralogues for the three central components suggests that the plastid PDC population is heterogeneous; further experimentation is needed to determine the biological significance.
      The plastid-localized heteromeric ACCase catalyzes the first committed step in de novo fatty acid synthesis, which occurs solely in plastids. ACCase is composed of biotin carboxylase, biotin carboxyl carrier protein (BCCP), α-carboxyltransferase (α-CT), and β-carboxyltransferase (β-CT) subunits. The pea chloroplast ACCase was found to elute at about 650–700 kDa in gel filtration analysis (
      • Sasaki Y.
      • Nagano Y.
      Plant acetyl-CoA carboxylase: structure, biosynthesis, regulation and gene manipulation for plant breeding.
      ). In this study, the Arabidopsis ACCase subunits were all found in the 1–2-MDa range (Table I). These include biotin carboxylase (At5g35360.1) (
      • Sun J.
      • Ke J.
      • Johnson J.L.
      • Nikolau B.J.
      • Wurtele E.S.
      Biochemical and molecular biological characterization of CAC2, the Arabidopsis thaliana gene coding for the biotin carboxylase subunit of the plastidic acetyl-coenzyme A carboxylase.
      ), α-CT (At2g38040.1), and the chloroplast-encoded β-CT (Atcg00500.1) as well as four BCCPs that were identified with distinct peptides. BCCP1 (At5g16390.1) has been well characterized (
      • Ke J.
      • Choi J.K.
      • Smith M.
      • Horner H.T.
      • Nikolau B.J.
      • Wurtele E.S.
      Structure of the CAC1 gene and in situ characterization of its expression. The Arabidopsis thaliana gene coding for the biotin-containing subunit of the plastidic acetyl-coenzyme A carboxylase.
      ,
      • Ke J.
      • Wen T.N.
      • Nikolau B.J.
      • Wurtele E.S.
      Coordinate regulation of the nuclear and plastidic genes coding for the subunits of the heteromeric acetyl-coenzyme A carboxylase.
      ). The other three BCCPs, namely At1g52670.1, At3g56130.1, and At3g15690.1, have similar molecular masses (∼25 kDa), but domain analysis revealed that they are missing the critical lysine residue for biotin attachment (data not shown). Nevertheless, the observation that these BCCPs elute at the same size range as the ACCase suggests that they associate with the ACCase. This is further supported by the isolation of these BCCPs together with BCCP1 from PII affinity chromatography where PII is a signaling protein that modulates ACCase activity (
      • Feria Bourrellier A.B.
      • Ferrario-Méry S.
      • Vidal J.
      • Hodges M.
      Metabolite regulation of the interaction between Arabidopsis thaliana PII and N-acetyl-l-glutamate kinase.
      ). Nevertheless, the functional roles of these BCCPs remain to be characterized.
      Additional proteins involved in the fatty acid biosynthesis pathway co-eluted with ACCase. KAS1 (At5g46290.1), an essential enzyme involved in the construction of unsaturated fatty acid carbon skeletons and acetyl-CoA synthetase (acetate-CoA ligase) (At5g36880.1), were also found at the HM fractions peaking at 3–5 and 1–2 MDa, respectively (Table I). Acyl carrier proteins (ACPs) carry the acyl chains during the synthesis of 16- and 18-carbon fatty acids. Several ACP isoforms are found in Arabidopsis and are expressed in a tissue-specific manner (
      • Bonaventure G.
      • Ohlrogge J.B.
      Differential regulation of mRNA levels of acyl carrier protein isoforms in Arabidopsis.
      ). ACP4 (At4g25050.1), the most abundant and most leaf-specific isoform (
      • Bonaventure G.
      • Ohlrogge J.B.
      Differential regulation of mRNA levels of acyl carrier protein isoforms in Arabidopsis.
      ), was the one observed in this study; it peaked at 0.8–1 MDa.

      DISCUSSION

       High Molecular Mass Protein-Protein and Protein-Nucleotide Complexes and Expanded Proteome Coverage

      Protein-protein and protein-nucleotide interactions play a crucial rule in orchestrating biological processes. Using gel filtration-based size fractionation, this study provides an overview of soluble chloroplast-localized assemblies between 0.8 and ∼5 MDa, the HM range, representing about 10–13% of the mass of the stromal proteome. This analysis complemented our previous, gel-based analysis that resolved stromal complexes up to ∼800 kDa (
      • Peltier J.B.
      • Cai Y.
      • Sun Q.
      • Zabrouskov V.
      • Giacomelli L.
      • Rudella A.
      • Ytterberg A.J.
      • Rutschow H.
      • van Wijk K.J.
      The oligomeric stromal proteome of Arabidopsis thaliana chloroplasts.
      ). When excluding the abundant Calvin cycle components, the HM range was dominated in biomass by 30, 50, and 70 S ribosome particles and associated factors as well as the multifunctional enzymes complexes PDC and ACCase involved in fatty acid metabolism. Furthermore, the plastid chromosome with interacting proteins separated from the bulk of ribosomes and was associated with a specific set of DNA-binding proteins, dominated by the heteromeric PEP.
      The distribution of proteins across the mass range could be quantified based on the number of adjusted SPCs using appropriate normalizations. Hierarchical clustering of the data set effectively grouped the proteins into biologically related functions and complexes, indicating that the complexes were stable during the gel filtration runs, and further benchmarking against known protein assemblies demonstrated that the clustering yielded biologically meaningful associations. Therefore, we could assign lesser or unknown proteins to various complexes even if targeted validation by e.g. co-immunoprecipitations will ultimately be needed.
      A second objective was to improve coverage of the chloroplast proteome and find proteins in underrepresented functional classes. Indeed, when comparing with previous chloroplast proteome analyses, we identified several low abundance proteins involved in RNA metabolism and ribosome assembly, mostly in the HM fractions. This indicates that many of the proteins involved in plastid gene expression are associated with large RNA-containing assemblies (ribosomes and RNA splice complexes) and the plastid chromosome. Further targeted analysis of these nucleotide-protein complexes is likely to reveal additional proteins. Affinity purifications that target specific binding domains (e.g. metals, ATP, and other cofactors; see e.g. Refs.
      • Ito J.
      • Heazlewood J.L.
      • Millar A.H.
      Analysis of the soluble ATP-binding proteome of plant mitochondria identifies new proteins and nucleotide triphosphate interactions within the matrix.
      ,
      • Schoonheim P.J.
      • Veiga H.
      • Pereira Dda C.
      • Friso G.
      • van Wijk K.J.
      • de Boer A.H.
      A comprehensive analysis of the 14-3-3 interactome in barley leaves using a complementary proteomics and two-hybrid approach.
      ,
      • Fuller B.
      • Stevens Jr., S.M.
      • Sehnke P.C.
      • Ferl R.J.
      Proteomic analysis of the 14-3-3 family in Arabidopsis.
      ) using highly purified chloroplast preparations will be needed to further improve proteome coverage.

