Proteome Analysis of Tobacco Bright Yellow-2 (BY-2) Cell Culture Plastids as a Model for Undifferentiated Heterotrophic Plastids Sacha Baginsky,* Asim Siddique, and Wilhelm Gruissem Institute of Plant Science and Functional Genomics Center Zurich, Swiss Federal Institute of Technology, ETH Zurich, 8092 Zurich, Switzerland Received April 23, 2004
We analyzed the proteome of undifferentiated plastids from a tobacco BY-2 cell culture by shotgun proteomics following multidimensional protein fractionation. The fractionation strategy initiated with the serial extraction of proteins from membranes which allowed us to distinguish soluble, peripheral, and integral membrane proteins. The majority of the identified proteins have a function in the cellular metabolism and most of them are active in amino acid synthesis pathways. A significant number of the identified proteins was not identified in chloroplast proteome analyses before. This suggests BY-2 plastid specific functions that differ from the major activities of chloroplasts. We have used the BY-2 plastid proteins reported here to assess the metabolic activities of undifferentiated heterotrophic plastids and compared the functional profile with that of differentiated heterotrophic amyloplasts. Comparative shotgun proteome analyses as reported here provide information about prevalent metabolic activities of different plastid types. Keywords: mass spectrometry • protein profiling • plastid differentiation
Introduction Plastids are a group of plant cell organelles that are responsible for essential biosynthetic and metabolic activities including photosynthetic carbon fixation and the synthesis of fatty acids, pigments, starch, and amino acids. Although plastids have lost their autonomy and transferred most of their genes to the nucleus1 they have retained the ability to develop and differentiate from proplastids in a signal-dependent and tissuespecific manner. Aspects of plastid morphology and function such as the structure, the pigment content (color) and the stage of development have been used for plastid classification.2 Probably the most fundamental distinction between different plastid types is heterotrophy versus autotrophy. Although autotrophy is restricted to photosynthetically active chloroplasts, several distinct heterotrophic plastid types can be found in different plant tissues e.g., elaioplasts, chromoplasts, amyloplasts, and etioplasts.2 These plastids are end products of a developmental program that is determined by the cell and tissue type (elaioplasts, amyloplasts, and chromoplasts) as well as by environmental factors2 (etioplasts). A special case among the heterotrophic plastids is the proplastid that is undifferentiated and represents an early stage of plastid development. Therefore, heterotrophy of this plastid type is potentially transient. * To whom correspondence should be addressed: Sacha Baginsky. Institute of Plant Sciences, Swiss Federal Institute of Technology, ETH Zentrum, LFW E51.1 Universita¨tstrasse 2, CH-8092 Zu ¨ rich, Switzerland. Phone: +41 1 632 3866; Fax: +41 1 632 10 79. E-mail: sacha.baginsky@ ipw.biol.ethz.ch.
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Journal of Proteome Research 2004, 3, 1128-1137
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Despite intensive research over the past decade, not much knowledge is available about the metabolic activities and the protein complement of different plastid types. Several proteome analyses with plant cell organelles were conducted during the last three years, including studies with mitochondria, peroxisomes, amyloplasts, and chloroplasts.3-14 Except for the analysis of the amyloplast proteome,7 all other plastid proteome studies have been conducted with autotrophic chloroplasts. These analyses confirmed that the proteomes of heterotrophic and autotrophic plastids differ considerably which is especially apparent for proteins that function in energy metabolism. Heterotrophic plastids import metabolites such as ATP and glucose 6-phosphate for essential biosynthetic activities, e.g., the synthesis of starch from ADP glucose, fatty acids from acetate and amino acids from inorganic nitrogen.2 These biosynthetic pathways have a high demand for energy and reducing equivalents. Substantial evidence has accumulated that heterotrophic plastids generate reducing equivalents by the oxidative branch of the pentose phosphate pathway that is initiated by glucose 6-phosphate.15 Glucose 6-phosphate as well as ATP is imported from the cytosol by well characterized plastidic glucose 6-phosphate/phosphate translocator and ATP/ ADP transporters.16 Most of the so far described metabolic functions are characteristic for fully differentiated plastids in specialized plant tissues. To date knowledge about prevalent metabolic activities in undifferentiated heterotrophic plastids is scarce. In the work presented here we analyzed the protein complement of heterotrophic BY-2 plastids as a model for undifferentiated plas10.1021/pr0499186 CCC: $27.50
2004 American Chemical Society
BY-2 Plastid Proteomics
tids.17 A detailed functional assignment of the identified proteins is presented and discussed in the context of the metabolic network that determines plastid functions in the plant cell.
Material and Methods Isolation of BY-2 Plastids. Tobacco (Nicotiana tabacum) BY-2 cells were grown in modified Murashige and Skoog medium as described previously.18 BY-2 plastids were isolated essentially as described19 with modifications. BY-2 cells were collected after 80-86 h from a suspension culture (∼250 g fresh weight), washed three times with 0.4 M mannitol pH 5.0 and digested in three volumes of enzyme solution (1% Onozuka RS cellulase and 0.1% pectolyase Y-23 containing 0.4 M mannitol, pH 5.6) at 30 °C for 2 h. Protoplasts were harvested by centrifugation at 1000 × g at 4 °C for 5 min followed by three washing steps with five volumes of ice cold 0.4 M mannitol, pH 5.0. The pellet was resuspended in plastid isolation buffer (0.4 M mannitol, 20 mM Tris-HCl, pH 7.6, 0.5 mM EDTA, 1.2 mM spermidine, 7 mM 2-mercapoethanol, 0.6% [w/v] poly(vinylpyrrolidone) [PVP] and 0.1% [w/v] BSA). The protoplasts were broken by passing the suspension several times through two layers of a 25 µm mesh under high pressure. The suspension of broken protoplasts was subsequently centrifuged at 1500 × g for 5 min at 4 °C to pellet the cell debris and nuclei. The supernatant was filtered again through two layers of a 25 µm mesh under gravitational force. Percoll was added to the filtrate to a final concentration of 15%. The proplastids were pelleted by centrifugation at 15 000 × g for 20 min at 4 °C. They were further purified from mitochondria, nuclei, and cellular debris by sucrose density gradient centrifugations (30 to 50 to 70% sucrose in 20 mM Tris-HCl, pH 7.6, 0.5 mM EDTA, 1.2 mM spermidine, and 7 mM 2-mercaptoethanol) for 40 min at 2000 × g. The