Analysis of the Soluble ATP-Binding Proteome of Plant Mitochondria

Analysis of the Soluble ATP-Binding Proteome of Plant Mitochondria Identifies New Proteins and Nucleotide Triphosphate Interactions within the Matrix Jun Ito, Joshua L. Heazlewood, and A. Harvey Millar* ARC Centre of Excellence in Plant Energy Biology and School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, 35 Stirling Highway, M316, Crawley 6009, WA, Australia Received August 9, 2006

Abstract: The interactions of ATP inside plant mitochondria were investigated by identifying the soluble nucleotide binding proteome captured using immobilized ATP. Selected proteins were separated by 1D SDS-PAGE and 2D IEF-SDS-PAGE and identified by ESI-Q-TOF MS/MS. A range of highly enriched proteins were identified from the mitochondrial proteome, including 14-3-3 proteins and RNA binding proteins, as well as proteins known to contain nucleotide binding domains and/or to be inhibited or stimulated by ATP. Keywords: plant mitochondria • Arabidopsis • ATP affinity • ATP synthesis • respiration

Introduction ATP is the main product of mitochondria through the coupling of respiratory electron transport to dissipation of a membrane electrochemical gradient through the process of oxidative phosphorylation. Substantial attention has been given to the efficiency of ATP production and the mechanism of its transport to the cytosol to couple energetically unfavorable reactions throughout the cell. In plants, efficiency has been an especially important topic of research, as it is the key feature of the nonphosphorylating NADH dehydrogenases and alternative oxidase, in that they are inefficient in terms of ATP production through the respiratory chain due to the lack of coupling of electron transport to proton extrusion from the matrix.1 ATP is formed by the coupling of proton extrusion to proton return through the F1F0 ATP synthase in the inner mitochondrial membrane that is linked to the synthesis of ATP from ADP and Pi in the matrix. ATP is trafficked across the inner membrane via an anion carrier termed the adenine nucleotide translocator that allows one ADP3- molecule in for every ATP4transported out, with a net negative charge translocation out of the matrix.2 Pi is replenished in the matrix by another anion carrier termed the phosphate carrier that is an electroneutral anti-porter of OH- out and Pi-.13 Beyond this outline of the role of ATP as an export product from plant mitochondria, ATP has been noted in enzyme * Address correspondence to A. Harvey Millar, ARC Centre of Excellence in Plant Energy Biology and School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, 35 Stirling Highway, M316, Crawley 6009, WA, Australia, Phone, +61 8 6488 7245; fax, +61 8 6488 4401; e-mail, [email protected]. 10.1021/pr060403j CCC: $33.50

 2006 American Chemical Society

kinetic and other functional assays to influence a range of plant mitochondrial functions as an important coenzyme and enzyme regulator. Succinate dehydrogenase activity is stimulated by ATP, and it is normal to include a ‘sparker’ amount of ATP in respiratory assays using succinate as a respiratory chain substrate to ensure maximal activity.4 The succinyl CoA ligase of plant mitochondria is different from its mammalian and yeast counterparts by being ADP-dependent rather than GDPdependent. This is the only substrate level phosphorylation known in the matrix reactions of the respiratory pathway, making ATP independent of the ATP synthase.5 Glycinedependent respiration always required the inclusion of glycine in buffers throughout the isolation procedure in order to maintain respiratory rates in the finally purified mitochondria, but it has been found that incubation of mitochondria with ATP restores their glycine-dependent respiratory rates.6 ATP markedly inhibits pyruvate dehydrogenase activity due to phosphorylation of the E1 R subunit.7 The 2-oxoglutarate dehydrogenase is activated by AMP and inhibited by ATP by mechanisms not involving phosphorylation.8 Mitochondrial malate dehydrogenase, citrate synthase and isocitrate dehydrogenase have all been noted to be inhibited by ATP in plant mitochondria extracts.9,10 In mitochondria from other organisms, ATP is also known to regulate cytochrome c oxidase activity11 and inhibit malic enzyme.12 Protein import and assembly of nuclear encoded proteins uses ATP for import across membranes and for chaperone functions performed by HSP90, -70, and -60.13 Mitochondrial genome expression relies on transcriptional and translational activities within the matrix that also use ATP and require the use and interconversion of a range of nucleotide triphosphates.14 Many of these reported roles for ATP in mitochondria represent early kinetics studies or the measured ATP-dependence of enzymatic reactions and have been made in a variety of plant and nonplant organisms. To understand at a more mechanistic level the role and interaction of enzymes and operations with ATP inside mitochondria, it is vital to have information on ATP interactions with particular gene products of defined sequence. This is best achieved by analysis of ATPbinding in a well-characterized system. In the wider aim to define protein-protein and protein-ligand interactions in proteomes,15 ATP binding is likely to be the central hub of a complex network of interactions of great significance to the operation of a proteome as a whole; this will be especially true Journal of Proteome Research 2006, 5, 3459-3469

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ATP Affinity Proteome of Plant Mitochondria

in mitochondria where ATP is both the major product and a known effector of enzyme functions. Arabidopsis is the primary model plant and has a fully sequenced nuclear and mitochondrial genome. Proteomics has been extensively used to analyze its mitochondrial protein complement in a gene-specific fashion revealing over 500 distinct gene products.16-18 The use of affinity chromatography is increasingly seen as a mechanism for subfractionating complex protein sets to purify functional groups that often contain low-abundance proteins that would be hard to identify without an affinity tagging approach.15,19 This approach has been used to select, for example, copper-binding proteins in mammals and plants20,21 and calcium-binding proteins in mammalian mitochondria.22 The use of ATP affinity chromatography has been largely restricted to purification of protein kinases.23 The broad structural similarity of ATP binding domains in many protein superfamilies suggests the adenine ring is buried deeply in a hydrophobic cleft, while the R, β, and γ phosphates interact with residues near the mouth of the cleft. The interaction of the adenine ring and R and β phosphates with conserved amino acid residues is important for ATP binding, hence γ phosphateimmobilized ATP is considered to best mimic presentation of the nucleotide triphosphate to interaction partners.24 The use of ATP affinity to more generally define nucleotide-affinity proteomes has been seldom reported in the literature to date. The two examples known to us are a relatively extensive investigation of the ATP binding proteins from mouse and human whole red blood cells that identified 72 mouse and 15 human ATP binding proteins from ATP-sepharose25 and a much more preliminary investigation of spinach chloroplasts that identified only 4 of a series of over 10 major protein bands that eluted from ATP-agarose.26 We have used affinity chromatography with several γ phosphate-linked ATP media to select nucleotide-binding proteins from the soluble fraction of Arabidopsis mitochondria and have detailed the benefits of this analysis in terms of depth of identification and new insights into plant mitochondrial composition and function.