       Metabolic Channeling in Fatty Acid Metabolism

      Metabolic functions were strongly underrepresented in the HM fraction but were otherwise dominated by fatty acid metabolism in terms of protein biomass, in particular the ≥5-MDa PDC and the 0.8–1-MDa heteromeric ACCase. We suggest that this bias toward fatty acid metabolism relates to the mixed hydrophobic and hydrophilic nature of the substrates and complexity of the cofactors and reactions. This complexity requires enclosure of the different intermediates within these MDa complexes, also designated as metabolic channeling (
      • Winkel B.S.
      Metabolic channeling in plants.
      ,
      • Jørgensen K.
      • Rasmussen A.V.
      • Morant M.
      • Nielsen A.H.
      • Bjarnholt N.
      • Zagrobelny M.
      • Bak S.
      • Møller B.L.
      Metabolon formation and metabolic channeling in the biosynthesis of plant natural products.
      ).

       DNA Binding: Chloroplast Chromatin

      Chloroplast DNA is packaged into nucleoids (organellar nuclei) consisting of multiple copies of the plastid genome complexed with proteins that are minimally characterized even if a number of them were identified from purified nucleoids or TACs (
      • Pfalz J.
      • Liere K.
      • Kandlbinder A.
      • Dietz K.J.
      • Oelmüller R.
      pTAC2, -6, and -12 are components of the transcriptionally active plastid chromosome that are required for plastid gene expression.
      ,
      • Sato N.
      • Terasawa K.
      • Miyajima K.
      • Kabeya Y.
      Organization, developmental dynamics, and evolution of plastid nucleoids.
      ,
      • Sakai A.
      • Takano H.
      • Kuroiwa T.
      Organelle nuclei in higher plants: structure, composition, function, and evolution.
      ). The majority of the nucleoid is tightly anchored to plastid membranes and requires detergent treatment, differential centrifugation, and possibly size exclusion column chromatography or co-immunoprecipitations for purification (
      • Pfalz J.
      • Liere K.
      • Kandlbinder A.
      • Dietz K.J.
      • Oelmüller R.
      pTAC2, -6, and -12 are components of the transcriptionally active plastid chromosome that are required for plastid gene expression.
      ,
      • Cannon G.C.
      • Ward L.N.
      • Case C.I.
      • Heinhorst S.
      The 68 kDa DNA compacting nucleoid protein from soybean chloroplasts inhibits DNA synthesis in vitro.
      ,
      • Krause K.
      • Maier R.M.
      • Kofer W.
      • Krupinska K.
      • Herrmann R.G.
      Disruption of plastid-encoded RNA polymerase genes in tobacco: expression of only a distinct set of genes is not based on selective transcription of the plastid chromosome.
      ). In contrast, our current study did not involve detergent treatments, and the extracted stromal proteome had negligible membrane contamination. Nevertheless, plastid DNA was clearly present in fractions >2 MDa, and we identified the four subunits of the PEP complex and a dozen DNA-binding proteins (e.g. involved in DNA repair and DNA organization), some of which were observed previously (
      • Pfalz J.
      • Liere K.
      • Kandlbinder A.
      • Dietz K.J.
      • Oelmüller R.
      pTAC2, -6, and -12 are components of the transcriptionally active plastid chromosome that are required for plastid gene expression.
      ). However, we clearly did not observe the relatively abundant membrane-associated MFP1 protein (At3g16000) (easily observed in thylakoid proteome analysis with high protein MOWSE scores; e.g. Ref.
      • Friso G.
      • Giacomelli L.
      • Ytterberg A.J.
      • Peltier J.B.
      • Rudella A.
      • Sun Q.
      • Wijk K.J.
      In-depth analysis of the thylakoid membrane proteome of Arabidopsis thaliana chloroplasts: new proteins, new functions, and a plastid proteome database.
      ) that anchors the plastid DNA to the membrane (
      • Jeong S.Y.
      • Rose A.
      • Meier I.
      MFP1 is a thylakoid-associated, nucleoid-binding protein with a coiled-coil structure.
      ). The DNA and associated proteins peaked in higher mass assemblies than the 70 S ribosomes, although there was some overlap. A more extensive proteome analysis of the membrane-associated nucleoid and follow-up functional analysis of the associated proteins, which also addresses the extent to which transcription and translation are coupled, are overdue.

