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Originally published In Press as doi:10.1074/jbc.M300245200 on April 24, 2003

J. Biol. Chem., Vol. 278, Issue 28, 26021-26030, July 11, 2003
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Phosphorylation of Formate Dehydrogenase in Potato Tuber Mitochondria*

Natalia V. Bykova {ddagger} §, Allan Stensballe ¶, Helge Egsgaard {ddagger}, Ole N. Jensen ¶ and Ian M. Møller ||

From the {ddagger}Plant Research Department, Risø National Laboratory, P. O. Box 49, DK-4000 Roskilde and the Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark

Received for publication, January 9, 2003 , and in revised form, March 7, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Two highly phosphorylated proteins were detected after two-dimensional (blue native/SDS-PAGE) gel electrophoretic separation of the matrix fraction isolated from potato tuber mitochondria. These two phosphoproteins were identified by mass spectrometry as formate dehydrogenase (FDH) and the E1{alpha}-subunit of pyruvate dehydrogenase (PDH). Isoelectric focusing/SDS-PAGE two-dimensional gels separated FDH and PDH and resolved several different phosphorylated forms of FDH. By using combinations of matrix-assisted laser desorption/ionization mass spectrometry and electrospray ionization tandem mass spectrometry, several phosphorylation sites were identified for the first time in FDH and PDH. FDH was phosphorylated on Thr76 and Thr333, whereas PDH was phosphorylated on Ser294. Both Thr76 and Thr333 in FDH were accessible to protein kinases, as demonstrated by protein structure homology modeling. The extent of phosphorylation of both FDH and PDH was strongly decreased by NAD+, formate, and pyruvate, indicating that reversible phosphorylation of FDH and PDHs was regulated in a similar fashion. At low oxygen concentrations inside the intact potato tubers, FDH activity was strongly increased relative to cytochrome c oxidase activity pointing to a possible involvement of FDH in hypoxic metabolism. Computational sequence analysis indicated that a conserved local sequence motif of pyruvate formate-lyase is found in the Arabidopsis thaliana genome, and this enzyme might be the source of formate for FDH in plants.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Reversible protein phosphorylation is one of the most important regulatory mechanisms in living cells (1), yet little is know about protein phosphorylation in mitochondria. The only well studied case is that of phosphorylation of the E1{alpha}-subunit of the pyruvate dehydrogenase (PDH)1 complex (2). The PDH complex contains both a kinase and a protein phosphatase, and phosphorylation inactivates the complex, which is reactivated by dephosphorylation. This means that the entry of carbon compounds from glycolysis into the citric acid cycle is turned off under conditions of high ATP concentration.

Although the PDH kinase and phosphatase system is the only fully characterized system for reversible protein phosphorylation in mitochondria, there are clear indications that others are present. Incubating intact mitochondria (3–5) or submitochondrial fractions like the outer membrane (6) or the inner membrane (7–9) with [{gamma}-32P]ATP gives labeling of up to 30 proteins (as detected by one-dimensional gel electrophoresis) indicating that kinases are present in these fractions and in contact with target proteins. The matrix appears to contain one of more protein phosphatases responsible for dephosphorylation of inner membrane phosphoproteins (8, 9).

Only eight mitochondrial phosphoproteins have been identified to date. In addition to the E1{alpha}-subunit of PDH, these are band {delta}'-subunits of the ATP synthase (10), HSP70 (11, 12) and MTSHP (13, 14), nucleoside diphosphate kinase (15, 16), E1 of the branched-chain {alpha}-ketoacid dehydrogenase (2), and the 18-kDa iron protein subunit of complex I in mammalian mitochondria (17). In a very recent study (18) a further 14 mitochondrial phosphoproteins were identified, all household proteins involved in the citric acid cycle and associated reactions, the respiratory chain complexes, in heat shock proteins, and the detoxification of reactive oxygen species.

Formate dehydrogenase (FDH) catalyzes the oxidation of formate to CO2 reducing NAD+ to NADH in the process. Formate may have a role in biosynthesis of numerous compounds, such as in energetic metabolism and in signal transduction pathways related to stress response. The enzyme is induced in potato leaves by a variety of stresses including hypoxia (19, 20). It is one of the most abundant proteins in the matrix of potato tuber mitochondria (19), but its function is unknown partly because the source of formate is unknown (19, 21).