Experimental Procedures Arabidopsis thaliana Suspension Cell Cultures. A heterotrophic Arabidopsis cell culture, established from callus of cv Erecta stem explants, has been maintained for over 14 years by weekly subculture into Murashige and Skoog basal medium supplemented with 3% (w/v) sucrose, 0.5 mg/L naphthaleneacetic acid, and 0.05 mg/L kinetin, pH 5.8.27 The cell cultures were maintained in Arabidopsis suspension cell culture medium in the dark at 22 °C in an orbital shaker (150 rpm). At 6 to 7 d, each flask (120 mL) contained 18-20 g of fresh weight of cells, and growth was approximately in the middle of the log phase. Subculture of 20 mL of culture to 100 mL of fresh medium began the cycle again.16 Mitochondrial Isolation. A total of 1.0-1.2 L of 7-d cell culture was filtered through Miracloth to remove media, and cells were then ruptured in a Waring blender. Blending of each 90-100 g aliquot was performed in 300 mL of grinding medium (0.3 M mannitol, 50 mM sodium pyrophosphate, 0.5% (w/v) bovine serum albumin (BSA), 0.5% (w/v) polyvinylpyrrolidone40, 2 mM EGTA, and 20 mM cysteine, pH 8.0). Filtered cell extract was separated by centrifugation at 1500g for 5 min at 4 °C, and the supernatant was centrifuged again at 24 000g for 15 min. The resultant organelle pellet was washed by repeating 3460

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technical notes the 1500 and 24 000g centrifugation steps at 4 °C in mannitol wash medium (0.3 M mannitol, 0.1% (w/v) BSA, and 10 mM TES (N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid)NaOH, pH 7.5). The final organelle pellet was resuspended in wash medium and loaded onto a Percoll step gradient consisting of 1:5:1 ratio, bottom to top, of 40% Percoll:25% Percoll: 18% Percoll in mannitol wash medium. The gradients were centrifuged for 45 min at 40 000g and 4 °C with centrifuge brake off, and mitochondria were present as an opaque band at the 25%:40% interface. This band was aspirated, concentrated, and washed by centrifugation at 24 000g and 4 °C for 15 min and was then loaded onto a self-forming Percoll gradient containing 35% Percoll in sucrose wash medium (0.3 M sucrose, 0.1% (w/v) BSA, and 10 mM TES-NaOH, pH 7.5). After centrifugation at 40 000g and 4 °C for 45 min with centrifuge brake off, mitochondria formed a band near the top of the gradient, and peroxisomal material banded near the bottom of the gradient. The mitochondrial band was aspirated and again washed and concentrated by two centrifugation steps at 24 000g and 4 °C for 15 min in sucrose wash medium (containing no BSA). This protocol is a slight variation on the one used by Millar et al.16 Purified mitochondrial proteins were quantified using Coomassie Plus protein assay reagent (Pierce) according to the manufacturer’s instructions and using bovine serum albumin (BSA) as the protein standard. Mitochondrial proteins were immediately transferred as 500 µg aliquots into sterile Eppendorf tubes and stored at -80 °C. Preparation of ATP-Acylamide Resin and Affinity Chromatography. Fifteen milligrams of γ-aminotridecyl-ATPacylamide resin (ATP-Binders Resin, Novagen) was weighed out and placed in a microcentrifuge tube. A volume of 1.2 mL of conditioning buffer (1.7% Tween-20 and 4 mM HEPES, pH 7.2) was added to the resin and mixed by vortexing for 30 s. The resin was incubated at 4 °C overnight, and the buffer was decanted using a pipet after pelleting the resin at 5000g for 2 min. Following this, the resin was washed twice by brief vortexing in 1.5 mL of deionized water and pelleted at 5000g for 2 min, and the water was decanted from the resin. The resin was then left to equilibrate in 1.5 mL of wash buffer (150 mM NaCl, 60 mM MgCl2, 2.5 mM MnCl2, 1 mM CaCl2, 0.05% Nonidet P-40, 1 mM dithiothreitol, 1 mM AMP, 1 mM ADP, 1 mM NADH, 0.2 mM activated sodium vanadate, 15 µL of Protease Inhibitor Cocktail (Novagen), and 25 mM HEPES, pH 7.2) for several minutes. After pelleting the resin at 5000g for 2 min and decanting the wash buffer from the resin, the lid of the Eppendorf tube was kept closed to prevent the resin from drying out. Three to four milligrams of mitochondria were freeze-thawed three times in liquid nitrogen to break apart mitochondrial membranes and release soluble proteins. Pelleting membranous material at 20 000g for 20 min at 4 °C retained approximately 1-2 mg of mitochondrial soluble proteins in the supernatant, which was then transferred to a separate Eppendorf tube. One volume of binding buffer (150 mM NaCl, 300 mM MgCl2, 12.5 mM MnCl2, 5 mM CaCl2, 0.25% Nonidet P-40, and 125 mM HEPES, pH 7.2) was added to 4 vol of protein sample; to this was added a nucleotide mixture to final concentrations of 1 mM AMP, 1 mM ADP, 1 mM NADH, pH 7.0, dithiothreitol to 1 mM, and 1/100 dilution of Protease Inhibitor Cocktail (Novagen). The sample mixture was then transferred to the equilibrated resin and incubated for 3 h at 4 °C with gentle orbital rotation. The sample/resin mixture was transferred to the sample cup of a spin filter and spun at 5000g for 2 min, and the flow-through proteins were transferred to a