yellow band of purified plastids that formed at the 50 to 70% sucrose interface was collected, washed twice with plastid isolation buffer (minus PVP and BSA) and loaded onto a linear sucrose gradient from 30 to 70% sucrose for further purification. Proplastids were washed two times with plastid isolation buffer, centrifuged at 500 × g and the pellet was stored at -80 °C for further studies. The purity of the preparation was established by the absence of mitochondrial fumarase and peroxisomal catalase activity. Fumarase activity was measured spectrophotometrically with L-malate as substrate (50 mM L-malate in 50 mM potassium phosphate buffer, pH 7.9) as described before.20 Catalase activity was measured by the decrease of H2O2 in the sample (0.01 M H2O2 in 0.1 M potassium phosphate buffer, pH 7.0), measured at 240 nm. Fractionation of Plastid Proteins. To minimize dynamic range limitations of mass spectrometric protein identifications we employed extensive protein fractionation techniques consisting of a serial protein extraction followed by liquid chromatography (LC) and 10% SDS-PAGE21 (sodium dodecyl sulfatepoly acryamide gel electrophoresis). Starting from intact plastids the proteins were first fractionated according to their differential solubility by a serial extraction procedure using four buffer compositions with an increasing solubilization capacity. After each step, insoluble material was precipitated by ultracentrifugation (100 000 × g for 45 min), washed twice, and subsequently used for the next extraction step. The buffer compositions of each step were as follows: First step: 40 mM Tris/HCl pH 8.0, 5 mM MgCl2, 1 mM DTT (dithiothreitol) and
research articles 2× protease inhibitor cocktail (Roche Diagnostics GmbH, Germany, 2× of supplier’s recommended concentration); second step: 8 M urea, 20 mM Tris-base, 5mM MgCl2, 20 mM DTT and 2× protease inhibitor; third step: 7 M urea, 2 M thiourea, 20 mM Tris-base, 40 mM DTT, 2% CHAPS (3-[(3cholamidopropyl)dimethylammonio]-1-propanesulfonate), 1% Brij 35 (polyoxyethylene (23) lauryl ether), 2× protease inhibitor; fourth step: 40 mM Tris base, 5% SDS, 40 mM DTT and 2× protease inhibitor. Soluble proteins and peripheral membrane proteins were further fractionated by liquid chromatography using ion exchange (IEX) chromatography on MonoQ (Bio Rad, Hercules, USA). Proteins bound to MonoQ were eluted in six steps ranging from 50 mM, 100 mM, 200 mM, 500 mM, 1 M to 2 M KCl in step 1 or step 2 buffer, respectively. Where indicated, highly abundant purine nucleotide binding proteins were subtracted from protein fractions by Blue Sepharose affinity chromatography in step 1 buffer prior to IEX chromatography. Bound proteins were eluted with step 1 buffer including 1.5 M KCl. Proteins from each fraction were further fractionated according to their molecular mass by 10% SDSPAGE.19 Integral membrane proteins were directly subjected to SDS-PAGE. The SDS-gels were cut into 10 sections and the proteins in each section were immediately subjected to in gel tryptic digest. Tryptic digestion was performed according to Shevchenko and colleagues22 using sequencing grade modified Trypsin (Promega, USA). Following digestion, peptides were lyophilized to dryness and stored at -80 °C. Mass Spectrometric Protein Identification. Prior to identification by mass spectrometry, tryptic peptides were separated by reversed phase-liquid chromatography (RP-LC) on C18 material. Peptides were separated either on a MTVC15C18w150 capillary column (Micro-Tech Scientific, Inc. Sunnyvale, USA) for electrospray ionization (ESI) or for nanospray ionization (NI) on laboratory made silica-capillary columns with an inner diameter of 75 µm (length 8 cm). Twenty µL (ESI) or 5 µL (NI) of peptides resolved in buffer A (5% acetonitrile, 0.5% formic acid or 0.5% acetic acid in Millipore water) were loaded onto the column and eluted with an increasing concentration of acetonitrile (buffer B: 0.5% formic acid or 0.5% acetic acid in acetonitrile) at a flow rate of 1-2 µL/min (ESI) or 300 nl/min (NI). Complex peptide mixtures were separated by increasing the acetonitrile concentration from 5 to 65% over 2 h followed by an increase up to 85% during additional 15 min. For less complex mixtures a shorter gradient of 40 plus 10 min was developed. RP-LC was coupled online to an LCQDecaXP ion trap mass spectrometer (Thermo Finnigan, San Jose) equipped with either an ESI-source or a nanospraysource. Analysis was performed in the positive ion mode and peptides were ionized with a spray voltage of 3-4 kV for ESI and 2.1-2.8 kV for NI and analyzed by MS full scan and four data dependent MS/MS-scans of the four most intense parentions. The dynamic exclusion function was enabled to allow two measurements of the same parent-ion during one minute followed by exclusion for one minute. Analysis and Interpretation of MS-Data. MS/MS data were analyzed by the SEQUEST software using the NCBI non redundant protein database (http://www.ncbi.nlm.nih.gov/). Cysteines were allowed to be either unmodified or carboxyamidomethylated. We manually interpreted each SEQUEST output by filtering the peptide hits using the following hierarchical criteria: Journal of Proteome Research • Vol. 3, No. 6, 2004 1129
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Figure 1. Isolation and purification of BY-2 plastids. Extracts from BY-2 cells were subjected to sucrose step gradient centrifugation (30%/50%/70% sucrose). The gradient was fractionated into 1 mL aliquots and for each fraction the fumarase and catalase activities were determined. Additionally, fractions 4 to 6 (Band A) and 11 to 13 (Band B) were pooled and probed with antibodies against plastid TOC75. TOC75 is a component of the translocon at the outer chloroplast envelope membrane [inset, pt: plastid marker (TOC75)].
1. A cross-correlation score (Xcorr) of at least 2.5. 2. An ion coverage (ratio between detected and expected yor b-ions) of more than 40%. 3. Only fragment-spectra from doubly charged parent-ions were considered 4. Long peptides with more than 50 theoretical fragment ions (b- and y-ions) were not taken into account. 5. Grouping of at least four peptides with an Xcorr value of at least 2.5 to the same protein was rated as a significant protein identification when at least one of them exhibited a ∆CN value (normalized difference in correlation score, giving the difference between the front-ranking hit and the following possible hits) higher than 0.1. 6. All MS/MS-spectra that were not grouped as described in 5. but matched the above-mentioned criteria (1.-4.) were visually examined for a correct peak assignment and evaluated by the following criteria: i. A gapless assignment of a series of y- or b-ions to peaks of high intensity. ii. A y-ion with an N-terminal proline corresponding to a high peak. iii. A neutral loss of 18 Da (loss of water) from y- or b-ions carrying the amino acids serine or threonine.