In the present study we use two-dimensional gel electrophoresis and mass spectrometry to show that a major part of the phosphorylation in potato tuber mitochondria that was ascribed previously to PDH is due, in fact, to labeling of FDH. We identify the site of FDH (and PDH) phosphorylation and describe some properties of FDH phosphorylation. Finally, we show that FDH activity is highest in intact sprouting tubers where the oxygen concentration is relatively low implying that FDH has a role in hypoxic metabolism.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plant Material, Isolation, and Subfractionation of Intact Mitochondria—Intact and functional highly purified mitochondria were isolated from potato tubers (Solanum tuberosum L.) using differential centrifugation followed by Percoll density gradient centrifugation and subfractionated into inner membrane vesicles and a matrix fraction essentially as described previously (22).

Phosphorylation Assay—Protein phosphorylation in intact plant mitochondria and in the mitochondrial subfractions was investigated by in vitro labeling analyses with [{gamma}-32P]ATP. Phosphorylation assays were carried out at 25 °C for 10 min in a volume of 200 µl containing 1 mg of protein, 0.3 M mannitol, 10 mM MOPS-KOH, pH 7.2, 5 mM MgCl2, 0.1 mM CaCl2, 1 mM phenylmethylsulfonyl fluoride, 150 µCi of [{gamma}-32P]ATP (AA0068, Amersham Biosciences), and 0.2 mM ATP. The reactions were terminated, and [{gamma}-32P]ATP was removed from the mitochondria by addition of ice-cold reaction buffer and centrifugation at 100,000 x g for 1 h or for the matrix fraction by precipitation with 80% ice-cold acetone containing 35 mM mercaptoethanol followed by centrifugation at 10,000 x g for 20 min.

Two-dimensional Blue Native/PAGE—In the first dimension blue native electrophoresis was used in order to separate protein complexes according to their size (23) followed by gradient 10–15% Tricine SDS-PAGE in the second dimension. For blue native gels 1 mg of matrix protein was supplemented with a buffer to give the following final concentrations: 750 mM aminocaproic acid, 0.5 mM EDTA, 50 mM Bis-Tris, pH 7.0 and 1.5% w/v n-dodecyl maltoside. Coomassie Blue solution, 15 µl of 5% (w/v) Serva Blue G, 750 mM aminocaproic acid, was added to the solution and loaded directly onto a 4.5% to 16% (w/v) acrylamide gradient blue native gels. BN-PAGE was carried out as described (23, 24).

Two-dimensional Isoelectric Focusing/Tricine SDS-PAGE—The isoelectric focusing was conducted with the IPGphor system (Amersham Biosciences) using 17-cm ReadyStrip IPG strips with a linear pH gradient range 5–8 according to the manufacturer's instructions (25). Mitochondrial proteins were solubilized in 300 µl of rehydration solution containing 7 M urea, 2 M thiourea, 2% w/v CHAPS, 2% w/v n-dodecyl maltoside, 20 mM dithiothreitol, 1% v/v IPG buffer, pH 3.0–10.0. For rehydration with matrix proteins, the solution contained 7 M urea, 2 M thiourea, 4% CHAPS, 20 mM dithiothreitol, 0.5% IPG buffer, pH 3.0–10.0. Tricine SDS-PAGE was carried out as described (26). The pI and molecular mass scales of the two-dimensional maps were calibrated using the ImageMaster two-dimensional Elite software (Amersham Biosciences). The 32P-containing phosphoproteins were visualized by PhosphorImaging using Quantity One software (Bio-Rad).