technical notes fresh tube. Three successive washes of the resin with 1 mL of wash buffer (150 mM NaCl, 60 mM MgCl2, 2.5 mM MnCl2, 1 mM CaCl2, 0.05% Nonidet P-40, 1 mM dithiothreitol, 1 mM AMP, 1 mM ADP, 1 mM NADH, 0.2 mM activated sodium vanadate, 10 µL of Protease Inhibitor Cocktail, and 25 mM HEPES, pH 7.2) removed non-ATP-binding proteins. After washing the resin, 150 µL of ice-cold elution buffer (20 mM ATP, 1 mM dithiothreitol, 1 mM AMP, 1 mM ADP, 1 mM NADH, 0.2 mM activated sodium vanadate, 0.05% Nonidet P-40, 1.5 µL of Protease Inhibitor Cocktail, and 25 mM HEPES, pH 7.2) was added to the resin and incubated for 20 min at 4 °C with gentle orbital rotation. The eluted protein fractions were collected by spin filtration at 5000g at 4 °C for 2 min, and the elution steps repeated 2 more times. Soluble mitochondrial protein samples eluted with NADH-, AMP-, ADP-, or ATP-only buffers were subjected to binding and washing steps as described above, except no AMP, ADP, or NADH were added to the binding and wash buffers, nor to the resin during the equilibration step. Elution steps for NADH-, AMP-, ADP-, and ATP-only samples were the same as described above, but the elution buffers for NADH-, AMP-, ADP-, and ATP-only samples consisted of 20 mM NADH, AMP, ADP, or ATP, respectively, and 1 mM dithiothreitol, 0.2 mM activated sodium vanadate, 0.05% Nonidet P-40, 1/100 dilution of Protease Inhibitor Cocktail (Novagen), and 25 mM HEPES, pH 7.2 per sample. Eluted proteins were desalted and concentrated by using Ultrafree centrifugal filter devices (Millipore) and quantified using Coomassie Plus protein assay reagent (Pierce). Preparation of γ-Aminooctyl-ATP-Sepharose Resin and Affinity Chromatography. Pretreatment of γ-aminooctylATP-sepharose resin (Jena Bioscience) prior to adding of protein sample was carried out by pipetting 500 µL of resin into a microcentrifuge tube, spinning it at 5000g for 2 min and decanting the 20% ethanol storage buffer from the resin using a pipet. One milliliter of 10 mM HEPES, pH 7.2, was added to the resin and mixed by vortexing for 30 s. Binding, washing, and eluting of ATP binding proteins was performed in the same manner and using the same solutions noted above in the ATPonly experiments without NADH, AMP, and ADP in buffers. SDS-PAGE and 2D Isoelectric Focusing (IEF)/SDS-PAGE Gels. SDS-PAGE gels used where 4% acrylamide stacking gels above 12% (w/v) acrylamide, 0.1% (w/v) SDS, or 5-16%(w/v) acrylamide, 0.1% (w/v) SDS in a large gel format (0.1 cm × 16 cm × 16 cm) and were run with Tris-glycine buffering systems. Gel electrophoresis was performed at 25 mA per gel and completed in 3 h. Isoelectric focusing (IEF) sample buffer consisted of 6 M urea, 2 M thiourea, 2% (w/v) CHAPS, 2% (v/v) ampholytes (pH 3-10), 2 mM tributylphosphine, and 0.001% (w/v) bromophenol blue. Aliquots of 330 µL were used to re-swell dried 180 mm, pH 3-10 nonlinear IPG strips (Immobiline DryStrips, APBiotech, Sydney) overnight, and then IEF was performed for 19.5 h reaching a total of 49 kVh at 20 °C on a flat-bed electrophoresis unit (Multiphor II, APBiotech, Sydney). IPG strips were then transferred to an equilibration buffer consisting of 50 mM Tris-HCl (pH 6.8), 4 M urea, 2% (w/v) SDS, 0.001% (w/v) bromophenol blue, and 100 mM β-mecaptoethanol and incubated for 20 min at room temperature with rocking. The equilibrated strips were then slotted into central single wells of 4% acrylamide stacking gels above 0.1 cm × 18.5 cm × 20 cm, 12% (w/v) acrylamide, 0.1% (w/v) SDS-polyacrylamide gels. Gel electrophoresis was performed at 100 V with circulating cooling (10 °C) and completed in 5 h.

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Five independent 2D gels were run, one each for 5 different ATP affinity traps each from a separate mitochondrial isolation. Quadrupole Time-of-Flight Mass Spectrometry (Q-TOF MS). Selected proteins were selected from the gel, washed, and in-gel-digested overnight at 37 °C according to Sweetlove et al.28 Peptides were extracted from the overnight digests by adding an equal volume of acetonitrile and shaking for 15 min at 8000 rpm on an orbital shaker. The solution was removed from the gel pieces, 10-20 µL of a 50% acetonitrile and 5% formic acid solution was added, and the shaking procedure was repeated. The solution was again removed and pooled, and the last step was repeated. Solvent was evaporated from each sample using a speedvac (Thermo Savant) for 20-30 min until nearly dried. Samples were hydrated in 16 µL of 5% acetonitrile and 0.1% formic acid prior to mass spectrometric analysis. Samples were analyzed using an Agilent 1100 series capillary LC system and an Applied Biosystems QSTAR Pulsar i LC/MS/MS system equipped with the IonSpray source running Analyst QS software (v1.0 SP8) with the instrument in positive ion mode. Each extracted peptide sample was loaded in turn with the Agilent 1100 series capillary LC system onto a 0.5 × 50 mm clipeus 5 µm C18 reverse-phase column (Higgins Analytical) with a C18 OPTI-GUARD guard column (Optimize Technologies) at 16 µL/min equilibrated with 5% acetonitrile and 0.1% formic acid. Peptides were eluted from the C18 reverse-phase column into the QSTAR Pulsar i by a 7 min acetonitrile gradient (5%-80%) at 16 µL/min under constant formic acid concentrations of 0.1%. During the period of ion detection, eluted peptides were analyzed by the mass spectrometer at 8 µL/min. The total analysis time for each sample was 23 min. The method used to analyze eluted ions employs the Information Dependent Acquisition (IDA) capabilities of Analyst QS and the rolling collision energy feature for automated collision energy determination based on the ions m/z (Sciex/AB). The method employed a 1 s TOF MS scan which automatically switched (using IDA) to a 2 s Product Ion scan (MS/MS) when a target ion reached an intensity of greater that 30 counts and its charge state was identified as 2+, 3+, or 4+. TOF MS scanning was undertaken on an m/z range of 4001600 m/z using a Q2 transmission window of 380 amu (100%). Product Ion scans were undertaken at m/z ranges of 70-2000 m/z at low resolution utilizing Q2 transmission windows of 50 (33%), 190 (33%), and 650 amu (34%). Data produced by this method were used for searching the Mascot search engine (Matrix Sciences) for protein identifications. The collisioninduced disassociation data (CID) from each sample were exported from Analyst QS using a purpose built script obtained from Matrix Sciences. The script was set up to centroid the survey scan ions (TOF MS) at a height percentage of 50% and a merge distance of 0.1 amu (for charge state determination), centroid MS/MS data at a height percentage of 50% and a merge distance of 2 amu, reject a CID if less than 10 peaks, and discard ions with charge equal and greater than 5+. Search parameters at Mascot employed a peptide tolerance of (1.2 Da and MS/MS tolerance of (0.6 Da, no variable modifications, and allow up to 1 miscleavage for trypsin digest, and the instrument type was set to ESI-QUAD-TOF. Searches were performed against a custom database of all Arabidopsis proteins, TAIR 6 release (30 700 sequences; 12 656 682 residues).