Results BY-2 Plastid Isolation and Protein Fractionation. The purity of the BY-2 plastid preparation was established by fumarase and catalase measurements, and supported by antibody detection of a plastid marker protein (Figure 1). After the first sucrose density gradient centrifugation, fumarase and catalase activity showed one major peak at the interphase between 30 and 50% sucrose (Band A, Figure 1) demonstrating that peroxisomes and mitochondria are enriched in Band A. In contrast, antibody detection of plastid TOC75 demonstrated that plastids are enriched in Band B (Figure 1, see inset). For further purification, Band B was recovered and loaded onto a linear density gradient ranging from 30 to 70% sucrose (data not shown). Plastids after the second density gradient were washed, concentrated and used for subsequent protein fractionation and proteome analysis. BY-2 plastid proteins were first separated by their differential solubility (Figure 2). This procedure is referred to as “serial extraction” (proteins are solubilized from membranes with 1130
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Figure 2. Fractionation strategy for BY-2 plastid proteins. The multidimensional chromatography separated proteins according to their differential physical and chemical properties.
Figure 3. Serial extraction of BY-2 plastid proteins. (A) Silver stain of the proteins of each fraction from the serial extraction procedure. Solubilization of proteins was accomplished with buffers of increasing solubilization capacity (Material and Methods). (B) Quantitative distribution of BY-2 proteins among the different fractions of the serial extraction procedure as determined by Bradford analysis39 (n.d.: not determined).
buffers of increasing solubilization capacity) and provides information about the extent of membrane association for every identified protein. Protein fractions were analyzed by SDSPAGE and subsequent silver staining (Figure 3A) and the relative distribution of soluble (OSMO), peripheral (8 M urea) and integral membrane proteins (CHAPS) was determined by Bradford analyses (Figure 3 B). The soluble proteins make up for 16.7% of the total BY-2 plastid proteins. This value indicates that the isolated BY-2 plastids were largely intact since the soluble proteins would have been lost from defective plastids. A fraction of 46.8% of the BY-2 plastid proteins is attached to membranes and was solubilized with a buffer containing 8 M urea (Figure 3 B). Integral membrane proteins that were extracted with 7 M urea, 2 M thiourea, 1% Brij 35 and 2% Chaps (Figure 3 A) make up for 36.4% of all BY-2 plastid proteins (Figure 3 B). It is notable, that most of the integral membrane proteins from BY-2 plastids were solubilized with the detergent combination 1% Brij35/2% Chaps (Figure 3 A, compare lanes “CHAPS” and “5% SDS”). In contrast, most of the integral membrane proteins from chloroplasts require 5% SDS for solubilization.13 These different properties of integral membrane proteins are most likely a consequence of alterations in the lipid composition of plastid membranes upon development and differentiation. In addition, BY-2 plastids lack a distinctive internal membrane system comparable to the thylakoid system in fully differentiated chloroplasts.23 Characterization of Identified Proteins. Following the serial extraction and multidimensional protein fractionation (Figure 2) protein mixtures were further fractionated by SDS-PAGE. Prior to mass spectrometry, we separated the tryptic peptides
BY-2 Plastid Proteomics
by reversed phase chromatography (C18) and analyzed them by electrospray ionization mass spectrometry (LC-ESI-MS/ MS) with an ion trap (LCQDecaXP, Finnigan, San Jose). Proteins were identified by SEQUEST searches using the NCBI non redundant protein database (http://www.ncbi.nlm.nih.gov/) and by MASCOT searches with the tobacco BY-2-EST database from Riken (http://mrg.psc.riken.go.jp/strc/). All identified proteins were assembled into a database in FASTA format and a BLAST search24 was performed against itself to eliminate redundant protein entries i.e., identical proteins from different organisms (all entries giving an E-value of zero were deleted). Identified proteins are listed in Tables 1 and 2 and further information on the identified proteins is available in the Supporting Information, Supplemental Tables 1-3. Table 1 lists all identified proteins that were manually assorted to their putative function. We included information about the “serial extraction” step from which each protein was identified, i.e., osmotic shock (O), urea (U), or CHAPS (C). By inference, this information provides information about the solubility of the identified proteins and their membrane attachment in vivo. Most of the enzymes active in amino acid and carbohydrate metabolism are soluble proteins that were identified in the osmotic shock fraction. Envelope proteins and putative transporters were identified from the urea and the CHAPS fractions consistent with their function at the envelope membrane. Proteins from all other functional categories include soluble, peripheral and integral membrane proteins in similar distribution. It is notable that most of the identified putative contaminants derived from the urea and the CHAPS fraction suggesting that their identification is a result of their attachment to the outer envelope membrane of the BY-2 plastids that occurred possibly during cell lysis. Proteins listed in Table 2 were identified from the BY-2 EST database from Riken (http://mrg.psc.riken.go.jp/strc/). We carefully analyzed the identified proteins in more detail to substantiate their plastid localization. In addition to the plastid-encoded proteins, we accepted all proteins as true plastid proteins that have (i) a predicted plastid transit peptide (ChloroP,25 Table 1 column “Localization”). (ii) a reported function in plastids and (iii) an Arabidopsis thaliana orthologue that has a clear plastid transit peptide (all information about these categories is available in the Supporting Information, Supplemental Table 2). The majority of all identified proteins (124 out of 168) fall into one of the above categories (Figure 4 A) and these proteins are thus most likely true plastid proteins (Supplemental Table 2). Twenty-two proteins could not be unambiguously assigned as true plastid proteins. We therefore analyzed whether orthologues of these proteins were identified in other plastid proteome studies9,10,12-14 to provide further evidence for their plastid localization or their specific interaction with the plastid outer membrane system. Seven of these proteins were identified in one other proteome analysis, 9 proteins in two other analyses and 2 proteins in three other studies, while only 5 proteins were not detected in chloroplasts previously (Figure 4B, Supplemental Table 3). Several proteins were identified that are most likely not of plastid origin. The majority of these proteins are ribosomal proteins or eukaryotic translation factors (Figure 4A, Supplemental Table 1). It is known that ribosomal proteins can associate with the outer envelope membrane of plastids therefore their detection in a plastid membrane preparation is not unexpected.7 In addition to ribosomal proteins, we detected several proteins that have a reported function in mitochondria
research articles and nuclei as well as the peroxisomal catalase (Figure 4 A, Table 1). Although dual targeting cannot be ruled out, the fact that these proteins rank among the most abundant proteins in other cell organelles suggests that they are true contaminants. Several proteomics studies reported contaminations of organelle preparations with highly abundant proteins from other cell organelles.4,8 It appears that fumarase and catalase assays for the detection of mitochondria and peroxisomes in a plastid preparation that were employed in this study (Figure 1) are insufficient to reveal minor contaminations with these cell organelles. Most of the identified true plastid proteins have a general function in the cellular metabolism and the majority is involved in the biosynthesis of amino acids. We have identified many tryptic peptides from glutamine synthetase, glutamate synthase, and glutamate dehydrogenase suggesting the high abundance of these enzymes (Table 1). These enzymes are active in the primary nitrogen fixation that is the basis for the synthesis of other amino acids.26 Consistently, several proteins from the arginine, the branched-chain amino acid and the aromatic amino acid biosynthesis were identified that utilize the reduced nitrogen (Table 1). It is conceivable that the BY-2 plastid provides the rapidly growing and dividing cell with the amino acids required for protein biosynthesis and other cellular functions. The identification of many proteins with a function in protein folding such as chaperones and heat shock related proteins together with the identification of CLP-protease subunits supports the view of a high protein turnover rate and the need for folding of newly synthesized proteins at a high rate. The CLP-protease is thought to perform a housekeeping function in plastids and is present in virtually all plant tissues. Its exact role is unknown, but chloroplast development is impaired in the absence of the CLP-protease.27 Besides dominant amino acid biosynthetic pathways, carbohydrate and starch metabolic activities are prevalent in BY-2 plastids (Table 1). We have identified several transporters and proteins of the plastid envelope membrane from BY-2 plastids. The most abundant of these proteins are the ATP/ADP transporter, the oxoglutarate/malate translocator and the outer envelope protein (OEP) 75 homologue. Although OEP 75 is a major component of the plastid protein import machinery,28-30 the ATP/ ADP transporter and the oxoglutarate/malate translocator are involved in metabolite exchange between the plastid and the cytosol.16,31 The identification of these transporters in the BY-2 plastid membranes reflects the energetic requirements of heterotrophic plastids. The ATP/ADP transporter imports cytosolic ATP that is generally required for the synthesis of various metabolites such as amino acids and starch in heterotrophic plastids.2 Oxoglutarate is the precursor for ammonia assimilation and recent reports suggested that the oxoglutarate/malate translocator imports carbon skeletons for net glutamate synthesis.16 In addition to the aforementioned transporters, we have detected the glucose 6-phosphate/phosphate translocator suggesting glucose 6-phosphate import into the BY-2 plastid. Glucose 6-phosphate initiates the oxidative pentose phosphate pathway that provides reducing equivalents for example for the synthesis of glutamate from oxoglutarate.16,32 The glucose 6-phosphate/phosphate translocator was not detected in autotrophic chloroplasts.13 Only a few plastid encoded proteins were identified although the plastome of tobacco is completely sequenced. It is notable that the genes for all identified plastid encoded proteins possess Journal of Proteome Research • Vol. 3, No. 6, 2004 1131
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Table 1. List of Proteins Identified by Electrospray Ionization Mass Spectrometry (LC-ESI-MS/MS) Identifier
acc. no
gi|7573355 gi|7488146 gi|7450428 gi|7437011 gi|7433550 gi|7431783 gi|7431781 gi|7431768 gi|7387849 gi|6685804 gi|6578124 gi|6319165 gi|6014908 gi|5931761 gi|5822270 gi|5701896 gi|482934 gi|419757 gi|399333 gi|3941322 gi|2982255 gi|2811029 gi|266463 gi|2492952 gi|232202 gi|2252472 gi|1711381 gi|1708993 gi|1477480 gi|127041 gi|1170938 gi|1168260 gi|114208 gi|114165 gi|1066499 gi|100437
CAB87661 T05416 T03270 T07937 T05362 T06228 S67499 T16982 O04974 O24578 AAF17705 AAF07191 Q42948 CAB56614 1QGNA CAA47373 CAA54043 S30145 P31300 AAC82334 AAC32115 O04866 P29696 Q42884 P30109 CAB10698 P52877 P53780 AAB67843 P23686 P43281 P46248 P25317 P23981 AAB41904 A35016