Tandem Mass Spectrometry Analysis of Tryptic Peptides—MS/MS analysis of peptides generated by in-gel digestion as described previously (27) was performed on a Finnigan LCQ (Classic) quadrupole ion storage mass spectrometer (San Jose, CA) equipped with a nanoelectrospray source (Protana Engineering, Odense, Denmark). Prior to MS analysis samples were purified on reverse phase POROS R2 or OligoR3 (20–30 µm bead size, PerSeptive Biosystems, Framingham, CA) nanocolumns (28, 29) and eluted with 1 µl of 50% (v/v) methanol, 5% (v/v) formic acid directly into the precoated borosilicate nanoelectrospray needles (Protana Engineering, Odense, Denmark).

Phosphopeptide Purification by Nanoscale Fe(III)-IMAC—Phosphopeptides present in tryptic digestion mixtures generated from phosphoproteins were purified by miniaturized Fe(III)-IMAC columns as described (29). Peptide samples were redissolved in 0.1 M acetic acid containing 10% (v/v) acetonitrile and slowly loaded onto the IMAC column (loading time 120–140 s). The column was then washed with 0.1 M acetic acid, 10% (v/v) acetonitrile in 0.1 M acetic acid, Milli-Q water. Peptides were eluted from nanoscale IMAC columns by using pH 10.5 solvent (Milli-Q water adjusted to pH 10.5 by addition of 25% (v/v) ammonia). The eluate was acidified to 5% (v/v) formic acid and loaded directly onto a nanoscale column (28) packed with Oligo R3 material. The column was rinsed with 5% formic acid and then eluted with saturated matrix solution (2,5-dihydroxybenzoic acid in 50% (v/v) acetonitrile, 5% formic acid). The matrix/analyte eluate was spotted as a series of nanoliter volume droplets onto the MALDI probe.

Peptide Mass Mapping by MALDI-TOF-MS—Delayed extraction MALDI mass spectra were recorded on a Reflex IV time-of-flight mass spectrometer (Bruker-Daltonics, Bremen, Germany) equipped with a nitrogen laser ({lambda} = 337 nm). Mass spectra were acquired in both positive ion linear mode and positive ion reflector mode. Instrument mass calibration was performed by using a tryptic peptide mixture derived from bovine lactoglobulin (external calibration). Internal mass calibration of peptide mass spectra using digestion products of trypsin or from the identified proteins resulted in mass errors of less than 50 ppm, typically 15–25 ppm.

Automated Nano-flow LC-MS/MS Analysis of Tryptic Peptides— Tryptic peptide mixtures were separated and analyzed using a nanoscale capillary high pressure liquid chromatography system (Ultimate, LC-Packings) interfaced directly to a Q-TOF tandem mass spectrometer (Q-TOF Ultima API, Micromass, UK). Peptide mixtures were separated on a C18 reverse phase column (75 µm (inner diameter) x 90.0 mm, Zorbax SB-C18 3.5 µm) during a 1.5-h gradient of 0–90% acetonitrile (v/v) containing 1% (v/v) formic acid, 0.6% (v/v) acetic acid, and 0.005% (v/v) heptafluorobutyric acid at a flow rate of 175 nl/min. The mass spectrometer was calibrated by using NaI (8-s scan time, m/z 400–2000).

Data Base Searching and Protein Sequence Analysis—Ion trap tandem mass spectrometry data were interpreted by using the Mascot MS-MS Ions Search software (www.matrix-science.com). The Sequest program (Thermoquest, San Jose, CA) was used to convert ion trap MS/MS spectra into DTA format. Only matching peptides with precursor ion mass tolerance of less that 1 Da and individual ion scores indicating identity to the protein were taken into consideration and used for estimation of a minimum percentage sequence coverage. Mass spectra obtained by automated nano-flow LC-MS/MS using data-dependent acquisition modes were analyzed using the MassLynx 3.5 software (Micromass, UK) and Mascot MS-MS Ions Search. MALDI mass spectra were analyzed by using the software package m/z (Proteometrics Ltd., New York). The differential peptide mass mapping and analysis of theoretical tryptic peptide sequences were performed using GPMAW 5.01 (Lighthouse Data, Odense, Denmark). Tandem mass spectra obtained from phosphopeptides were manually inspected.

Computer Modeling of S. tuberosum FDH—Sequence of S. tuberosum FDH was from NCBI-CAA79702 (19). The three-dimensional structural homology model was constructed with Swiss model 3.5 protein modeling server and the Swiss-PdbViewer version 3.7b2 (30, 31).