Results Total soluble protein samples from purified mitochondrial samples (1 mg protein) were incubated with 15 mg of ATPJournal of Proteome Research • Vol. 5, No. 12, 2006 3461


Figure 1. ATP affinity enrichment of soluble Arabidopsis mitochondrial proteins on ATP-acrylamide resin. SDS-PAGE separated protein samples of total protein, unbound protein, buffer supernatant from three successive wash steps (wash 1-3), and the supernatant after addition of 20 mM ATP to the ATPacrylamide resin (ATP eluate). Proteins are visualized by colloidal Coomassie staining.

acrylamide gel (ProteoEnrich ATP Binders, Novagen) in binding buffer, and following this, a series of wash steps under highsalt concentration (150 mM NaCl) removed the unbound protein samples by successive centrifugation of the resin, removal of the supernatant, and refreshing of the wash buffer. Supernatants of samples treated in this fashion and separated by SDS-PAGE are shown in Figure 1, with successive dilution of the protein content through each wash step (1-3). The binding and wash buffers contained 1 mM AMP, 1 mM ADP, and 1 mM NADH to reduce binding of non-ATP-binding proteins to the resin during the binding and washing steps. Finally, the resin was resuspended in an elution buffer containing 20 mM ATP. Inclusion of ATP lead to displacement of substantial amounts of proteins from the resin that were visualized as a complex set of proteins by SDS-PAGE. These proteins had a distinct banding pattern compared to the total soluble protein and the unbound/flow-through samples. Approximately 2.5% of the protein initially incubated with the resin was collected in the ATP-eluted sample (∼25 µg from 1 mg total protein). To determine if ADP, AMP, and NADH were displacing specific proteins during the binding and elution phase, we conducted a series of experiments without these reagents in the wash buffer (Figure 2). Samples A-D were bound to ATP acrylamide and washed three times in the absence of any nucleotides. After this third wash, the supernatants were concentrated and separated by SDS-PAGE to reveal low levels of proteins in the wash solutions. Addition of 20 mM NADH, AMP, or ADP to samples A-C, respectively, yielded only a low amount and a small number of protein bands in the superna3462


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Figure 2. Specificity of ATP affinity enrichment of soluble Arabidopsis mitochondrial proteins on ATP-acrylamide resin. SDS-PAGE separated protein samples of the third wash (wash 3) of five independent incubations of soluble Arabidopsis mitochondrial proteins with ATP-acrylamide resin (A-E), and the protein banding pattern from elution with 20 mM NADH (A), 20 mM AMP (B), and 20 mM ADP (C), compared to 20 mM ATP (D). This is also compared to lanes E, where 20 mM ATP was used to elute proteins after the three other nucleotides (1 mM NADH, AMP, and ADP) were present during the three successive wash steps (wash 1-3). Proteins are visualized by colloidal Coomassie staining.

tants after incubation and centrifugation. The sample with ADP provides the only pattern distinct from the wash steps, with two bands above the 66 kDa marker and a prominent band at ∼50 kDa, and weaker bands at 30 and 22 kDa (the higher bands were successfully analyzed by LC-MS/MS and identified as the heat shock protein 70 proteins At4g24280 and At4g37910 and the alanine aminotransferase At1g17290, see supplementary data 1 in Supporting Information). In contrast, 20 mM ATP elution from sample D yielded an abundant complex sample. Sample E was treated as Figure 1 with 1 mM NADH, AMP, and ADP present in all the binding and wash steps and final elution with ATP. No significant difference was seen in the banding patterns between the final samples in D and E on SDS-PAGE gels. With samples prepared in the manner shown in Figure 2, lane E, a series of 24 regions from a Coomassie-stained gel were cut from 12% polyacrylamide SDS-PAGE gel separations (Figure 3A). These were in-gel-digested with trypsin and prepared for LC-MS/MS analysis. In addition, a different γ phosphatetethered media was tested based on a 10-mer flexible linker to sepharose (Jena Bioscience). ATP displacement of proteins from the 13-mer linker acrylamide and 10-mer linker sepharose media were compared on 4-15% (w/v) polyacrylamide gradient gels (Figure 3B) to maximize the separation of proteins in the complex region noted between 50 and 100 kDa seen in Figure 3A. A broad degree of similarity in the protein banding pattern was observed when ATP eluates from the two media were compared (Figure 3B). But there were several differences

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higher in the ATP eluate compared to total soluble protein (Table 1). This was defined as greater than near equal abundance when 250 µg of total protein was compared to 75 µg of ATP-purified protein. Some of these proteins were greater than 20-30-fold higher in abundance in the ATP eluate gels. The remainder were abundant in the ATP eluate but also present in the total soluble protein set at equal or greater amounts (Table 2). More minor spots on the ATP-eluted gels that overlapped with major regions of abundant protein in the soluble protein gels were not analyzed.

Figure 3. ATP affinity-enriched soluble Arabidopsis mitochondrial proteins on ATP-acrylamide and ATP-sepharose selected for analysis by mass spectrometry. (A) ATP-acrylamide protein set on 12% polyacrylamide gel; (B) ATP-acrylamide and ATPsepharose sets on 5-16% polyacrylamide gradient gels. Numbers on left of lanes are molecular mass. Numbers on right of lanes are band numbers for mass spectrometry analysis and appear in Tables 1 and 2.

around the 43 and 30 kDa marker points, as well as differences in the abundance of specific protein bands (Figure 3B). A selection of 24 protein regions from the gel of the ATPacrylamide samples and 21 protein regions from the gel of the ATP-sepharose samples were excised and digested in-gel with trypsin and prepared for LC-MS/MS analysis. To further explore the complex nature of the ATP eluate fractions, IEF/SDS-PAGE gels were performed to compare 250 µg of total soluble protein sample before affinity chromatography with 75 µg of ATP-acrylamide-bound fraction protein (Figure 4). Spot abundance was compared between gels to locate proteins that were differentially abundant in the ATPeluted sample, and proteins that were near equally abundant in both samples. This process was repeated five times on separate gels from independent ATP elutions from different mitochondrial preparations (supplementary data 3 in Supporting Information). In total, 53 spots were selected from single spot excision and in-gel digestion followed by LC-MS/MS from selected gels; in 53 spots, 54 proteins were identified with confidence. Of these, 34 proteins were highly enriched in the ATP-eluted sample (Table 1) and 20 were abundant proteins in the ATP-eluted samples but were in overlapping positions with major proteins in the total soluble proteome (Table 2). The functional significance of these two groups is explored in more detail below. Because of the very different protein patterns on the soluble and ATP eluate gels, it was not possible to use standard 2D gel comparison tools that rely on largely similar protein patterns with a small number of changes. Instead, manual matching was performed, and spots were assigned to the two groups based on abundance being >3-fold

