A survey of the plant mitochondrial proteome in relation to development

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<ul><li><p>Juliette Bardel1</p><p>Mathilde Louwagie2</p><p>Michel Jaquinod2</p><p>Agns Jourdain1</p><p>Sylvie Luche3</p><p>Thierry Rabilloud3</p><p>David Macherel4</p><p>Jrme Garin2</p><p>Jacques Bourguignon1</p><p>1Laboratoire de PhysiologieCellulaire Vgtale,DRDC, CEA-Grenoble,Grenoble, France</p><p>2Laboratoire de Chimiedes protines, DRDC,CEA-Grenoble, France</p><p>3Laboratoire de BionergtiqueCellulaire et Pathologique,DRDC, CEA-Grenoble, France</p><p>4UMR Physiologie Molculairedes Semences,Universit dAngers/INH/INRA,Angers, France</p><p>A survey of the plant mitochondrial proteomein relation to development</p><p>To expand the functional analysis of plant mitochondria, we have undertaken the build-ing of the proteome of pea mitochondria purified from leaves (green and etiolated),roots and seeds. In the first stage, we focused our proteomic exploration on the solubleprotein complement of the green leaf mitochondria. We used traditional two-dimen-sional polyacrylamide gel electrophoresis, in combination with size exclusion chroma-tography as a third dimension, to identify the major proteins and further resolve theirmacromolecular complexity. The two-dimensional map of soluble proteins of greenleaf mitochondria revealed 433 spots (with Coomassie blue staining) and around 73%of the proteins (in mass) were identified using three different approaches: Edmandegradation, matrix-assisted laser desorption/ionization mass spectrometry andelectrospray ionization tandem mass spectrometry. Quite a lot of the polypeptideswere present in multiforms which indicated the presence of isoforms or the occurrenceof post-translational modifications. Among these proteins, we uncovered an abundantfamily that was identified as aldehyde dehydrogenases, representing approximately7.5% of the soluble proteins. The comparative analysis of soluble mitochondrial pro-teomes led to the identification of a number of proteins which were specifically presentin root or in seed mitochondria, thus revealing the impact of tissue differentiation at themitochondrial level.</p><p>Keywords: Aldehyde dehydrogenase / Mass spectrometry / Metabolism / Plant mitochondria /Two-dimensional gel electrophoresis PRO 0210</p><p>1 Introduction</p><p>Mitochondria are semiautonomous organelles whose uni-versally recognised function is to provide cellular ATP bythe process of oxidative phosphorylation. Recent devel-opments in cell biology, genetics and medical sciencehave placed mitochondria in the foreground as keyplayers in fields as diverse as evolution, cell death and agrowing number of severe pathologies [14]. Comparedto their animal counterparts, plant mitochondria carryother primordial biosynthetic functions related to theautotrophic status of plants and their inability to avoidstress factors. Prominent features of plant mitochondriainclude the presence of additional NAD(P)H dehydro-genases and an alternative oxidase in the inner mem-</p><p>brane [5, 6] that confer an increased flexibility in energytransfer. In photosynthetic tissues, mitochondria are ob-ligatory partners of chloroplasts with respect to CO2light-dependent assimilation, due to their implication inthe photorespiratory pathway [7]. Their contributioninvolves the oxidation of glycine at very high ratesbecause of a tremendous accumulation of the glycinecleavage system (or glycine decarboxylase complex) inthe matrix space [8].