gi|99742 gi|7489279 gi|7484671 gi|7433617 gi|7431231 gi|7109469 gi|6690395 gi|5123836 gi|3121731 gi|2833379 gi|2499497 gi|1351271 gi|120714
A41370 T07790 T09153 T09541 T06401 AAF36733 AAF24124 CAB45387 O04916 Q42581 Q42961 P48496 P09317
gi|7441880 gi|7331143 gi|461944 gi|4583546 gi|2829901 gi|1729864 gi|1710807 gi|134103
T08899 AAF60293 Q04960 CAB40381 AAC00609 P54411 P08926 P21240
gi|729409 gi|6831644 gi|133424 gi|133248
P41342 Q42362 P06271 P19683
gi|7450157 gi|6018208 gi|4204861 gi|1708313 gi|1515105 gi|1076758
S74252 AAF01789 AAD11550 P51818 CAA68885 S49340
1132
organism
fraction
no. of peptidesa
localizationb
proteins identified from BY2 plastids amino acid synthesis Arabidopsis thaliana O 2 Y Arabidopsis thaliana O/U 2 Y Nicotiana tabacum O 3 reported Pt Chlamydomonas reinhardtii C 2 Y Arabidopsis thaliana O 2 reported Pt Glycine max O 10 reported Pt Nicotiana tabacum U/C 46 reported Pt Nicotiana plumbaginifolia U/C 7 reported Pt Lycopersicon pennellii O/U 7 Y Zea mays U 2 Y Canavalia lineata O 10 Y Solanum tuberosum O 2 Y Nicotiana tabacum O 3 Y Nicotiana plumbaginifolia U 9 reported Pt Nicotiana tabacum O 5 reported Pt Nicotiana sylvestris O 6 Y Nicotiana tabacum U 2 reported Pt Arabidopsis thaliana O 27 Y Capsicum annuum O 9 Y Medicago truncatula O 2 Y Picea mariana C 4 reported Pt Alnus glutinosa O 21 Y Solanum tuberosum O 5 reported Pt Lycopersicon esculentum O 5 Y Nicotiana tabacum U/C 4 co Arabidopsis thaliana O 2 Y Spinacia oleracea O 4 Y Arabidopsis thaliana U 2 Y Arabidopsis thaliana O 28 Y Arabidopsis thaliana U/C 8 co Lycopersicon esculentum U/C 8 Arabidopsis thaliana O 7 Y Nicotiana tabacum U/C 3 co Nicotiana tabacum O 5 Y Medicago sativa U/C 29 Y Solanum tuberosum O 12 Y carbohydrate metabolism Arabidopsis thaliana O 7 Y Solanum tuberosum O 35 Y Spinacia oleracea O 31 Y Capsicum annuum O 44 Y Lycopersicon esculentum O/U/C 8 Y Arabidopsis thaliana O 11 Y Arabidopsis thaliana O 2 Y Nicotiana tabacum O 6 Y Solanum tuberosum U 2 reported Pt Arabidopsis thaliana U 2 reported Pt Nicotiana tabacum O 13 Y Spinacia oleracea O 11 Y Ustilago maydis O 19 reported Pt chaperones Spinacia oleracea C 6 Y Lycopersicon esculentum O/U 10 Y Cucumis sativus U 3 1 Arabidopsis thaliana U 2 Y Arabidopsis thaliana U 2 reported Pt Avena sativa U 4 1, 2 Pisum sativum O 19 Y Arabidopsis thaliana U/C 12 Y gene expression Nicotiana tabacum U/C 18 Y Musa acuminata O 2 reported Pt Nicotiana tabacum U 2 pt-encoded Nicotiana sylvestris O 7 Y heat shock-related proteins Arabidopsis thaliana O 2 1, 2 Philodina roseola U/C 2 1, 2 Triticum aestivum U 6 1, 2 Arabidopsis thaliana C 2 1, 3 Arabidopsis thaliana U 2 1, 2 Secale cereale U/C 15 Y
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protein
diaminopimelate decarboxylase-like protein probable phosphoglycerate dehydrogenase probable histidinol-phosphate transaminase tryptophan synthase beta chain glycine hydroxymethyltransferase Fe-dependent glutamate synthase glutamate synthase glutamate dehydrogenase 2-isopropylmalate synthase B adenylosuccinate synthetase ornithine carbamoyltransferase branched-chain amino acid aminotransferase dihydropicolinate synthase acetolactate synthase small subunit cystathionine gamma-synthase glutamate-ammonia ligase glutathione reductase ketol-acid reductoisomerase cysteine synthase gamma-glutamylcysteine synthetase probable NADH-glutamate synthase acetylornithine aminotransferase 3-isopropylmalate dehydrogenase chorismate synthase 1 glutathione S-transferase argininosuccinate lyase phosphoserine aminotransferase cystathionine beta-lyase carbamoyl phosphate synthetase large chain S-adenosylmethionine synthetase 1 S-adenosylmethionine synthetase 2 aspartate aminotransferase probable glutathione S-transferase EPSP synthase 1 NADH-dependent glutamate synthase cystathionine gamma-lyase 2-dehydro-3-deoxy-phosphoheptonate aldolase transaldolase glucose-6-phosphate isomerase transketolase malate dehydrogenase putative enolase phosphoglucose isomerase NAD-malate dehydrogenase aconitate hydratase ribose-phosphate pyrophosphokinase 1 phosphoglycerate kinase triosephosphate isomerase glyceraldehyde 3-phosphate dehydrogenase dnaK-type molecular chaperone HSC70-9 chaperonin 21 DNAJ protein homolog GrpE protein putative 10kd chaperonin T-complex protein epsilon subunit rubisco subunit binding-protein alpha subunit rubisco subunit binding-protein beta subunit elongation factor TU 30S ribosomal protein S8 DNA-directed RNA polymerase beta chain 31 kDa ribonucleoprotein heat shock protein 91 82-90 kD heat shock protein heat shock protein 90 heat shock protein 81-3 heat shock protein 90A heat-shock protein 82K
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BY-2 Plastid Proteomics Table 1. (Continued) Identifier
acc. no
organism
fraction
no. of peptidesa
localizationb
protein
proteins identified from BY2 plastids (continued) hypothetical or unknown proteins gi|7529745 CAB86930 Arabidopsis thaliana U 2 co putative protein gi|7489244 T07050 Solanum tuberosum O/U 13 Y hypothetical protein R1 gi|7486826 T04912 Arabidopsis thaliana C 2 reported Pt hypothetical protein T10I14.140 gi|7486039 T02491 Arabidopsis thaliana U 2 1, 2 hypothetical protein/putative ABC transporter gi|7485588 T01258 Arabidopsis thaliana U 4 Y hypothetical protein gi|7447845 T04985 Arabidopsis thaliana U 2 reported Pt hypothetical protein T16L1.170 gi|7438086 S76557 Synechocystis sp O 4 reported Pt hypothetical protein gi|7362740 CAB83110 Arabidopsis thaliana U 2 putative protein gi|6723394 CAB66403 Arabidopsis thaliana U 3 Y putative protein gi|6714365 AAF26055 Arabidopsis thaliana U 2 Y unknown protein gi|6358779 AAF07360 Arabidopsis thaliana U 2 Y unknown protein gi|5042434 AAD38273 Arabidopsis thaliana U 2 Y hypothetical protein gi|3915961 P09976 Nicotiana tabacum U 4 pt-encoded YCF2 hypothetical 267 kDa protein gi|2811028 O04658 Arabidopsis thaliana U/C 2 hypothetical 47.9 kDa protein gi|231660 P12222 Nicotiana tabacum U 6 pt-encoded YCF1 hypothetical 226 kDa protein gi|1076722 S49173 Hordeum