Table 1 contains the LC-MS/MS identifications of proteins enriched on 2D gels (>3-fold) and shows the presence or absence of these same proteins in the 1D ATP-polyacryamide and ATP-sepharose gel analyses. Of the 34 proteins in this group, 18 were identified using both media on 1D gels and were enriched on the 2D separations, while the remainder were found in at least one of the 1D gel separations and the 2D gel separations. These identified proteins represent a distinct set of functional categories including a high proportion of heat shock proteins. Only 12 of the 34 had been previously found on gel-based separations and analysis of mitochondrial proteins from Arabidopsis, while a further 9 had been found in the proteome but only in nongel complex mixture LC-MS/MS analysis (Table 1). A final 12 were new identifications not previously made by proteomic analysis of mitochondria. The 9 previously found only by LC-MS/MS include RNA binding proteins, the LON protease, and several Fe-S biogenesis proteins, all low-abundance proteins that have been selectively enriched by the affinity chromatography. Remarkably, in some cases, these were abundant Coomassie-stained protein spots in 25-75 µg of protein of the ATP eluate samples (Figure 4 and supplementary data 3 in Supporting Information). Of the 12 new identifications, two notable ones have previously been claimed to be Arabidopsis mitochondrial proteins on the basis of GFP-fusion expression and co-localization with mitochondria in vivo. These are an RRM containing RNA binding protein (At1g74230)29 and a glycolate dehydrogenase (At5g06580).30 The identification of an unknown protein (At3g52570.1) marks the first time this has been found in plant mitochondria. It has not been reported to have been identified in any other mass spectrometry analysis in Arabidopsis (www.suba.bcs.uwa.edu.au). Bioinformatic analysis shows it is predicted to localize to the mitochondria by several subcellular prediction programs including Predotar, TargetP, Mitoprot2, and iPSORT. It contains esterase and hydrolyase interpro domains (IPR000379, IPR000073), but it is not closely related to a known function protein. It is only known from three ESTs, making it the product of a rare transcript in Arabidopsis. Table 2 contains the LC-MS identifications of major proteins found on 2D gels that eluted off ATP affinity media by ATP (Figures 1, 2, and 4), but also appear to be significantly abundant proteins in the soluble proteome (Figure 4). Placement of proteins in Table 2 versus Table 1 was determined by both their comigration with major proteins in Figure 4 and their known abundance from previous proteome studies.16-18 The presence or absence of these same proteins in the 1D ATPpolyacryamide and ATP-sepharose gel analyses are also shown. Of the 20 proteins in this group, all are previously known from MS studies of mitochondria. Further HSPs are found that complete the set of mitochondrial HSPs found in Table 1. Spot 33 from Figure 4 could not be identified with significant matches and is not included in Table 2. Journal of Proteome Research • Vol. 5, No. 12, 2006 3463


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Figure 4. Comparsion of ATP affinity-enriched soluble Arabidopsis mitochondrial proteins on ATP-acrylamide with total soluble mitochondrial proteins on IEF-SDS/PAGE. Total soluble mitochondrial protein (250 µg) (A) was compared to 75 µg of ATP affinitypurified protein (B). Numbers on left are molecular mass. Numbers on figures are spot numbers for mass spectrometry analysis and appear in Tables 1 and 2. Equal abundance indicates a 3-fold increase in abundance during affinity purification.

Discussion Heat shock proteins are the classical ATP binding proteins expected to be observed in this kind of study. Here, we have identified HSPs from the 60, 70, and 90 classes in Tables 1 and 2. Their activity involves cycles of binding and stepwise release of substrate proteins catalyzed by ATP hydrolysis, and they each contain ATP binding sites and ATPase motifs.13 The LON protease is also ATP-dependent and contains a classical LON active site responsible for ATP hydrolysis. The elongation factor Tu promotes binding of aminoacyl-tRNAs to ribosomes. This is typically a GTP-dependent event, and Tu factors are primarily responsible for the ATPase and GTPase activity of ribosomes.31 The mitochondrial Tu proteins found here contain ATP/GTP binding sites conserved with other Tu proteins in plants and animals.32 Plant annexins are also classical GTPase/ ATPases with strong affinity for ATP,33 these proteins are cytosolic, but coat membranes, and this annexin (At1g35720) has been found as a low-abundance protein in the proteome of many Arabidopsis organelles.34-37 This selection of ATPases is highly represented in the ATP affinity-purified sets of Tables 1 and 2. Most notable among the new identifications in Table 1 are three 14-3-3 proteins. These proteins belong to a family of 15 in Arabidopsis with diverse roles in protein regulation, complex scaffolding, and forming a guidance complex for protein targeting to organelles.38 The 14-3-3 proteins are known to bind to short amino acid motifs containing phosphoserine or phosphothreonine residues on target proteins, but their interaction with ATP is unclear. Previous use of ATP-acylamide has purified 14-3-3 from rat spleen samples,39 but it is not clear if 14-3-3 proteins coeluted with ATP binding proteins through protein-protein interactions with other ATP-binding proteins, or bind to ATP themselves. None of the plant 14-3-3 proteins contain targeting information in their primary sequences, and most are considered to be located in multiple locations in 3464


Arabidopsis cells based on GFP-targeting studies.40,41 Proteomic studies have previously reported that most of the 14-3-3 GRFs are present in whole plants,42 and further that 14-3-3 GRF1, -6, -7, and -8 are in plastids36 and 14-3-3 GRF1 and -8 are in the nucleus.43 The data for 14-3-3 GRF3, -5, and -10 in or on mitochondria presented here are not in conflict with these previous claims based on direct MS analysis of 14-3-3 localization. Further strong support of a role of two of these, GRF3 and GRF10, in mitochondria was gained by our analysis of the coexpression of all 15 of the 14-3-3 proteins within the Arabidopsis nuclear gene set using public microarray data with the Botany Array Resource Expression Angler tool.44 This showed that both GRF3 and GRF10 coexpressed with a set of genes that were enriched for genes encoding proteins within the known mitochondrial proteome (Figure 5). By random we would expect ∼2-3% of a set of Arabidopsis genes to be in the known mitochondrial proteome. Most of the 14-3-3 proteins coexpress with gene sets containing 0-12% genes whose products are destined for mitochondria. However, GRF3 coexpressed with a set of genes of which 68% (19 of 28 with coexpression coefficient >0.6) and GRF10 with a set of genes of which 38% (18 of 48 with coexpression coefficient >0.6) were genes whose products are known to be located in mitochondria by MS studies (Figure 5). This makes GRF3 and GRF10 stand out as mitochondrial-related in their expression profiles and is significant further support for the enrichment and mass spectra data placing them in mitochondria presented in Table 1. Work in barley using antibodies has shown that 14-3-3 proteins are present in mitochondria and that they function in regulation of the F1F0 ATP synthase through their phosphorylation-dependent interaction with the F1 β-subunit.45 Our proteomic data pinpoints two 14-3-3 proteins in Arabidopsis that may be involved in analogous processes, but these 14-3-3 proteins might be present in mitochondria due to chaperone roles or involvement in other regulatory processes.