</p><p>More recently, it has been shown that plant mitochondriaare also implicated in the synthesis of essential cofactorssuch as biotin [9], folate [10], lipoate [11, 12] and vitaminC [13]. These examples emphasise the functional com-plexity of plant mitochondria which cover a great varietyof biochemical reactions, from housekeeping to bio-genesis, in a coordinated fashion with the cellular en-vironment. However, in contrast to the vast and growingnumber of mitochondrial functions, relatively few mito-chondrial proteins have been characterised. The recentdevelopment of proteomics [1416] opens the path to-ward a deeper exploration of mitochondrial functionsthrough their protein complement. Previous work dealingwith organelle proteome analysis has proven the useful-ness of the approach, for instance in the case of humanmitochondria [17], macrophage phagosome [18], thy-lakoid proteins of chloroplast [19] and chloroplast enve-lope membranes [20].</p><p>Correspondence: Dr. Jacques Bourguignon, Laboratoire dePhysiologie Cellulaire Vgtale, UMR 5019, CEA / CNRS / Uni-versit Joseph Fourier, Dpartement de Biologie Molculaire etStructurale, CEA-Grenoble, 17 rue des Martyrs, 38054 Grenoblecedex 9, FranceE-mail: jacques.bourguignon@cea.frFax: +33-4-38-78-5091</p><p>Abbreviations: ALDH, aldehyde dehydrogenase; FDH, formatedehydrogenase; GCS, glycine cleavage system; MDH, malatedehydrogenase; MnSOD, manganese superoxide dismutase;RNP, RNA binding protein; SCADH, short-chain alcohol dehydro-genase; SHMT, serine hydroxymethyltransferase; SVC, succi-nyl-CoA ligase</p><p>880 Proteomics 2002, 2, 880898</p><p> WILEY-VCH Verlag GmbH, 69451 Weinheim, 2002 1615-9853/02/0707880 $17.50+.50/0</p></li><li><p>Proteomics 2002, 2, 880898 Plant mitochondria proteomics and development 881</p><p>In the present work, we have undertaken a comprehen-sive functional analysis of plant mitochondria by buildingthe proteome of mitochondria isolated from differenttissues and organs from pea. In the first stage, wefocusedon the soluble protein complementwhosemacro-molecular complexity was further resolved by combiningsize exclusion chromatography with the traditional two-dimensional polyacrylamide gel electrophoresis separa-tion. We report here the 2-D map of soluble proteins ofmitochondria isolated frompea leaves (green or etiolated),roots and seeds together with the identification of themajor proteins, results which are discussed in a physio-logical context.</p><p>2 Materials and methods</p><p>2.1 Isolation of pea mitochondria andpreparation of a soluble mitochondrialprotein extract (matrix extract)</p><p>Mitochondria were isolated and purified from leaves of12-day old pea plants as described by Douce et al. [21].The procedure is composed of differential centrifugationfollowed by two steps of purification on Percoll/polyvinyl-pyrrolidone gradient. In the case of the purification ofmitochondria from etiolated leaves and roots, the pro-cedure used for the purification of the potato tuber mito-chondria was employed [21] with two steps of purificationon Percoll gradient. The mitochondria were isolated from16 h imbibed pea seeds according to the proceduredescribed by Benamar, Tallon and Macheral (manuscriptin preparation). These purified mitochondria were sub-mitted to an osmotic shock by dilution (1:20) in a buffercontaining 40mM MOPS (pH 7.4) and 1mM DTT and thesoluble proteins were released from the mitochondria bythree freeze-thaw cycles. After removal of mitochondrialmembrane by ultracentrifugation at 38000 rpm for 1 h(Beckman SW-40 rotor), the supernatant containing themitochondrial soluble proteins was concentrated on a3 kDa Macrosep (Pall, Portsmouth, UK) to a concentrationof 4050mg/mL.</p><p>2.2 Fractionation of the soluble mitochondrialproteins</p><p>The soluble protein extract (200 mg) prepared from pealeaf mitochondria was applied to a Sephacryl S-300 col-umn (Amersham Biosciences, Uppsala, Sweden) equili-brated with a buffer containing 50 mM Tris (pH 7.4) andDTT 1 mM. The proteins were then eluted, as function oftheir molecular mass, with the same buffer at 4C with aflow rate of 0.3 mL/min (fraction size, 3 mL), the glycine</p><p>cleavage system components and the serine hydroxy-methyltransferase being used as standards to calibratethe column.