       RNA Processing, Splice, and Degradation Machinery

      Most of the plastid-encoded genes in higher plants are organized as operons, which are generally transcribed as polycistronic transcriptional units (
      • Barkan A.
      Proteins encoded by a complex chloroplast transcription unit are each translated from both monocistronic and polycistronic mRNAs.
      ,
      • Westhoff P.
      • Herrmann R.G.
      Complex RNA maturation in chloroplasts. The psbB operon from spinach.
      ). The resulting primary transcripts are modified to generate functional RNAs by RNA cleavage of pre-existing RNAs, RNA stabilization and degradation, intron splicing, and RNA editing. In addition to known factors involved in RNA metabolism, we discovered several new proteins in the HM range that, based on their functional domains (e.g. CRM and PPR), are likely to be involved in RNA metabolism. Group II intron ribonucleoprotein complexes were found enriched at size ranges of about 500–800 kDa (
      • Kroeger T.S.
      • Watkins K.P.
      • Friso G.
      • van Wijk K.J.
      • Barkan A.
      A plant-specific RNA-binding domain revealed through analysis of chloroplast group II intron splicing.
      ,
      • Jenkins B.D.
      • Barkan A.
      Recruitment of a peptidyl-tRNA hydrolase as a facilitator of group II intron splicing in chloroplasts.
      ) even if they can also be found in higher mass ranges. We identified Arabidopsis homologues for most known group II intron splicing proteins, and most of them migrate at 1–2 MDa but were also found at higher mass ranges, suggesting a possible coupling between transcription and RNA processing. Furthermore, these RNA-interacting proteins also form associations with RNA-containing high molecular weight assemblies such as ribosomes and PEP or other RNA processing complexes. More detailed biochemical analysis, including protein-RNA interaction analysis using Ribonucleoprotein Immuno Precipitation-Chip (
      • Barkan A.
      Genome-wide analysis of RNA-protein interactions in plants.
      ) as well as targeted affinity purifications combined with protein mass spectrometry, will be required to fully understand the organization of RNA processing and how it interfaces with transcription as well as protein translation and protein assembly.