Measuring the Oxygen Concentration in Intact Potato Tubers—Tubers of 40–200 g fresh weight (average weight 109 g after peeling; 4–6 cm in diameter) stored at 4 °C were incubated at room temperature (22–24 °C) in darkness for 2–3 weeks and had started sprouting.

An OX100 microelectrode from Unisense, Århus, Denmark, with a tip diameter of 100 µm was calibrated in air-saturated water immediately before use. A tuber was immobilized on a platform, and the electrode was inserted into the tuber using a micromanipulator. Two different insertion methods were used. (a) The electrode was inserted 25 mm into the tuber stopping for 1 min to take a reading just below the surface and at 5, 10, 15, 20, and 25 mm depth in the same hole. (b) Alternatively, the oxygen concentration at each depth was measured by sticking the electrode in at a new position. The read-out usually stabilized well before the minute was up, and the values were normally similar with the two insertion methods indicating that leakage of air into the tuber along the electrode did not occur. The oxygen concentration was calculated assuming that the water in equilibrium with air contains 270 µM oxygen at 22 °C (32).

After the oxygen concentration measurements the tuber was peeled and homogenized to give a soluble fraction where the activity of FDH and CCO was measured (see below).

Enzyme Activity Measurements in Whole Tuber Extracts—Each tuber was homogenized separately using a Braun juice extractor and 17 ml of medium/100 g fresh weight containing 0.9 M mannitol, 30 mM MOPS, 3 mM EDTA, 0.3% (w/v) bovine serum albumin, and 25 mM cysteine, pH 7.2. Large particles were removed by filtration through nylon cloth (mesh 120 µm) and centrifugation at 1600 x g for 10 min. After decantation and volume measurement, the supernatant was frozen in liquid nitrogen and stored at –80 °C until use.

CCO activity was measured as described previously (33). FDH activity was measured as NADH production at 340 nm in a medium containing 0.3 M sucrose, 20 mM MOPS, pH 7.2, 1 µg/ml antimycin A, 15 mM formate, 1 mM NAD+, and 0.025% (v/v) Triton X-100. The reaction was started by the addition of sample, and it was linear for at least 5 min.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Separation of FDH and PDH on Two-dimensional Gels—BN-gel electrophoresis separates protein complexes according to size, and the complexes can subsequently be separated into their component polypeptides by SDS-PAGE in the second dimension (23, 26, 34). On BN two-dimensional gels the 32P-labeled matrix fraction from potato tuber mitochondria shows a very large and heavily labeled spot at 42–45 kDa, which stretches from around 200 kDa down to <100 kDa in the 1st dimension (Fig. 1, A and B). MS identification showed that the E1{alpha}-subunit of PDH was only found in spots 1 and 2, whereas FDH was found in all parts of the smear indicating that this enzyme was also labeled (Table I). The E1{alpha} component of pea PDH forms a catalytically active unit of 160 kDa (2), and this may migrate intact under the conditions in the first dimension. FDH appears to migrate as a tetramer of the 40-kDa mature subunit (19).



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  		FIG. 1.
Two-dimensional resolution of mitochondrial matrix protein complexes by Blue Native/Tricine SDS-PAGE. A, Coomassie staining; B, PhosphorImager. The numbers on the left refer to the molecular masses of standard proteins. The numbered arrows indicate protein spots identified by mass spectrometry and are listed in Table I. Note that the heavily labeled spot below spot 3 is malate dehydrogenase, which is a dimer of around 70 kDa.

 

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  		TABLE I
Identification of two- and one-dimensional separated protein spots and bands

Sequence tags were identified either by nano-ESI-MS/MS analysis using nanoelectrospray source (spots 1, 3-11, and 17-19) or by automated nano-flow LC-MS/MS analysis of tryptic peptides (spots 2 and 12-16) followed by searching MS/MS data sets against nonredundant NCBI data base using the MASCOT search engine (Matrixscience).