</p><p>2.3 Sample preparation for 2-D gels andIPG focusing</p><p>Soluble mitochondrial proteins (500 g) were diluted in asolution containing at the final concentration: 7 M urea,2.0 M thiourea, 4% w/v CHAPS, 20 mM DTT and 0.4%w/v carrier ampholytes (Pharmalytes 310; AmershamBiosciences) [22]. Proteins were separated by 2-DE, es-sentially as previously described on home-made pH 48gradients [23].</p><p>2.4 Equilibration, SDS dimension and staining</p><p>After equilibration as previously described [17], the stripswere sealed on top of the 1.5 mm thick 2-D gel (Bio-Radvertical system; Hercules, CA, USA) with the help of1% low melting agarose in 0.2% SDS, 0.15 M N,N-bis(hydroxyethyl)N,N,N-tris(hydroxymethyl)aminomethane(Bis-Tris), 0.1 M HCl buffer supplemented with bromo-phenol blue as a tracking dye. SDS-PAGE was carriedout at constant power (15 W per gel) with cooling at10C, until the tracking dye reached the bottom of thegel. Gels were then incubated in a solution of 10% aceticacid and 50% methanol and stained with the same solu-tion containing 2.5 g/L Coomassie Blue (R-250).</p><p>2.5 Protein identification by N-terminalsequencing</p><p>When N-terminal sequencing was performed, the 2-Dgels were blotted onto PVDF membranes (ProBlott mem-branes; Applied Biosystems, Foster City, CA, USA) usingthe Bio-Rad TransBlot Cell according to the manufac-turers instructions. After transfer, at 90 V for 3 h, mem-branes were washed twice for 5 min with deionized waterand then stained for 15 min with the coloration solutioncontaining: 0.1% w/v Coomassie Blue R-250, 10% v/vacetic acid and 40% v/v ethanol. The membranes weredestained with a solution of 30% v/v ethanol. Proteinspots blotted on a PVDF membrane and stained withCoomassie blue were excised and sequenced on an ABI492 Procise sequencer (Applied Biosystems).</p><p>2.6 Protein digestion</p><p>The protein spots of interest were excised from Coomas-sie blue stained 2-D gels and washed with 25 mM ammo-nium hydrogenocarbonate (NH4HCO3) (pH 8.0) for 2 h,</p></li><li><p>882 J. Bardel et al. Proteomics 2002, 2, 880898</p><p>and then with 50% v/v acetonitrile, 25 mM NH4HCO3for another 2 h. This procedure was repeated until the gelpieces were destained and complete dehydration wasperformed with a vacuum centrifuge (Eppendorff Speed-Vac). Three to 5 L of a solution containing 0.1 to 0.5 g oftrypsin (porcine sequencing grade modified, Promega),25 mM NH4HCO3 (pH 8.0) and 10% v/v acetonitrile wasadded to the tube and the digestion proceeded at 37Cfor 3 to 4 h.</p><p>2.7 MALDI analysis</p><p>Mass spectra of the tryptic digests were acquired on aBiflex (Bruker-Daltonic, Bremen, Germany) MALDI-TOFmass spectrometer equipped with a gridless delayedextraction. The instrument was operated in the reflectormode. 0.5 L of each digest solution (in 25 mMNH4HCO3/10% acetonitrile) was deposited directly ontothe sample probe on a dry thin layer of matrix. The matrixwas prepared by mixing four volumes of saturated solu-tion of -cyano-4-hydroxy-trans-cinnamic acid (CCA) inacetone with three volumes of solution of nitrocellulose(10 mg/mL) in acetone/isopropanol (1:1). Deposits werewashed with 5 L of 0.1% v/v TFA before analysis. Amass list of peptides was obtained for each proteindigest. This peptide mass fingerprint was then sub-mitted to an appropriate software to identify the proteins(MS-Fit, available online at http://prospector.ucsf.edu/ucsfhtml3.4/msfit. htm, or ProFound, available online athttp:// When aprotein could not be identified from its tryptic peptidemass map, the tryptic digest was extracted twice with a50% v/v acetonitrile solution containing 25 mM NH4HCO3.The digest solution and the extracts were then pooled,dried in a vacuum centrifuge, and desalted with ZipTipC18 (Millipore, Bedford, MA, USA) before the electrospraytandem mass spectrometry analysis.