       Ribosomes and Ribosome Biogenesis

      The most abundant proteins in the HM data set were ribosomal proteins, and indeed, the mass spectrometry analysis identified nearly all known or predicted ribosomal proteins as well as most known and many potential ribosome-associated proteins. Interestingly, several of the ribosomal proteins were represented by multiple gene products; in some cases, one gene was plastid-encoded, whereas the other was nucleus-encoded, and in other cases, both homologues were plastid-encoded but differed greatly in protein abundance. The resulting ribosome heterogeneity is likely to have functional consequences and may represent adaptation to particular developmental states, cell types, or stresses; these observations warrant further investigation. Functional assignments of ribosome-associated proteins include translation, co-translational modifications, and ribosome biogenesis, indicating that SEC fractionated ribosomes in various assembly and functional states (Fig. 4).
      The identification of 12 Arabidopsis orthologues of bacterial ribosome assembly factors in the HM-A fractions suggests that ribosome assembly in chloroplasts resembles that of the prokaryotic system. Most of these factors exhibited similar low accumulation levels (50–100-fold lower than the average ribosome abundance). So far, only two chloroplast-localized RA-GTPases in Arabidopsis were characterized, namely AtObgC and the YqeH orthologue AtNOA. Analysis of their SEC elution profiles in this study established that AtObgC associates with 30 and 50 S particles and that AtNOA1 interacts with 30 and 70 S ribosomes, consistent with bacterial studies (Fig. 4). Most bacterial RA-GTPases are essential to bacterial viability (
      • Caldon C.E.
      • Yoong P.
      • March P.E.
      Evolution of a molecular switch: universal bacterial GTPases regulate ribosome function.
      ,
      • Caldon C.E.
      • March P.E.
      Function of the universally conserved bacterial GTPases.
      ,
      • Britton R.A.
      Role of GTPases in bacterial ribosome assembly.
      ). Similarly, functional analysis of AtObgC and AtNOA1 mutants has revealed that both proteins are essential for plastome synthesis and chloroplast development (
      • Bang W.Y.
      • Hata A.
      • Jeong I.S.
      • Umeda T.
      • Masuda T.
      • Chen J.
      • Yoko I.
      • Suwastika I.N.
      • Kim D.W.
      • Im C.H.
      • Lee B.H.
      • Lee Y.
      • Lee K.W.
      • Shiina T.
      • Bahk J.D.
      AtObgC, a plant ortholog of bacterial Obg, is a chloroplast-targeting GTPase essential for early embryogenesis.
      ,
      • Chigri F.
      • Sippel C.
      • Kolb M.
      • Vothknecht U.C.
      Arabidopsis OBG-like GTPase (AtOBGL) is localized in chloroplasts and has an essential function in embryo development.
      ,
      • Moreau M.
      • Lee G.I.
      • Wang Y.
      • Crane B.R.
      • Klessig D.F.
      AtNOS/AtNOA1 is a functional Arabidopsis thaliana cGTPase and not a nitric-oxide synthase.
      ,
      • Flores-Pérez U.
      • Sauret-Güeto S.
      • Gas E.
      • Jarvis P.
      • Rodríguez-Concepción M.
      A mutant impaired in the production of plastome-encoded proteins uncovers a mechanism for the homeostasis of isoprenoid biosynthetic enzymes in Arabidopsis plastids.
      ). Targeted biochemical and genetic analyses of other Arabidopsis orthologues of bacterial ribosome biogenesis proteins, including RA-GTPases (EngA, Era, Hflx, and YqeH) and other factors (RimM, RbfA, RsmD, YrdC, and SpoU) as well as plant-specific maturation factors (DAL and IOJAP), will provide insights on their specific roles in chloroplast ribosome biogenesis and the consequences for plastid protein homeostasis. The proteome analysis in this study has opened up this challenging topic for further investigation in Arabidopsis.
      Proteins with CRM domains have been shown to participate in the assembly of catalytic ribonucleoprotein complexes, namely group II intron particles and the 50 S ribosomal subunit (
      • Barkan A.
      • Klipcan L.
      • Ostersetzer O.
      • Kawamura T.
      • Asakura Y.
      • Watkins K.P.
      The CRM domain: an RNA binding module derived from an ancient ribosome-associated protein.
      ). So far, all the characterized CRM domain proteins in plants (CRS1, CAF1, and CAF2) associate with RNA in vivo and are involved in group II intron splicing (
      • Till B.
      • Schmitz-Linneweber C.
      • Williams-Carrier R.
      • Barkan A.
      CRS1 is a novel group II intron splicing factor that was derived from a domain of ancient origin.
      ,
      • Ostheimer G.J.
      • Williams-Carrier R.
      • Belcher S.
      • Osborne E.
      • Gierke J.
      • Barkan A.
      Group II intron splicing factors derived by diversification of an ancient RNA-binding domain.
      ,
      • Asakura Y.
      • Barkan A.
      A CRM domain protein functions dually in group I and group II intron splicing in land plant chloroplasts.
      ,
      • Asakura Y.
      • Bayraktar O.A.
      • Barkan A.
      Two CRM protein subfamilies cooperate in the splicing of group IIB introns in chloroplasts.
      ). Here we also identified a CRM domain protein (At4g39040.1) migrating at 1–2 MDa (50 S ribosomes); it is potentially a ribosome maturation factor because its E. coli homologue Yhby was found to tightly associate with pre-50 S ribosomes harboring immature 23 S rRNAs (
      • Barkan A.
      • Klipcan L.
      • Ostersetzer O.
      • Kawamura T.
      • Asakura Y.
      • Watkins K.P.
      The CRM domain: an RNA binding module derived from an ancient ribosome-associated protein.
      ).
      The Arabidopsis CSP41B and PNPase have multiple functions in plastid gene expression, including 23S rRNA maturation (
      • Beligni M.V.
      • Mayfield S.P.
      Arabidopsis thaliana mutants reveal a role for CSP41a and CSP41b, two ribosome-associated endonucleases, in chloroplast ribosomal RNA metabolism.
      ,
      • Walter M.
      • Kilian J.
      • Kudla J.
      PNPase activity determines the efficiency of mRNA 3′-end processing, the degradation of tRNA and the extent of polyadenylation in chloroplasts.
      ,
      • Yehudai-Resheff S.
      • Hirsh M.
      • Schuster G.
      Polynucleotide phosphorylase functions as both an exonuclease and a poly(A) polymerase in spinach chloroplasts.
      ,
      • Yehudai-Resheff S.
      • Portnoy V.
      • Yogev S.
      • Adir N.
      • Schuster G.
      Domain analysis of the chloroplast polynucleotide phosphorylase reveals discrete functions in RNA degradation, polyadenylation, and sequence homology with exosome proteins.
      ,
      • Slomovic S.
      • Portnoy V.
      • Yehudai-Resheff S.
      • Bronshtein E.
      • Schuster G.
      Polynucleotide phosphorylase and the archaeal exosome as poly(A)-polymerases.
      ). Their SEC elution profiles show that they elute mainly in the 1–2-MDa fraction, suggesting interactions with the 50 S particle, although this does not rule out associations with other RNA-containing complexes. Another endonuclease, RNase J, is found to be crucial for 16 S rRNA processing in B. subtilis (
      • Britton R.A.
      • Wen T.
      • Schaefer L.
      • Pellegrini O.
      • Uicker W.C.
      • Mathy N.
      • Tobin C.
      • Daou R.
      • Szyk J.
      • Condon C.
      Maturation of the 5′ end of Bacillus subtilis 16S rRNA by the essential ribonuclease YkqC/RNase J1.
      ). The elution profile of its Arabidopsis orthologue (>1 MDa) indicates interactions with various ribonucleoprotein complexes including 30 and 70 S particles, consistent with bacterial studies (
      • Britton R.A.
      • Wen T.
      • Schaefer L.
      • Pellegrini O.
      • Uicker W.C.
      • Mathy N.
      • Tobin C.
      • Daou R.
      • Szyk J.
      • Condon C.
      Maturation of the 5′ end of Bacillus subtilis 16S rRNA by the essential ribonuclease YkqC/RNase J1.
      ). Overall, these findings support the role of CSP41B, PNPase, and RNase J in ribosome assembly and maturation. RNA helicases have been implicated in various RNA processing functions, including rRNA maturation during ribosome biogenesis (
      • Linder P.
      Dead-box proteins: a family affair–active and passive players in RNP-remodeling.
      ). Four Arabidopsis DEAD box RNA helicases were seen in the fractions at mass ranges >1 MDa and should be considered potential candidates for ribosome biogenesis. Interestingly, RH3 is specifically up-regulated in Arabidopsis clpr2-1 mutants with reduced plastid-localized ClpPR protease levels, and this mutant exhibits a delay in ribosome assembly and/or defects in RNA metabolism (
      • Zybailov B.
      • Friso G.
      • Kim J.
      • Rudella A.
      • Rodríguez V.R.
      • Asakura Y.
      • Sun Q.
      • van Wijk K.J.
      Large scale comparative proteomics of a chloroplast Clp protease mutant reveals folding stress, altered protein homeostasis, and feedback regulation of metabolism.
      ).