vulgare O 2 reported Pt hypothetical protein nucleotide metabolism gi|6939228 AAF31730 Arabidopsis thaliana O 5 Y putative phosphoribosylformylglycinamidine synthase gi|4874278 AAD31343 Arabidopsis thaliana O/U 7 Y member of the phosphoribosyl pyrophosphate synthetase family gi|1172754 Q05728 Arabidopsis thaliana O/U 6 Y phosphoribosylformylglycinamidine cyclo-ligase other metabolism gi|7525018 NP_051044 Arabidopsis thaliana U/C 17 pt-encoded ATPase alpha subunit gi|7438127 T02431 Nicotiana tabacum O/U/C 15 Y acetyl-CoA carboxylase gi|7431333 T03406 Nicotiana tabacum U/C 2 reported Pt probable isocitrate dehydrogenase gi|6856558 AAF29977 Tagetes erecta U 2 reported Pt isopentenyl pyrophosphate:dimethyllallyl pyrophosphate isomerase gi|585449 P37222 Lycopersicon esculentum O 5 reported Pt NADP-dependent malic enzyme gi|585421 P38418 Arabidopsis thaliana U 4 Y lipoxygenase gi|585010 P37294 Synechocystis sp U 2 reported Pt phytoene synthase gi|5532608 AAD44809 Nicotiana tabacum O/U 11 Y 6,7-dimethyl-8-ribityllumazine synthase gi|4567203 AAD23619 Arabidopsis thaliana U 4 reported Pt putative beta-hydroxyacyl-ACP dehydratase gi|3721540 BAA33531 Nicotiana tabacum U/C 26 Y sulfite reductase gi|3157931 AAC17614 Arabidopsis thaliana O 2 reported Pt similar to pyrophosphate-dependent phosphofructokinase beta subunit gi|3023817 Q43793 Nicotiana tabacum U 5 Y glucose-6-phosphate 1-dehydrogenase gi|2498077 P93554 Saccharum officinarum O 7 reported Pt nucleoside diphosphate kinase I gi|2497542 Q40546 Nicotiana tabacum U 2 Y pyruvate kinase isozyme G gi|2497541 Q40545 Nicotiana tabacum O 10 Y pyruvate kinase isozyme A gi|2454184 AAB86804 Arabidopsis thaliana U 4 Y pyruvate dehydrogenase E1 beta subunit gi|2340166 AAB67319 Arabidopsis thaliana U 2 glutathione S-conjugate transporting ATPase gi|1583601 2121278A Capsicum annuum U 17 Y zeta carotene desaturase gi|1552717 AAB08578 Nicotiana tabacum U/C 2 squalene synthase porphyrin and chlorophyll metabolism gi|3334149 O22436 Nicotiana tabacum U 2 Y MG-protoporphyrin IX chelatase gi|2493809 Q42840 Hordeum vulgare U 2 Y coproporphyrinogen III oxidase gi|1170242 P42045 Hordeum vulgare U/C 2 Y ferrochelatase gi|1097877 2114378A Lycopersicon esculentum U 2 Y aminolevulinate dehydratase proteases gi|6456169 AAF09157 Arabidopsis thaliana U 3 Y putative aminopeptidase gi|4887543 CAB43488 Arabidopsis thaliana U 2 pt-encoded ATP-dependent Clp protease subunit ClpP gi|4105131 AAD02267 Spinacia oleracea U 8 Y ClpC protease gi|399213 P31542 Lycopersicon esculentum O 13 Y ATP-dependent Clp protease ATP-binding subunit clpA homologue CD4B gi|399212 P31541 Lycopersicon esculentum U/C 12 Y ATP-dependent Clp protease ATP-binding subunit CLPA homologue CD4A gi|2493734 P74361 Synechocystis sp U 4 reported Pt CLPB protein gi|2077957 CAA73318 Arabidopsis thaliana U 4 reported Pt putative FTSH protease gi|116527 P12210 Nicotiana tabacum O 2 reported Pt ATP-dependent Clp protease proteolytic subunit redox gi|4996602 BAA78552 Nicotiana tabacum O 2 Y thylakoid-bound ascorbate peroxidase gi|3121825 O24364 Spinacia oleracea O 8 Y peroxiredoxin BAS1 gi|134672 P11796 Nicotiana plumbaginifolia U 10 1, 3, 4 superoxide dismutase (Mn) gi|100284 A39267 Nicotiana plumbaginifolia O 20 reported Pt superoxide dismutase (Fe) starch metabolism gi|4874272 AAD31337 Arabidopsis thaliana U 4 1, 2 strong similarity to gb|Y09533 involved in starch metabolism gi|4584503 CAB40743 Solanum tuberosum O 4 Y starch branching enzyme II gi|2833389 Q43846 Solanum tuberosum O 4 Y soluble glycogen [starch] synthase (SS III) gi|267196 Q00775 Solanum tuberosum O 2 Y granule-bound glycogen [starch] synthase gi|21579 CAA36612 Solanum tuberosum O 16 Y starch phosphorylase gi|130173 P04045 Solanum tuberosum O 15 Y alpha-glucan phosphorylase, L 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Table 1. (Continued) Identifier
gi|7489185 gi|7488227 gi|7450791 gi|6016683 gi|464848 gi|462013 gi|1351202 gi|113217
acc. no
T03843 T04622 T09555 AAF01510 P33628 P35016 P28551 P23343
gi|7489246 T07405 gi|7267551 CAB78032 gi|7229675 gi|6226239 gi|4101473 gi|2499535 gi|124429 gi|100424
AAF42936 O24381 AAD01191 Q41364 P23525 S17917
gi|2668492 BAA23769 gi|1061420 AAA81348 gi|1049255 AAA80347 gi|6730768 gi|6714392 gi|477819 gi|4210330 gi|3928138 gi|3334123 gi|2689078 gi|2565305 gi|2117355 gi|1842188 gi|1172555 gi|114421 gi|1076621 gi|100961
AAF27157 AAF26081 B48529 CAA11552 CAA10267 Q96250 AAB88906 AAB82711 S51590 CAA69726 P42055 P17614 S46306 S17947
gi|7649159 gi|7451352 gi|6056371 gi|4883746 gi|266989 gi|1549222 gi|122101
AAF65769 T06379 AAF02835 AAD31625 Q01474 BAA13463 P04915
gi|1093432 2104177A
organism
no. of fraction peptidesa localizationb
protein
proteins identified from BY2 plastids (continued) structural maintenance and plastid positioning Nicotiana tabacum C 6 Y prohibitin Arabidopsis thaliana U 6 co prohibitin-like protein Arabidopsis thaliana U/C 2 Y fibrillarin Arabidopsis thaliana U 3 1 putative clathrin heavy chain Picea abies U/C 6 1 tubulin alpha chain Catharanthus roseus U/C 10 1, 2 endoplasmin homolog Glycine max U/C 8 1 tubulin beta chain Daucus carota U/C 2 1 actin 1 transporters and envelope proteins Solanum tuberosum U/C 3 co oxoglutarate/malate translocator Arabidopsis thaliana C 2 reported Pt outer envelope membrane protein OEP75 precursor homologue Arabidopsis thaliana U 2 Y glucose 6 phosphate/phosphate translocator Solanum tuberosum U/C 13 Y plastidic ATP/ADP-transporter Arabidopsis thaliana U 5 1, 2, 3 K+-efflux antiporter-1 Spinacia oleracea U/C 2 Y 2-oxoglutarate/malate translocator Spinacia oleracea U/C 4 Y 37 KD inner envelope membrane protein Solanum tuberosum U/C 6 co ADP,ATP carrier protein proteins with a reported function in other cell organelles putative membrane contaminants Arabidopsis thaliana U/C 3 Y metal-transporting P-type ATPase Vicia faba U/C 2 co p-type H+-ATPase Zea mays U/C 3 co H+-pyrophosphatase putative mitochondrial contaminants Arabidopsis thaliana U 2 Y putative alanine aminotransferase