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Table 1. Proteins Identified as Highly ATP Affinity-Enriched from Arabidopsis Mitochondrial Soluble Protein Samples by LC-MS/MSa spot/band nos. (no. 2D exp)

1D 8 12 16 11

ATP-A 1D grad 5 10

10

12 12

10 10 13

4 21

22

2

13

2D

(exp. ATP-S 2D) 1D 6 11 15

peptide Ion matches gene locus

6 24 30 23

(4) (4) (5) (5)

At3g52200.1 At3g17240.1 At5g03290.1 At4g00570.1

10

(5)

13, 14 13, 14 50

(5) (5) (4)

11 11

At1g17290.1 At1g72330.1 At3g45300.1

21

(5)

2

At2g26080.1

52

(5)

1

(4)

2

At1g74260.1

39

(3)

17

At3g55010.1

18

(4)

At2g13560.1

At2g35120.1

At5g06580.1

11 9 8

9 7 5

8 9 19

(5) (5) 3

10 7 6

At2g2800.1 At5g09590.1 At4g24280.1

6 7 7 20

3 4

(5) (5) (5) (3)

4 5

16

3 4 5 38

18

At3g07770.1 At2g04030.1 At5g56030.1 At5g55200.1

4

2

2

(3)

3

At5g26860.1

3

42

(3)

15

13

51

(5)

14

At4g02930.1

20

16

44

(5)

18

At1g74230.1

24

24

47

(5)

At4g13850.1

18

14

10

At1g62750.1

37

(5)

At5g61030.1

9

(5)

At3g49240.1

20

17

45,53

(4)

At5g38480.1

19

16

46

(4)

At5g16050.1

20

17

45,53

(4)

At1g22300.1

17 21 14 19

14 20 12 16

43 49 27 40

(5) (5) (5) (5)

21 13

At1g35720.1 At1g51390.1 At5g65720.1 At3g52570.1

functional class/ protein name

1D

ATP-A 1D grad

2D

TCA and Associated Enzymes PDC E2 subunit 23 15 15 Lipoamide dehydrogenase 15 11 25 NAD isocitrate subunit 3/4 9 6 Malate oxidoreductase 6 26 (Malic enzyme) MOX-4 Malate oxidoreductase 4 21 (Malic enzyme) MOX-2 Enzymes Involved in Metabolism Alanine aminotransferase 18 16 23, 18 Alanine aminotransferase 12 9 7, 7 Isovaleryl-CoA 2 7 dehydrogenase Glycine decarboxylase 18 13 P protein Glycine decarboxylase 10 3 5 H protein Glycinamide ribonucleotide 13 3 transformylase Phosphoribosylformylglycin4 amidine cyclo-ligase Glycolate dehydrogenase 9 2 Protein Fate/Heat Shock Proteins Chaperonin 60 19 16 22 Heat shock protein 70-5 37 14 36 Heat shock protein 9 3 6 70-like protein Heat shock protein 89-1 26 17 29 Heat shock protein 88-1 24 17 12 Heat shock protein 81-2 16 17 Cochaperone GRPE 11 4 6 family protein LON protease 8 27 10 Translational Apparatus Mitochondrial elongation 11 2 factor Tu Mitochondrial elongation 15 16 14 factor Tu RNA Binding/Processing Proteins Glycine-rich RNA binding 16 12 13 protein Glycine-rich RNA binding 4 3 2 protein Glycine-rich RNA binding 3 4 1 protein Pentatricopeptide repeat18 11 containing protein 14-3-3 Proteins GRF 3 14-3-3 protein GF 12 5 3,8 14 psi GRF 5 14-3-3 protein GF 13 12 6 14 upsilon GRF 10 14-3-3 protein GF 8 7 2,3 14 epsilon Other Proteins 24 7 10 Ca2+-dependent annexin NFU5 Fe-S centre protein 9 5 4 Cysteine desulfurase 4 4 17 Expressed protein 2 5 11

Mascot ATP-S

MW (Da)

pI

492 1082 261 1157

68862 54000 40624 66000

5.2 7 6.8 7.1

18 38 5

953

926, 671 573, 462 283 544

14 7 4

145

nucleotide binding site Binds NAD Binds NAD Binds NAD

m m m (LC only) m (LC only)

69000 5.1 Binds NAD

m (LC only)

59000 6.4 59000 6.4 45000 7.6 FAD

m new m

113000 6.6 P-phosphate 18000 4.1

100

3

154

7

84

MP

151111 4.9

m (LC only) m new

41000 5.1 ATP dependent

new

62000 6.9 Binds NAD

new

1098 1712 253

4 47 18

62000 4.8 ATP binding site 73000 5.4 ATP binding site 76000 4.8 ATP binding site

m m new

982 504 586 145

17 18 19

90000 88000 80002 33000

m m new m (LC only)

401

5

57

5 ATP binding site 4.6 ATP binding site 4.7 ATP binding site 7

103866 5.3 ATP dependent

m (LC only)

86000 5.3 ATP/GTP binding m

539

7

441

11

47

49000 6.7 ATP/GTP binding m

29000 4.4 RNA binding

new


m

48


m (LC only)

387


new

266

29000 4.4 ATP binding

new

281


new

152


new

36000 29200 50000 37000

other m (LC only) m (LC only) new

375 134 665 303

6 1

5 ATP/GTP binding 4.6 7 6.5

a 1D and 1D grad numbers represent band numbers in Figure 3 for ATP-acylamide (ATP-A), 2D numbers are spot numbers from Figure 4 and supplementary Data 3, numbers in parentheses are the number of separate 2D experiments where this spot was found enriched, and 1D ATP-S are band numbers from Figure 3 from the ATP-sepharose gel. Gene locus is the physical locus number for the Arabidopsis gene match, and the annotation of this gene is given in functional class/protein name. The peptide ion matches give a numerical indication of the number of first ranked nonredundant MS/MS spectra matching to this gene product in the 1D and 2D gel separation analyses; all matches in the 1D gels had significant Mascot score matches. The Mascot score is noted in the score in the 2D gel spot identifications and relates to the information in supplementary data files. MW and pI are presented for each match. Searching of PFAM and literature indicated nucleotide binding information, and this is noted in comment form for each protein. MP represents prior evidence that this specific gene match represents an Arabidopsis Mitochondrial Protein (MP); m ) mitochondrial protein identified on gel separations; m (LC only) ) mitochondrial protein identified only from complex mixture LC coupled with MS; new ) no prior claim as a mitochondrial protein by mass spectrometry; other ) suspected contaminant. Data for the MP column was derived from the database of mitochondrial identifications in Arabidopsis at www.suba.bcs.uwa.edu.au.