       Translation and Co-translational Modifications

      The isolation of ribosomes together with translational factors and proteins involved in post-translational modifications suggests that translating ribosomes were captured. Our analysis clearly provided support for ribosome association of various N-terminal modifying enzymes (MAP and PDF) as well as targeting/folding proteins (TF and cpSR54). The E. coli TF has been shown to provide a folding cavity for the nascent protein emerging from the ribosome tunnel (
      • Ullers R.S.
      • Houben E.N.
      • Brunner J.
      • Oudega B.
      • Harms N.
      • Luirink J.
      Sequence-specific interactions of nascent Escherichia coli polypeptides with trigger factor and signal recognition particle.
      ,
      • Rutkowska A.
      • Mayer M.P.
      • Hoffmann A.
      • Merz F.
      • Zachmann-Brand B.
      • Schaffitzel C.
      • Ban N.
      • Deuerling E.
      • Bukau B.
      Dynamics of trigger factor interaction with translating ribosomes.
      ,
      • Maier T.
      • Ferbitz L.
      • Deuerling E.
      • Ban N.
      A cradle for new proteins: trigger factor at the ribosome.
      ). We did not find any obvious growth phenotype for a TF-null mutant in Arabidopsis,
      H. Rutschow and K. J. van Wijk, unpublished data.
      which is perhaps not surprising because a clear phenotype in E. coli is only seen in combination with deletion of the DnaK chaperone (
      • Ullers R.S.
      • Houben E.N.
      • Brunner J.
      • Oudega B.
      • Harms N.
      • Luirink J.
      Sequence-specific interactions of nascent Escherichia coli polypeptides with trigger factor and signal recognition particle.
      ). It has been shown for E. coli that during protein synthesis in particular the RPL23 protein serves as a platform for the association of enzymes, targeting factors and chaperones that act upon the nascent polypeptide that emerges from the exit tunnel (
      • Kramer G.
      • Boehringer D.
      • Ban N.
      • Bukau B.
      The ribosome as a platform for co-translational processing, folding and targeting of newly synthesized proteins.
      ,
      • Kramer G.
      • Rauch T.
      • Rist W.
      • Vorderwülbecke S.
      • Patzelt H.
      • Schulze-Specking A.
      • Ban N.
      • Deuerling E.
      • Bukau B.
      L23 protein functions as a chaperone docking site on the ribosome.
      ). Interestingly, Arabidopsis has two identical chloroplast-encoded L23 proteins, and future studies should determine how these two paralogues contribute to protein synthesis and homeostasis.

      Acknowledgments

      Part of this work was carried out by using the resources of the Computational Biology Service Unit of Cornell University, which is partially funded by the Microsoft Corp. We thank Dr. Qi Sun for valuable support. We thank several other members of the van Wijk laboratory, Boris Zybailov, Giulia Friso, Wojciech Majeran, Yuka Asakura, and Heidi Rutschow, for support and discussions.