 



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  		FIG. 2.
Two-dimensional separation of intact potato tuber mitochondria and their matrix fraction by isoelectric focusing/Tricine SDS-PAGE. A, intact mitochondria, Coomassie staining; B, intact mitochondria, PhosphorImager; C, matrix, Coomassie staining; D, matrix, PhosphorImager. Denaturing IEF was carried out using a linear IPG strip of pH 5–8. The numbers above the gel images indicate pI values, and the numbers on the left indicate molecular masses of standard proteins. The numbered arrows indicate protein spots identified by mass spectrometry (Table I).

 


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  		FIG. 7.
Regulation of phosphorylation of FDH and PDH in intact mitochondria and matrix fraction by various metabolites as analyzed by one-dimensional gel electrophoresis and PhosphorImaging. A, relative labeling; B, PhosphorImager; C, Coomassie staining. Lane a, control mitochondria (isolated mitochondria suspended in phosphorylation medium and incubated for 10 min); lane b, phosphorylation of isolated mitochondria in the presence of 15 mM formate; lane c, phosphorylation of mitochondria in the presence of 5 mM pyruvate; lane d, phosphorylation of mitochondria in the presence of 15 mM formate and 1 mM NAD+; lane e, control matrix fraction (incubated in phosphorylation medium for 10 min); lane f, phosphorylation of matrix proteins in the presence of 15 mM formate; lane g, phosphorylation of matrix proteins in the presence of 15 mM formate and 1 mM NAD+; lane h, phosphorylation of matrix proteins in the presence of 1 mM NAD+; lane i, phosphorylation of matrix proteins in the presence of 1 mM NADH. The reactions were terminated by addition of sample buffer containing 0.1% SDS and then heat-denatured at 100 °C for 5 min. The same amount of protein 120 µg per lane in equal volumes was loaded on a gradient 10–15% linear acrylamide gel and separated by Tricine SDS-PAGE. The 32P-labeled protein bands were analyzed by PhosphorImaging, and quantification of 32P incorporation was done using Quantity One software (Bio-Rad). The numbers on the gel image refer to the protein bands identified by nano-ESI-MS/MS analysis shown in Table I.

 


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  		FIG. 9.
The correlation between FDH and CCO activity in potato tuber extracts and the concentration of oxygen inside the intact tubers. The oxygen concentration inside an intact tuber was measured by insertion of a microelectrode. FDH and CCO activities were measured in an extract from the same tuber. Each point represents one tuber. The lines were fitted using linear regression analysis. A, correlation between FDH activity and oxygen concentration. B, correlation between CCO activity and oxygen concentration. C, changes in FDH activity relative to aerobic respiration (expressed as CCO activity).

 
On IEF/SDS-PAGE gels PDH and FDH again migrate in the same area (Fig. 2, A and B), but both are now found in several discrete spots. Only PDH was identified in spots 4–6 and only FDH in spots 3, 8, 10, 11, 16, 18, and 19 (Table I). Spots 1, 2, 7, 9, 12–15, and 17 contained both proteins. For FDH the two largest and most alkaline spots 18 and 19 were not labeled, indicating that only a minor part of FDH was labeled under the isolation and assay conditions used in this study.

Identification of Site of Phosphorylation on PDH—Mammalian mtPDH is phosphorylated on three serines, only one of which is conserved in plant mtPDH. However, the site of plant mtPDH phosphorylation has not been identified previously. By using automated nano-flow LC-MS/MS, we identified a 14-amino acid-long phosphopeptide, Tyr290–Arg303 (Fig. 3) in the tryptic peptide mixture derived from protein spot 13 (Fig. 2). The same phosphopeptide was found in protein spots 1 and 12–14 (Figs. 1 and 2). The MS/MS scan in Fig. 3B derived from the peptide eluting at the time point is indicated with an arrow in Fig. 3A. The mass difference between the doubly protonated precursor ion ((M + 2H+)2+, m/z 837.94) and the ion of m/z 788.94 is a signature for the loss of phosphoric acid from the phosphopeptide via a gas-phase {beta}-elimination mechanism. The mass difference between the peptide fragment ions y9 and y10 (69 Da) corresponds to the dehydroalanyl residue formed by loss of phosphoric acid from phospho-Ser294 (35). Thus, Ser294 in PDH is phosphorylated.