Arabidopsis thaliana U 2 Y putative mitochondrial LON ATP-dependent protease Solanum tuberosum U/C 2 1 ubiquinol-cytochrome-c reductase Arabidopsis thaliana U 13 Y 2-oxoglutarate dehydrogenase Catharanthus roseus U 14 co mitochondrial elongation factor TU Arabidopsis thaliana U/C 2 co ATP synthase gamma chain Brassica rapa C 2 co cytochrome c oxidase subunit II Tritordeum sp. U 3 Y glycine decarboxylase P subunit Solanum tuberosum C 2 Y mitochondrial processing peptidase Betula pendula U 2 Y mitochondrial phosphate translocator Solanum tuberosum U/C 8 co 34 kDa outer mitochondrial membrane protein porine Nicotiana plumbaginifolia U/C 23 Y ATP synthase beta chain Nicotiana tabacum C 2 co cytochrome b5 Zea mays C 2 co protein URF13 putative nuclear contaminants Euphorbia esula U/C 10 co histone H2A Pisum sativum U/C 2 co SAR DNA-binding protein 2 Arabidopsis thaliana U 2 co nucleolar protein Campodea tillyardii C 2 co histone H3 Arabidopsis thaliana C 6 co GTP-binding protein SAR1B Nicotiana tabacum U/C 2 co NtSar1 protein Physarum polycephalum O/U/C 30 co histone H4 putative peroxisomal contaminant Helianthus annuus U 2 co catalase
a Only peptides with a significant SEQUEST score (i.e., Xcorr > 2.5, dCn > 0.1) were taken into account. Protein hits were only accepted if at least two peptides were detected that comply with these criteria. b Localization prediction was performed with ChloroP25 as described. Reports on plastid localization were retrieved from the NCBI protein database (http://www.ncbi.nlm.nih.gov). c Putative protein functions were manually assigned. Proteins with a reported function in other cell organelles were considered as putative contaminants. Provided is the identifier (“Identifier”, the database accession number (“acc. no.”), the organism (“organism”), the number of identified tryptic peptides (“no. of peptides”), protein localization [“Localization” (Y: transit peptide by ChloroP24, reported Pt: reported chloroplast function or Arabidopsis orthologue with a transit peptide, co: putative contaminant, 1: detected in ref 13, 2: detected in ref 14; 3: detected in ref 12; 4: detected in ref 9)] and the information from which extraction step the protein was identified (“Fraction”), i.e., osmotic shock (O), urea (U), or CHAPS (C).
a promoter that is recognized by the nucleus encoded plastid RNA polymerase (NEP, Table 3). While ycf1, clpP, and atpB contain an additional promoter element for the plastid encoded plastid RNA polymerase (PEP), ycf2, and rpoB are exclusively transcribed from a NEP promoter.33-35 The NEP RNA polymerase has a predominant role for the transcription of the plastome in undifferentiated plastids when the PEP components are not expressed.33,34 We identified several peptides of YCF1 and YCF2 from fractions with a molecular weight above 200 kDa as judged by SDS-PAGE. This is consistent with the 1134
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molecular weight predicted for YCF1 (226 kDa) and YCF2 (267 kDa), suggesting that the complete reading frame is transcribed and translated in BY-2 plastids.36 Protein Profiling Reveals Prevalent Functions of Different Plastid Types. Shotgun proteomics is biased toward the detection of abundant proteins and therefore provides some limited quantitative information. A comparative analysis of shotgun proteomics data from different plastid types is therefore suitable to reveal prevalent metabolic activities and plastidtype specific functions. We have conducted BLAST searches
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Table 2. Tandem Mass Spectrometry (MS/MS) Data Were Searched against the BY-2 EST Database from the Riken Plant Science Center with MASCOT (peptide mass tolerance 1.5, fragment ion tolerance 0.8)a identified EST
7106f1 2121f1 191f1 1483f1 4271f1 2970f1 7005f1 2251f1 746f1 6996f1 1271f1 2332f1 5211f1 6919f1 8125f1 8084f1 8212f1
Mowse score
34 77 36 37 42 85 41 34 34 36 39 36 37 37 34 47 42
peptides matched
1 1 1 1 1 2 2 2 2 1 3 3 3 2 2 1 3
BLAST E value
10-96
4.00 × 1.00 × 10-91 3.00 × 10-81 7.00 × 10-69 5.00 × 10-49 7.00 × 10-49 9.00 × 10-49 5.00 × 10-46 7.00 × 10-30 8.00 × 10-29 1.00 × 10-23 9.00 × 10-16 9.00 × 10-15 8.00 × 10-08 8.00 × 10-08 9.00 × 10-08 3.00 × 10-06
protein
NADH-dependent glutamate synthase dnaK-type molecular chaperone hsp70 cysteine synthase MADS box protein similarity to ubiquitin carboxyl-terminal hydrolase family 2 endoplasmin homologue (Grp94) putative protein; protein id: At4 g29480.1 Na+/H+ antiporter, isoform 1 neutral ceramidase PNCBP putative protein; protein id: At4 g08830.1 expressed protein; protein id: At3 g15610.1 cytochrome P450-like protein putative protein; protein id: At4 g13370.1 non-LTR retroelement reverse transcriptase-like protein cytochrome-b5 reductase - like protein hypothetical protein similar to putative retroelements
a All peptide identifications with a Mowse score above 34 are presented [provided the identified EST has homology to a protein with an Evalue < E-4 (http:// mrg.psc.riken.go.jp/strc/)].
Table 3. Promoter Elements Responsible for the Regulation of Plastid Transcriptiona
ycf1 ycf2 clpP atpA atpB rpoB
Figure 4. Localization of the BY-2 plastid proteins. (A) Proteins are designated as true plastid proteins if they (i) have a plastid transit peptide (ChloroP25), (ii) have a reported function in plastids, (iii) have an orthologue in Arabidopsis with a clear transit peptide, and (iv) are plastid encoded. Identified proteins were designated as putative contaminants if they had a reported function in other cell organelles and membrane systems. For twenty-two proteins we could not decide between these two possibilities (column marked with *). These proteins were analyzed for homologues in the published chloroplast proteome analyses (B). Abbreviations: in 1: homologue detected in one other proteome study; in 2: homologue detected in two other proteome studies; in 3: homologue detected in three other proteome studies; n.d.: not detected.