      Supplementary Material

      REFERENCES

      1. Buchanan B. Gruissem W. Jones R.L. Biochemistry and Molecular Biology of Plants. American Society of Plant Physiologists, Rockville, MD2000
        • Emanuelsson O.
        • Nielsen H.
        • Brunak S.
        • von Heijne G.
        Predicting subcellular localization of proteins based on their N-terminal amino acid sequence.
        J. Mol. Biol. 2000; 300: 1005-1016
        • van Wijk K.J.
        Plastid proteomics.
        Plant Physiol. Biochem. 2004; 42: 963-977
        • Jarvis P.
        • Robinson C.
        Mechanisms of protein import and routing in chloroplasts.
        Curr. Biol. 2004; 14: R1064-1077
        • Soll J.
        • Schleiff E.
        Protein import into chloroplasts.
        Nat. Rev. Mol. Cell Biol. 2004; 5: 198-208
        • Kessler F.
        • Schnell D.J.
        The function and diversity of plastid protein import pathways: a multilane GTPase highway into plastids.
        Traffic. 2006; 7: 248-257
        • Ferro M.
        • Salvi D.
        • Brugiere S.
        • Miras S.
        • Kowalski S.
        • Louwagie M.
        • Garin J.
        • Joyard J.
        • Rolland N.
        Proteomics of the chloroplast envelope membranes from Arabidopsis thaliana.
        Mol. Cell. Proteomics. 2003; 28: 325-345
        • Kieselbach T.
        • Schröder W.P.
        The proteome of the chloroplast lumen of higher plants.
        Photosynth. Res. 2003; 78: 249-264
        • Zybailov B.
        • Rutschow H.
        • Friso G.
        • Rudella A.
        • Emanuelsson O.
        • Sun Q.
        • van Wijk K.J.
        Sorting signals, N-terminal modifications and abundance of the chloroplast proteome.
        PLoS One. 2008; 3: e1994
        • Baginsky S.
        • Kleffmann T.
        • von Zychlinski A.
        • Gruissem W.
        Analysis of shotgun proteomics and RNA profiling data from Arabidopsis thaliana chloroplasts.
        J. Proteome Res. 2005; 4: 637-640
        • Peltier J.B.
        • Cai Y.
        • Sun Q.
        • Zabrouskov V.
        • Giacomelli L.
        • Rudella A.
        • Ytterberg A.J.
        • Rutschow H.
        • van Wijk K.J.
        The oligomeric stromal proteome of Arabidopsis thaliana chloroplasts.
        Mol. Cell. Proteomics. 2006; 5: 114-133
        • Yamaguchi K.
        • von Knoblauch K.
        • Subramanian A.R.
        The plastid ribosomal proteins. Identification of all the proteins in the 30 S subunit of an organelle ribosome (chloroplast).
        J. Biol. Chem. 2000; 275: 28455-28465
        • Yamaguchi K.
        • Subramanian A.R.
        The plastid ribosomal proteins. Identification of all the proteins in the 50 S subunit of an organelle ribosome (chloroplast).
        J. Biol. Chem. 2000; 275: 28466-28482
        • Yamaguchi K.
        • Subramanian A.R.
        Proteomic identification of all plastid-specific ribosomal proteins in higher plant chloroplast 30S ribosomal subunit.
        Eur. J. Biochem. 2003; 270: 190-205
        • Zanetti M.E.
        • Chang I.F.
        • Gong F.
        • Galbraith D.W.
        • Bailey-Serres J.
        Immunopurification of polyribosomal complexes of Arabidopsis for global analysis of gene expression.
        Plant Physiol. 2005; 138: 624-635
        • Carroll A.J.
        • Heazlewood J.L.
        • Ito J.
        • Millar A.H.
        Analysis of the Arabidopsis cytosolic ribosome proteome provides detailed insights into its components and their post-translational modification.
        Mol. Cell. Proteomics. 2008; 7: 347-369
        • Suzuki J.Y.
        • Ytterberg A.J.
        • Beardslee T.A.
        • Allison L.A.
        • Wijk K.J.
        • Maliga P.
        Affinity purification of the tobacco plastid RNA polymerase and in vitro reconstitution of the holoenzyme.
        Plant J. 2004; 40: 164-172
        • Thelen J.J.
        • Miernyk J.A.
        • Randall D.D.
        Partial purification and characterization of the maize mitochondrial pyruvate dehydrogenase complex.
        Plant Physiol. 1998; 116: 1443-1450
        • Sasaki Y.
        • Nagano Y.
        Plant acetyl-CoA carboxylase: structure, biosynthesis, regulation and gene manipulation for plant breeding.
        Biosci. Biotechnol. Biochem. 2004; 68: 1175-1184
        • Phinney B.S.
        • Thelen J.J.
        Proteomic characterization of a Triton-insoluble fraction from chloroplasts defines a novel group of proteins associated with macromolecular structures.
        J. Proteome Res. 2005; 4: 497-506
        • Hu Q.
        • Noll R.J.
        • Li H.
        • Makarov A.
        • Hardman M.
        • Graham Cooks R.
        The Orbitrap: a new mass spectrometer.
        J. Mass Spectrom. 2005; 40: 430-443
        • Scigelova M.
        • Makarov A.
        Orbitrap mass analyzer—overview and applications in proteomics.
        Proteomics. 2006; 6: 16-21
        • Dunkley T.P.
        • Watson R.
        • Griffin J.L.
        • Dupree P.
        • Lilley K.S.
        Localization of organelle proteins by isotope tagging (LOPIT).
        Mol. Cell. Proteomics. 2004; 3: 1128-1134
        • Dong M.
        • Yang L.L.
        • Williams K.
        • Fisher S.J.
        • Hall S.C.
        • Biggin M.D.
        • Jin J.
        • Witkowska H.E.
        A “tagless” strategy for identification of stable protein complexes genome-wide by multidimensional orthogonal chromatographic separation and iTRAQ reagent tracking.
        J. Proteome Res. 2008; 7: 1836-1849
        • Andersen J.S.
        • Wilkinson C.J.
        • Mayor T.
        • Mortensen P.
        • Nigg E.A.
        • Mann M.
        Proteomic characterization of the human centrosome by protein correlation profiling.
        Nature. 2003; 426: 570-574
        • Majeran W.
        • Zybailov B.
        • Ytterberg A.J.
        • Dunsmore J.
        • Sun Q.
        • van Wijk K.J.
        Consequences of C4 differentiation for chloroplast membrane proteomes in maize mesophyll and bundle sheath cells.
        Mol. Cell. Proteomics. 2008; 7: 1609-1638
        • Shevchenko A.
        • Tomas H.
        • Havlis J.