PPR proteins are low-abundance proteins involved in RNA interactions in the matrix, regulating the expression of the mitochondrial genome post-transcriptionally and are involved in RNA editing and exon splicing.46 We have previously identified 10 PPRs in complex mixture LC-MS/MS analysis of the

mitochondrial proteome;17 the protein identified here in Table 1 (At3g49240) is a new protein of this class not found in our previous studies and appears to be selectively enriched by ATP binding. With only 4 ESTs reported for this gene, it is a rather low-abundance protein in plants. Journal of Proteome Research • Vol. 5, No. 12, 2006 3465

technical notes


Table 2. Proteins Identified as Moderately ATP Affinity-Enriched from Arabidopsis Mitochondrial Soluble Protein Samples by LC-MS/MSa spot/band nos. (no. 2D exp)

1D

ATP-A 1D grad

15

13

2D

(exp. 2D)

peptide ion matches ATP-S 1D grad

29 31

(5) (3)

11

gene locus

functional class/ protein name

1D

ATP-A 1D grad

TCA and Associated Enzymes At2g20420.1 Succinyl CoA ligase β-subunit 8 11 At1g48030.1 Lipoamide dehydrogenase

2D 22 4

Mascot 768 125

5 14

2 12

22 26, 27

(5) (5)

13

15

13

35

(5)

14

At2g05710.1 Aconitate hydratase 17 15 21 781 At2g44350.1 Citrate synthase 20 14 18, 15 959, 617 Enzymes Involved in Metabolism At5g07440.1 Glutamate dehydrogenase 2 12 12 18 777

13

36

(4)

14

At5g18170.1 Glutamate dehydrogenase

25 41 41 48

(4) (3) (3) (3)

16 10

At3g48000.1 At3g45770.1 At3g59760.1 At4g34200.1

32

(4)

12

24

(4)

17

(5)

11

15,16

(5)

8

28

(4)

12 17 17 11

12

10

10

8

13

9

ATP-S 1D grad

21 12 27

12

413

4 4 2 4

124 128 84 179

10 12

At1g63940.1 Monodehydroascorbate reductase

4

146

9

At3g54660.1 Glutathione reductase

7

276

Aldehyde dehydrogenase ETR1 enoyl thioester reductase Cysteine synthase Phosphoglycerate dehydrogenase

7 6 2 8

19

Membrane Respiratory Complex Proteins At5g08670.1 ATP synthase β-subunit 13 4 20

811

18

At5g66760.1 SDH R subunit

7, 12

315, 495

24

At1g51980.1 MPP R-1 subunit 6 Protein Fate/Heat Shock Proteins At3g23990.1 Heat shock protein 60-3b 16 10 chaperonin At2g33210.1 Heat shock protein 60-2 22 9 chaperonin At3g12580.1 Heat shock protein 70 4 3

9

341

24

790

16

12

500

16

10

19

11

9

11

(5)

10

11

9

12

(3)

10

9

7

34

(4)

4

107

9

7

7

(5)

7

At4g37910.1 Heat shock protein 70-4

27

26

1013

49

7

4

20

(4)

5

Translational Apparatus At1g45332.1 Mitochondrial elongation 9 3 factor

16

570

21

33

MW (Da)

pI

nucleotide binding site

45345 6.7 ATP grasp domain 54000 7.4 NAD-binding (IPR002938) 108000 6.7 52000 6.9 ATP inhibition

MP m m m m

44698 6.5 NAD binding (IPR000205) 44500 6.8 NAD binding (IPR000205) 58588 7.5 NAD-cofactor 40800 7.2 46000 8.5 63000 6.5 NAD binding (IPR000205) 52500 7.3 FAD binding domain IPR001327 60000 8 FAD binding domain IPR001327

m

59676 6.6 nucleotidebinding domain, PF00306 69600 6.2 FAD binding (IPR002938, IPR003953, IPR001327) 54000 6.3 -

m

m m m m m m m

m

m

61280 5.7 ATP binding site 61000 6.6 ATP binding site 71000 4.9 ATP binding site 73000 5.2 ATP binding site

m

83000 6.3 GTP binding (IPR006297)

m

m

m

a 1D and 1D grad numbers represent band numbers in Figure 3 for ATP-acylamide (ATP-A), 2D numbers are spot numbers from Figure 4 and supplementary Data 3, numbers in parentheses are the number of separate 2D experiments where this spot was found enriched, and 1D ATP-S are band numbers from Figure 3 from the ATP-sepharose gel. Gene locus is the physical locus number for the Arabidopsis gene match, and the annotation of this gene is given in functional class/protein name. The peptide ion matches give a numerical indication of the number of first ranked nonredundant MS/MS spectra matching to this gene product in the 1D and 2D gel separation analyses; all matches in the 1D gels had significant Mascot score matches. The Mascot score is noted in the score in the 2D gel spot identifications and relates to the information in supplementary data files. MW and pI are presented for each match. Searching of PFAM and literature indicated nucleotide binding information, and this is noted in comment form for each protein. MP represents prior evidence that this specific gene match represents an Arabidopsis mitochondrial protein (MP) from www.suba.bcs.uwa.edu.au.; m ) mitochondrial protein identified on gel Separations.

The major metabolic enzymes that have been selected in Table 1 do not reflect the most abundant proteins in these classes in the matrix of mitochondria. In most cases, evidence of interaction with ATP has been reported. ATP is a potent inhibitor of the pyruvate dehydrogenase complex due to the phosphorylation of this protein complex by an associated protein kinase.7 Mitochondrial malic enzyme is known to be inhibited by ATP in most eukaryotic systems. In humans, where this has been best investigated, the X-ray crystal structure reveals two ATP molecules can bind to the enzyme at the active site and an exo site, and it appears competitive binding in the active site is responsible for the inhibition.12 The claimed activation of the enzyme by ATP in some species may be due to binding at the exo site.12 Isocitrate dehydrogenase is known to be inhibited by ATP in kinetic studies of the plant mitochondrial enzyme from castor bean.10 The restoration of the glycine decarboxylase complex (GDC) activity, consisting of four subunits (P-, H-, L-, and T-proteins) in pea leaf mitochondria by ATP in the absence of glycine is 3466


believed to be due to the ATP-dependent activation of the T-protein.6 However, we have found that specific isoforms of P- and H-proteins of GDC are prominent ATP-binding proteins in Arabidopsis mitochondria, suggesting that further ATPdependent activation sites may exist in GDC. The strong binding of alanine aminotransferase and isovaleryl-CoA dehydrogenase to ATP is not known, to our knowledge, and warrants further investigation. Further metabolic enzymes were reported in Table 2. Mitochondrial citrate synthase is known to be inhibited by ATP, and indeed, this was the mechanism that was first used to differentiate the mitochondrial and the peroxisomal forms of the enzyme, as the latter is not inhibited by ATP.9 Succinyl-CoA ligase synthesizes ATP in the matrix and is a reversible enzyme with ATP-binding capabilities; the plant enzyme is well-characterized as an ATP- rather than GTPdependent enzyme.5 The catalytic F1 unit (R3 β3γδ) of the ATP synthase complex (F0F1) is the site of ATP synthesis from ADP, Pi, and Mg2+ in mitochondria.47 It is generally accepted that the β subunit

technical notes

Ito et al.