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  		FIG. 3.
Identification of a site of phosphorylation in the E1{alpha}-subunit of the PDH complex by automated nano-flow LC-MS/MS analysis. A, data-dependent analysis of peptide mixture using 15% of in situ digested two-dimensional IEF/SDS gel spot (spot 13 in Fig. 2) was performed during a 1.5-h gradient of 0–90% acetonitrile. The top panel depicts the base peak chromatogram of MS/MS experiments performed during the analysis in information-dependent acquisition mode, and the lower panel represents the base peak chromatogram of the survey scans only. The arrow indicates the MS/MS scan of fragmented phosphopeptide. B, identification of phosphorylated peptide YHGH(pS)MSDPGSTYR (amino acids 290–303). Tandem MS obtained using collision-induced dissociation (CID) displays significant fragment ion signals originating by partial or complete neutral loss of phosphoric acid from the phosphoserine residues via a {beta}-elimination process as well as internal fragmentation. Metastable loss of H3PO4 (–49 Da) from the doubly charged precursor ion at m/z value 837.94 phosphopeptide is indicated.

 

Identification of the Phosphorylation Sites on FDH—Initial phosphorylation analysis of FDH was performed by using a combination of nanoscale Fe(III)-IMAC for enrichment of tryptic phosphopeptides and OligoR3 column for phosphopeptide concentration and desalting (29). Identical phosphopeptide mass maps were obtained for FDH protein spots 1 and 2 on BN/SDS-PAGE (Fig. 1A and Table I). Fig. 4A shows a MALDI mass spectrum of an IMAC-retained phosphopeptide from FDH. The ion at m/z 2215.0 is the unphosphorylated, singly charged 19-amino acid peptide Gly70–Lys88, whereas the ion at 2295.0 Da is the phosphorylated form of this peptide (HPO3, +80 Da). The peptide Gly70–Lys88 has two candidate phosphorylation sites, viz. residues Tyr73 and Thr76. Closer inspection of the mass spectrum reveals several ion signals indicative of {beta}-elimination of phosphoric acid (see figure legend), making Thr76 the prime candidate for phosphorylation because Tyr(P) cannot undergo {beta}-elimination.



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  		FIG. 4.
Identification of a site of phosphorylation in mitochondrial FDH. A, MALDI-TOF MS peptide mass map after phosphopeptide enrichment by nanoscale IMAC. One peptide was found to have detectable levels of +80 Da modification that are indicative of phosphorylation. The candidate phosphopeptide ion signal was accompanied by metastable decomposition product ion corresponding to gas-phase {beta}-elimination of phosphoric acid when analyzed in the reflector TOF mode. Due to both dephosphorylation and dehydration Thr becomes a dehydro-2-aminobutyryl residue. The apparent mass difference (86 Da) between the precursor ion and the metastable ion observed in the mass spectra actually corresponds to a 98-Da difference (loss of phosphoric acid). The mass discrepancy is due to a different ion kinetic energy of the metastable ions, which therefore do not follow the same calibration curve as the intact precursor ions (29). B, the candidate phosphopeptide was subsequently sequenced by nanoelectrospray tandem mass spectrometry in a quadrupole ion trap mass spectrometer. The resulting mass spectrum (precursor ion (M + 3H)3+ = 766.24) displayed a number of sequence-specific peptide fragment ion signals identifying the peptide GHQYIV(pT)PDKEGPDCELEK (amino acids 70–88). The y- and b-ion series of different charge states (+1, +2, and +3) exhibited further fragmentation by {beta}-elimination of phosphoric acid from y142+, y173+, y172+, and y182+* (m/z values 800.31, 668.49, 1002.2, and 1061.8, respectively). The phosphothreonine 76 assignment was confirmed by a complementary b-98 ion series b82+,b132+, b152+, b162+, and b182+ (m/z values 439.76, 701.47, 839.65, 905.93, and 1026.2, respectively). The peaks denoted y* and y° are the result of ammonia (–17 Da) and water (–18 Da) losses from corresponding ion, respectively. The peaks with ammonia losses are y182+* and y12* (m/z values 1061.8 and 1400.3, respectively) and with water loss is y52+° (m/z value 330.91). One of these ions y182+* also contains dehydro-2-aminobutyryl residue (in position y13) instead of phosphothreonine.