with the BY-2 plastid proteins reported here to identify homologues that were detected in previously published chloroplast proteome analyses (Figure 5 and Supplemental Table 3). In a report on the complete chloroplast proteome, we identified 116 homologues, 98 homologues in dedicated envelope membrane studies and only 11 in dedicated thylakoid lumen proteome analyses (Figure 5, Supplemental Table 3). Forty-seven BY-2 plastid proteins have no homologue in the chloroplast proteome and are thus candidates to indicate prevalent functions of BY-2 plastids (Supplemental Table 3). A comparison of the BY-2 plastid protein complement with that of heterotrophic amyloplasts7 revealed plastid specific differences between the two proteomes (Figure 6). The protein profile of BY-2 plastids suggests a predominant function of this organelle in metabolic pathways, e.g., amino acid biosynthesis,
NEP
PEP
+ + + + + +
+ + + + -
a The assignment of promoter elements was retrieved from information available in the literature [refs 29-31]. NEP: nucleus encoded plastid RNA polymerase, PEP: plastid encoded plastid RNA polymerase. Ycf1 and ycf2: hypothetical proteins 1 and 2; clpP: CLP-protease subunit P, atpA, atpB: R, β subunit of ATP synthase; rpoB: β subunit of the plastid encoded plastid RNA polymerase
Figure 5. BY-2 plastid specific proteins. Identified proteins were BLAST-searched against the proteins reported in different chloroplast proteome analyses.9,10,12-14 The numbers indicate the overlap between the designated studies as well as those proteins for which no homologue was detected in the chloroplast proteome (see also Supplemental Table 3).
carbohydrate metabolism, and other metabolic activities. The majority of all identified BY-2 plastid proteins are active in amino acid biosynthetic pathways that are of minor account in amyloplasts (Table 1 and Figure 6). Prevalent amyloplast proteins are storage proteins and enzymes involved in starch metabolism7 (Figure 6). Interestingly, many amyloplast proteins are of unknown function. The comparison of protein profiles from different plastid types is useful to assess prevalent enzymatic activities and plastid specific metabolic functions. Journal of Proteome Research • Vol. 3, No. 6, 2004 1135
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Figure 6. Functional comparison of BY-2 plastid and amyloplast proteins.7 Proteins were manually assorted to their putative function. To minimize the impact of technical differences between the two studies, membrane proteins and transporters were not considered. Only those proteins that were identified from complete amyloplast preparations were used for the classification.7
Discussion Our study provides a first insight into the proteome composition of the undifferentiated plastids from BY-2 tobacco cells. Several reports suggest that BY-2 plastids have functional and morphological properties that resemble those of undifferentiated proplastids from meristematic cells. For example, studies of the transcription system of BY-2 plastids showed, that plastid encoded genes are transcribed from NEP consensus promoter elements.18,37 The NEP RNA polymerase is the predominant transcription enzyme in proplastids and responsible for the activation of the plastid encoded transcription machinery.33,34 Our proteome analysis supports this view since all plastid encoded proteins identified in this study have a NEP promoter element34,35 (Table 3). Several peptides from the two large open reading frames ycf1 and ycf2 were identified (data not shown). These two reading frames are indispensable for plastid development.36 Although the molecular function for YCF1 and YCF2 is unknown to date, it is conceivable that these two hypothetical proteins play a key role in plastid differentiation from proplastids.36 BY-2 plastids have retained the ability to develop and differentiate in a hormone dependent manner. Auxin-depletion from the growth medium induces changes of BY-2 plastid morphology and the accumulation of starch in the plastids. From these observations Miyazawa and colleagues concluded that BY-2 plastids differentiate into amyloplast-like organelles.23,38 A comparison of proteins identified from BY-2 plastids with those reported for wheat amyloplasts revealed significant differences between these two plastid types, suggesting functional adaptations of the plastid proteome during differentiation7 (Figure 6). The comparison of different plastid types on the basis of their protein profiles as presented here for the BY-2 plastid and the amyloplast (Figure 6) allows distinguishing them and provides insights into plastid metabolic functions. Provided the significant differences between the functional profiles of the amyloplast and the BY-2 plastid protein complement (Figure 6), the relatively large number of homologues in the chloroplast proteome was unexpected (Figure 5). Chloroplasts, unlike amyloplasts and BY2 plastids are autotrophic organelles. Although we did not specifically enrich membrane proteins, 98 of the identified BY-2 plastid proteins have a 1136
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homologue in one of the dedicated envelope proteome studies. Furthermore, 116 BY-2 plastid proteins have a homologue in the proteome of complete chloroplasts (Figure 5). These proteins perform housekeeping functions, e.g., metabolite exchange between the plastid and the cytosol, carbohydrate and amino acid metabolism (Supplemental Table 3) and are thus essential for the viability of the plant cell, independent from the plastid-type. At present, the limited number of proteome analyses with different plastid types from the same source makes a cross species comparison of proteomes from different plastid types necessary. Thus, conclusions about plastid-specific functions [potentially residing in the 47 BY-2 plastid proteins that have no homologue in the chloroplast proteome (Figure 5 and Supplemental Table 3)] must be drawn with great caution. Nevertheless, as more proteome studies become available, comparative protein profiling will become a valuable strategy to obtain deeper insights into plastid development and function. The rationale of a shotgun proteome study as reported here is the identification of all metabolic and regulatory pathways that are active in an organelle. Since shotgun proteomics is biased toward abundant proteins and thus provides some limited quantitative information, we assume that the identified proteins belong to the most abundant proteins of the organelle.11 By inference, the detection of many proteins of a metabolic pathway is indicative of prevalent pathway activity. Expanding this conclusion to pathway cross talk, the identification of the prevalent enzymes present in BY-2 plastids by our shotgun approach reflects the heterotrophic growth of the cell culture and its elevated amino acid biosynthetic activity. The identification of many peptides from the oxoglutarate/malate transporter suggests that BY-2 plastids import oxoglutarate, the precursor of primary nitrogen fixation at a high rate. It is conceivable that a high plastidic concentration of oxoglutarate is required when primary nitrogen fixation is highly active.2,16,31 Furthermore, the identified ATP/ADP translocator and the glucose 6-phosphate/phosphate translocator are responsible for the import of metabolites that are generally required for the energy metabolism of heterotrophic plastids. This energy is necessary for the biosynthesis of several compounds, among them amino acids and starch.2,16
Concluding Remarks The data presented here provide a first overview over the proteome of undifferentiated plastids from a tobacco cell culture. Although the tobacco genome is not completely sequenced, our study shows that a proteome analysis based on tandem mass spectrometry is feasible. Database independent analysis tools, e.g., de novo sequencing software are required to improve protein detection from tobacco BY-2 plastids. Abbreviations: NEP: nucleus encoded plastid RNA polymerase; PEP: plastid encoded plastid RNA polymerase; BY-2: bright yellow-2; BCAA: branched-chain amino acids
Acknowledgment. We would like to thank the staff of the Functional Genomics Center Zurich for their dedicated help and Ken Matsuoka for providing the BY-2 EST database. The research was supported by funds from the ETH Zurich and SEP Life Sciences to W.G. We also acknowledge the generous fellowship support of the VELUX Foundation to A.S. Supporting Information Available: Cytosolic ribosomal proteins, Localization of identified proteins, and homo-
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logues of the identified BY2 proteins in other plastid proteome studies (3 tables). This material is available free of charge via the Internet at http://pubs.acs.org.
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