Figure 5. Percentage of genes encoding mitochondrial proteins within the sets of Arabidopsis genes that coexpress with the 15 Arabidopsis 14-3-3 genes. The most significantly coexpressed genes with 14-3-3 genes using Expression Angler from the Botany Array Resource (bbc.botany.utoronto.ca/ntools/cgi-bin/ntools_expression_angler.cgi) were analyzed using the SUBA mass spectrometry database (www.suba.bcs.uwa.edu.au) in organelles to show the proportion of genes that coexpress with each 14-3-3 that have been experimentally found in the mitochondrion. Coexpressed lists for each 14-3-3 protein were limited to r > 0.6, up to a maximum of 50 genes, in this analysis (number above each bar shows list size in each analysis). GRF1 At4g09000, GRF2 At1g78300, GRF3 At5g38480, GRF4 At1g35160, GRF5 At5g16050, GRF6 At5g10450, GRF7 At3g02520, GRF8 At5g65430, GRF9 At2g42590, GRF10 At1g22300, GRF11 At1g34760, GRF12 At1g26480, GRF13 At1g78220, GRF14 At1g22290, GRF15 At2g10450.

undergoes various conformational changes, from an “open” state that binds weakly to nucleotides, to a “tight” state that has a very strong affinity for ATP, and finally to a “loose” state, which has a lower affinity for ATP and from which ATP can be released.48 Furthermore, structural analysis has shown that mammalian F1 contains a P-loop in the β catalytic subunit (amino acid residues 156GGAGVGKT163), which is involved in binding to ATP, and that the β subunit binds tightly to ATP in the presence of Mg2+.47 Here, we have demonstrated that plant ATP synthase β subunit also binds to ATP in the presence of Mg2+. Plant complex III (b/c1 complex) of the electron transport chain is unique among eukaroytes in that it contains the mitochondrial processing peptidase (MPP).49 MPP consists of R and β subunits, which cooperate in cleaving off the Nterminal presequence of the precursor proteins at a single and specific site shortly after its import into the mitochondria. MPP specifically recognizes a large variety of mitochondrial precursor proteins and requires the presence of metal ions, such as Ca2+ and Mn2+, for endopeptidase activity.49 While there is no evidence of ATP-dependence by MPP for the import of precursor proteins into the matrix in plants, ADP and ATP have been shown to bind to and exert strong control on the conformational and functional properties of R and β subunits, respectively, of MPP in Neurospora crassa.50 Our results here indicate that only the R subunit of plant MPP binds to ATP, but not ADP, as it was absent as a protein band on 1D gels of ADPeluted mitochondrial proteins (Figure 2). Among the identified proteins were two enzymes of purine synthesis. Phosphoribosylformylglycinamide cycle-ligase is an ATP-dependent enzyme of purine synthesis that has been shown to be dually targeted to mitochondria and chloroplasts in plants.51 In contrast, glycinamide ribonucleotide transformylase is not ATP-dependent, but may well bind to ATP due to its substrate-binding site that has affinity for its substrate, a modified ribose-phosphate.52 The large series of dehydrogenases and reductases in Table 2 all have nucleotide-binding sites that may be responsible for the ATP-binding ability of these enzymes, although, interestingly, they could not be displaced from the ATP column by NADH (Figure 2). A range of dehydrogenases, including glutamate dehydrogenase and aldehyde dehydrogenase, were

also isolated from human cells with ATP affinity capture.25 In the case of glutamate dehydrogenase, studies in mammals have shown allosteric regulation by GTP and ATP,53 but early studies in plants did not address ATP/GTP regulation, and to our knowledge, no further studies have considered this issue.54 The antioxidant defense proteins found here for the ascorbate/ glutathione cycle (glutathione reductase and monodehydroascorbate reductase) are the same protein isoforms we have previously shown are dually targeted to chloroplasts and mitochondria in plants, but have only previously been found in complex mixture LC-MS analysis.55 Their nucleotide-binding sites may be responsible for their binding to ATP, but to our knowledge, they do not have a known history of binding purine triphosphates. This analysis has provided a series of novel pathways for research, notably, the identification of a range of new lowabundance proteins in the mitochondrial proteome which have the potential to be influenced by ATP or other nucleotides and the first identification of specific 14-3-3 proteins in plant mitochondria. It also illustrates the complication of distinguishing affinity-enriched proteins from the potential nonspecific elutions for affinity media. We have sought to address this through the proof of ATP dependence of elution (Figure 2), the use of multiple affinity media for the same ligand (Figure 3 and Tables 1 and 2), and the use of 2D gels to compare total protein and affinity-enriched samples (Figure 4). In combination, these provide a level of confidence in the identifications made. This work was limited to soluble proteins and has not considered the ATP affinity of mitochondrial membrane proteins. Such work would require the use of detergents and would require the use of approaches other than IEF SDS-PAGE to confirm specific from nonspecific elutions.

Acknowledgment. This research was supported by the Australian Research Council (ARC) Centre of Excellence in Plant Energy Biology. J.I. is funded by an Australian Postgraduate Award, A.H.M. by an ARC Queen Elizabeth II Fellowship, and J.L.H. by an ARC Australian Postdoctoral Research Fellowship. Supporting Information Available: Detailed information of the mass spectra matching, individual peptide scores, and sequence coverage diagrams that form the basis of the 2D Journal of Proteome Research • Vol. 5, No. 12, 2006 3467

technical notes


gel identifications in Tables 1 and 2 data are provided in supplementary data 1. Generic format data files (Mascot generic) generated from the primary mass spectra based on the script explained above are provided as supplementary data 2; these can be directly reanalysed at www.matrixscience.com by the reader. All 5 independent IEF-SDS-PAGE gels run of the ATP-eluted protein population, referred to in Tables 1 and 2 are provided in supplementary data 3. This material is available free of charge via the Internet at http://pubs.acs.org.

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Journal of Proteome Research • Vol. 5, No. 12, 2006 3469

Analysis of the Soluble ATP-Binding Proteome of Plant Mitochondria

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