</p><p>2.8 ESI-MS/MS</p><p>Aquadrupole time-of-flight (Q-TOF) instrument (Micromass,Manchester, UK) was usedwith a Z-spray ion-source work-ing in the nanospray mode. Approximately 3 to 5 L of thedesalted sample was introduced into a needle (mediumsample needle; MDS Protana, Odense, Denmark) to runMS and MS/MS experiments. The capillary voltage was setto an average of 1000 V, and the sample cone to 50 V.Glufibrinopeptide was used to calibrate the instrument inthe MS/MS mode. MS/MS spectra were transformed usingMaxEnt3 (MassLynx; Micromass). Amino acid sequences,sequence tags,orpeptide ion fragments thatcouldbedeter-mined were used to screen the protein and expression tag(EST) databases with dedicated software: Pepfrag (http://</p><p>prowl1.rockefeller.edu/prowl/pepfrag.html), Scan (http://dna.stanford.edu/scan), BLAST (http://ncbi.nlm. nih.gov/blast/blast.cgi) or FASTA3 (http://www.ebi.ac.uk/fasta3/)for searching homologies.</p><p>2.9 Bioinformatic analysis</p><p>When indicated, the prediction of mitochondrial targetingfor protein sequences issued from database homologiessearch was performed with four different programs avail-able on the internet: MitoProt (http://www.mips.biochem.mpg.de/cgi-bin/proj/medgen/mitofilter) [24], Predotar V0.5(http://www.inra.fr/Internet/Produits/Predotar/), PSORT(http://psort.nibb.ac.jp) [25], and TargetP V1:01 (http://www.cbs.dtu.dk/services/TargetP/) [26].</p><p>3 Results</p><p>3.1 Exploration of the proteome of pea leafmitochondria toward a 3-D analysis:gel filtration as the first dimension</p><p>Highly purified mitochondria were isolated from pealeaves according to Douce et al. [21] using a two-roundisopycnic purification on Percoll gradients. The solubleproteins were released by a combination of osmoticshock and freeze-thaw cycles and separated from mem-brane proteins by high speed centrifugation. The solublemitochondrial proteome was analysed by high resolution2-D PAGE that combines IEF on immobilized pH gradi-ents in the first dimension with SDS-PAGE as the seconddimension. Initial experiments done on wide range IPGstrips (pH 310) revealed that almost all the proteinsexhibited pIs between 4 and 8 (results not shown). Sucha distribution led us to prepare strips in this range toincrease the resolution during the first dimension. The2-DE map of the soluble proteins (500 g) from pea leafmitochondria is presented in Fig. 1. When the gel wasstained with Coomassie blue, 433 spots were detectedwith the PDQuest 2-D gel analysis software (Bio-Rad).The spots were quantified and the values obtained rang-ed from 972 to 1143997 arbitrary detection units for atotal integrity density of 17975406. This means that a pro-tein which represents 0.005% of the initial input can bevisualised. Since the information concerning pea proteinsin databases was limited (286 proteins in SWISS-PROTand 485 in TrEMBL, May 2002), our strategy was to useN-terminal Edman degradation as the primary tool foridentification of protein spots. After electroblotting onPVDF membranes, the major visible spots were analysedyielding a set of peptide sequence tags between 8 and 24residues that are listed in Table 1.</p></li><li><p>Proteomics 2002, 2, 880898 Plant mitochondria proteomics and development 883</p><p>Table 1. Pea proteins identified from the 2-D gel analyses of the soluble proteins of green leaf mitochondria (pI 4.0 to 8.0)</p><p>Name or description SpotNo.</p><p>Accessionno.(organism)</p><p>Edman* or ESI-MS/MS MALDI pIobs</p><p>MMobs(kDa)</p><p>sequence % of identity/% of similarity</p><p>Cov.(%)</p><p>Proteins involved in the photorespiratory pathway</p><p>Glycine cleavage system P protein(GCS P)</p><p>4721, 4722,4723</p><p>P26969(Pisum)</p><p>ISVEALKPSDTF* 100 () 6.4 104</p><p>Glycine cleavage system H protein(GCS H)</p><p>386, 10, 17</p><p>P16048(Pisum)</p><p>SNVLDGLKYA* 100 () 4.2 14</p><p>Glycine cleavage...</p></li></ul>


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