        • Olsen J.V.
        • Mann M.
        In-gel digestion for mass spectrometric characterization of proteins and proteomes.
        Nat. Protoc. 2006; 1: 2856-2860
        • Zybailov B.
        • Sun Q.
        • van Wijk K.J.
        Workflow for large scale detection and validation of peptide modifications by RPLC-LTQ-Orbitrap: application to the Arabidopsis thaliana leaf proteome and an online modified peptide library.
        Anal. Chem. 2009; 81: 8015-8024
        • Sun Q.
        • Zybailov B.
        • Majeran W.
        • Friso G.
        • Olinares P.D.
        • van Wijk K.J.
        PPDB, the Plant Proteomics Database at Cornell.
        Nucleic Acids Res. 2009; 37: D969-D974
        • Zybailov B.
        • Friso G.
        • Kim J.
        • Rudella A.
        • Rodríguez V.R.
        • Asakura Y.
        • Sun Q.
        • van Wijk K.J.
        Large scale comparative proteomics of a chloroplast Clp protease mutant reveals folding stress, altered protein homeostasis, and feedback regulation of metabolism.
        Mol. Cell. Proteomics. 2009; 8: 1789-1810
        • Thimm O.
        • Bläsing O.
        • Gibon Y.
        • Nagel A.
        • Meyer S.
        • Krüger P.
        • Selbig J.
        • Müller L.A.
        • Rhee S.Y.
        • Stitt M.
        MAPMAN: a user-driven tool to display genomics data sets onto diagrams of metabolic pathways and other biological processes.
        Plant J. 2004; 37: 914-939
        • Liu H.
        • Sadygov R.G.
        • Yates 3rd., J.R.
        A model for random sampling and estimation of relative protein abundance in shotgun proteomics.
        Anal. Chem. 2004; 76: 4193-4201
        • Zybailov B.
        • Coleman M.K.
        • Florens L.
        • Washburn M.P.
        Correlation of relative abundance ratios derived from peptide ion chromatograms and spectrum counting for quantitative proteomic analysis using stable isotope labeling.
        Anal. Chem. 2005; 77: 6218-6224
        • Old W.M.
        • Meyer-Arendt K.
        • Aveline-Wolf L.
        • Pierce K.G.
        • Mendoza A.
        • Sevinsky J.R.
        • Resing K.A.
        • Ahn N.G.
        Comparison of label-free methods for quantifying human proteins by shotgun proteomics.
        Mol. Cell. Proteomics. 2005; 4: 1487-1502
        • Lu P.
        • Vogel C.
        • Wang R.
        • Yao X.
        • Marcotte E.M.
        Absolute protein expression profiling estimates the relative contributions of transcriptional and translational regulation.
        Nat. Biotechnol. 2007; 25: 117-124
        • Zybailov B.
        • Mosley A.L.
        • Sardiu M.E.
        • Coleman M.K.
        • Florens L.
        • Washburn M.P.
        Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae.
        J. Proteome Res. 2006; 5: 2339-2347
        • Thelen J.J.
        • Muszynski M.G.
        • David N.R.
        • Luethy M.H.
        • Elthon T.E.
        • Miernyk J.A.
        • Randall D.D.
        The dihydrolipoamide S-acetyltransferase subunit of the mitochondrial pyruvate dehydrogenase complex from maize contains a single lipoyl domain.
        J. Biol. Chem. 1999; 274: 21769-21775
        • Sharma M.R.
        • Wilson D.N.
        • Datta P.P.
        • Barat C.
        • Schluenzen F.
        • Fucini P.
        • Agrawal R.K.
        Cryo-EM study of the spinach chloroplast ribosome reveals the structural and functional roles of plastid-specific ribosomal proteins.
        Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 19315-19320
        • Beligni M.V.
        • Yamaguchi K.
        • Mayfield S.P.
        Chloroplast elongation factor ts pro-protein is an evolutionarily conserved fusion with the s1 domain-containing plastid-specific ribosomal protein-7.
        Plant Cell. 2004; 16: 3357-3369
        • Asakura Y.
        • Barkan A.
        Arabidopsis orthologs of maize chloroplast Splicing factors promote splicing of orthologous and species-specific group II introns.
        Plant Physiol. 2006; 142: 1656-1663
        • Ueda T.
        • Matsuda N.
        • Anai T.
        • Tsukaya H.
        • Uchimiya H.
        • Nakano A.
        An Arabidopsis gene isolated by a novel method for detecting genetic interaction in yeast encodes the GDP dissociation inhibitor of Ara4 GTPase.
        Plant Cell. 1996; 8: 2079-2091
        • Liljas A.
        Structural Aspects of Protein Synthesis. World Scientific, Singapore2004
        • Ramakrishnan V.
        Ribosome structure and the mechanism of translation.
        Cell. 2002; 108: 557-572
        • Albrecht V.
        • Ingenfeld A.
        • Apel K.
        Characterization of the snowy cotyledon 1 mutant of Arabidopsis thaliana: the impact of chloroplast elongation factor G on chloroplast development and plant vitality.
        Plant Mol. Biol. 2006; 60: 507-518
        • Bhadula S.K.
        • Elthon T.E.
        • Habben J.E.
        • Helentjaris T.G.
        • Jiao S.
        • Ristic Z.
        Heat-stress induced synthesis of chloroplast protein synthesis elongation factor (EF-Tu) in a heat-tolerant maize line.
        Planta. 2001; 212: 359-366
        • Rao D.
        • Momcilovic I.
        • Kobayashi S.
        • Callegari E.
        • Ristic Z.
        Chaperone activity of recombinant maize chloroplast protein synthesis elongation factor, EF-Tu.
        Eur. J. Biochem. 2004; 271: 3684-3692
        • Caldon C.E.
        • Yoong P.
        • March P.E.
        Evolution of a molecular switch: universal bacterial GTPases regulate ribosome function.
        Mol. Microbiol. 2001; 41: 289-297
        • Caldon C.E.
        • March P.E.
        Function of the universally conserved bacterial GTPases.
        Curr. Opin. Microbiol. 2003; 6: 135-139
        • Owens R.M.
        • Pritchard G.
        • Skipp P.
        • Hodey M.
        • Connell S.R.
        • Nierhaus K.H.
        • O'Connor C.D.
        A dedicated translation factor controls the synthesis of the global regulator Fis.
        EMBO J. 2004; 23: 3375-3385
        • Qin Y.
        • Polacek N.
        • Vesper O.
        • Staub E.
        • Einfeldt E.
        • Wilson D.N.
        • Nierhaus K.H.
        The highly conserved LepA is a ribosomal elongation factor that back-translocates the ribosome.
        Cell. 2006; 127: 721-733
        • Pfennig P.L.
        • Flower A.M.
        BipA is required for growth of Escherichia coli K12 at low temperature.
        Mol. Genet. Genomics. 2001; 266: 313-317
        • Beckering C.L.
        • Steil L.
        • Weber M.H.
        • Völker U.
        • Marahiel M.A.
        Genomewide transcriptional analysis of the cold shock response in Bacillus subtilis.
        J. Bacteriol. 2002; 184: 6395-6402
        • Eymann C.
        • Homuth G.
        • Scharf C.
        • Hecker M.
        Bacillus subtilis functional genomics: global characterization of the stringent response by proteome and transcriptome analysis.
        J. Bacteriol. 2002; 184: 2500-2520
        • Krishnan K.
        • Flower A.M.
        Suppression of Delta bipA phenotypes in Escherichia coli by abolishment of pseudouridylation at specific sites on the 23S rRNA.
        J. Bacteriol. 2008; 190: 7675-7683
        • Wang F.
        • Zhong N.Q.
        • Gao P.
        • Wang G.L.
        • Wang H.Y.
        • Xia G.X.
        SsTypA1, a chloroplast-specific TypA/BipA-type GTPase from the halophytic plant Suaeda salsa, plays a role in oxidative stress tolerance.
        Plant Cell Environ. 2008; 31: 982-994
        • Sharma M.R.
        • Dönhöfer A.
        • Barat C.
        • Marquez V.
        • Datta P.P.
        • Fucini P.
        • Wilson D.N.
        • Agrawal R.K.
        PSRP1 is not a ribosomal protein, but a ribosome binding factor that is recycled by RRF and EF-G.
        J. Biol. Chem. 2010; 285: 4006-4014
        • Kramer G.
        • Boehringer D.
        • Ban N.
        • Bukau B.
        The ribosome as a platform for co-translational processing, folding and targeting of newly synthesized proteins.
        Nat. Struct. Mol. Biol. 2009; 16: 589-597
        • Serero A.
        • Giglione C.
        • Meinnel T.
        Distinctive features of the two classes of eukaryotic peptide deformylases.
        J. Mol. Biol. 2001; 314: 695-708
        • Giglione C.
        • Serero A.
        • Pierre M.
        • Boisson B.
        • Meinnel T.
        Identification of eukaryotic peptide deformylases reveals universality of N-terminal protein processing mechanisms.
        EMBO J. 2000; 19: 5916-5929
        • Dirk L.M.
        • Williams M.A.
        • Houtz R.L.
        Eukaryotic peptide deformylases. Nuclear-encoded and chloroplast-targeted enzymes in Arabidopsis.
        Plant Physiol. 2001; 127: 97-107
        • Franklin A.E.
        • Hoffman N.E.
        Characterization of a chloroplast homologue of the 54-kDa subunit of the signal recognition particle.
        J. Biol. Chem. 1993; 268: 22175-22180
        • Li X.
        • Henry R.
        • Yuan J.
        • Cline K.
        • Hoffman N.E.
        A chloroplast homologue of the signal recognition particle subunit SRP54 is involved in the posttranslational integration of a protein into thylakoid membranes.
        Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 3789-3793
        • Nilsson R.
        • Brunner J.
        • Hoffman N.E.
        • van Wijk K.J.
        Interactions of ribosome nascent chain complexes of the chloroplast-encoded D1 thylakoid membrane protein with cpSRP54.
        EMBO J. 1999; 18: 733-742
        • Rutschow H.
        • Ytterberg A.J.
        • Friso G.
        • Nilsson R.
        • van Wijk K.J.
        Quantitative proteomics of a chloroplast SRP54 sorting mutant and its genetic interactions with CLPC1 in Arabidopsis.
        Plant Physiol. 2008; 148: 156-175
        • Ferbitz L.
        • Maier T.
        • Patzelt H.
        • Bukau B.
        • Deuerling E.
        • Ban N.
        Trigger factor in complex with the ribosome forms a molecular cradle for nascent proteins.
        Nature. 2004; 431: 590-596
        • Nishio K.
        • Hirohashi T.
        • Nakai M.
        Chloroplast chaperonins: evidence for heterogeneous assembly of alpha and beta cpn60 polypeptides into a chaperonin oligomer.
        Biochem. Biophys. Res. Commun. 1999; 266: 584-587
        • Sharkia R.
        • Bonshtien A.L.
        • Mizrahi I.
        • Weiss C.
        • Niv A.
        • Lustig A.
        • Viitanen P.V.
        • Azem A.
        On the oligomeric state of chloroplast chaperonin 10 and chaperonin 20.
        Biochim. Biophys. Acta. 2003; 1651: 76-84
        • Schroda M.
        • Vallon O.
        • Whitelegge J.P.
        • Beck C.F.
        • Wollman F.A.
        The chloroplastic GrpE homolog of Chlamydomonas: two isoforms generated by differential splicing.
        Plant Cell. 2001; 13: 2823-2839
        • Kaczanowska M.
        • Rydén-Aulin M.
        Ribosome biogenesis and the translation process in Escherichia coli.
        Microbiol. Mol. Biol. Rev. 2007; 71: 477-494
        • Connolly K.
        • Culver G.
        Deconstructing ribosome construction.
        Trends Biochem. Sci. 2009; 34: 256-263
        • Altschul S.F.
        • Madden T.L.
        • Schäffer A.A.
        • Zhang J.
        • Zhang Z.
        • Miller W.
        • Lipman D.J.
        Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.
        Nucleic Acids Res. 1997; 25: 3389-3402
        • Britton R.A.
        Role of GTPases in bacterial ribosome assembly.
        Annu. Rev. Microbiol. 2009; 63: 155-176
        • Scott J.M.
        • Haldenwang W.G.
        Obg, an essential GTP binding protein of Bacillus subtilis, is necessary for stress activation of transcription factor sigm▵B).
        J. Bacteriol. 1999; 181: 4653-4660
        • Lin B.
        • Thayer D.A.
        • Maddock J.R.
        The Caulobacter crescentus CgtA(C) protein cosediments with the free 50S ribosomal subunit.
        J. Bacteriol. 2004; 186: 481-489
        • Sato A.
        • Kobayashi G.
        • Hayashi H.
        • Yoshida H.
        • Wada A.
        • Maeda M.
        • Hiraga S.
        • Takeyasu K.
        • Wada C.
        The GTP binding protein Obg homolog ObgE is involved in ribosome maturation.
        Genes Cells. 2005; 10: 393-408