 

To confirm this hypothesis the Fe(III)-IMAC enriched phosphopeptide sample was next subjected to amino acid sequencing by nanoelectrospray ion trap tandem mass spectrometry using the triply protonated precursor ion (m/z 766.24) (Fig. 4B). About 10 fragment ions can be matched to the peptide, and in all cases the masses match a sequence containing dehydro-2-aminobutyryl residue instead of Thr76. The peptide gave quite a high prediction score 0.82 for phosphorylation when tested with the NetPhos 2.0 software (Table II). Thus, these experiments strongly indicate that Thr76 in FDH is phosphorylated.


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  		TABLE II
Identification of predicted tryptic phosphopeptides for formate dehydrogenase from potato tuber mitochondria

The MS data were obtained either by nano-ESI-MS/MS or LC-MS/MS analysis (Figs. 4 and 5). Potential phosphorylation sites were predicted by the NetPhos 2.0 software (score value in the range 0-1 with the assignment above 0.5 threshold). The tryptic peptide sequences are given. Their positions and the modified amino acid position given are relative to the start of the FDH protein (accession number in NCBI CAA79702 [GenBank] ) with transit peptide intact. The theoretical tryptic peptide sequences were analyzed by GPMAW 5.01 (Lighthouse Data, Odense, Denmark) software for protein sequence processing.

 



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  		FIG. 5.
Identification of a site of phosphorylation in mitochondrial FDH by automated nano-flow LC-MS/MS analysis. The graph represents the data collected during the CID MS/MS analysis of the triply charged precursor ion (m/z value 820.3), which generated a y-ion series (y4–y14) and confirmed the identity of the peptide as 326YMPNQAMTPHISGTTIDAQLR346. The phosphorylation site Thr-333 was assigned by the 83-Da mass difference between the y13 and y14 ions of C-terminal peptide fragments, corresponding to dehydro-2-aminobutyryl residue, which was formed by {beta}-elimination of phosphoric acid from phosphothreonine 333. The peak at m/z value 758.32 (indicated with an asterisk) represents the internal fragment ion PNQAMTP that has lost its H3PO4 group (98.0 Da).

 
Fig. 5 shows the tandem mass spectrometry fragmentation pattern of yet another phosphopeptide Tyr326–Arg346 revealed by nano-flow LC-MS/MS analysis of the tryptic peptide mixture obtained from FDH spot 2 on BN/SDS-PAGE (Fig. 1A and Table I). The 21-amino acid peptide Tyr326–Arg346 is heavily modified; it contains two oxidized methionines and two deamidated amino acids (converting Asn329 into Asp329 and Gln330 into Glu330). The phosphorylation site Thr333 was assigned by the 83-Da mass difference between the y13 and y14 fragment ions, corresponding to a dehydro-2-aminobutyryl residue. No fewer than 10 y-ions can be matched. Thus, Thr333 in FDH is phosphorylated. The other modified amino acids (oxidation and deamidation) will be discussed under the "Discussion." The phosphopeptide Tyr326–Arg346 was found in protein spots 2, 15, and 16 (Table I). In none of the three cases did we detect a PDH phosphopeptide despite the fact that spots 2 and 15 contained both FDH and PDH (see above). Thus, spots 2 and 15 probably contained unphosphorylated PDH and phosphorylated FDH.

In summary, we have identified two sites of phosphorylation on FDH, Thr76 and Thr333. To get an idea of their location in the native FDH conformation, we applied a structural homology three-dimensional model of S. tuberosum FDH (Fig. 6). S. tuberosum FDH shares high sequence and structural similarity to the bacterial FDH from Pseudomonas sp. 101. A backbone alignment of the mitochondrial FDH and bacterial FDH is shown in Fig. 6A and underlines their similarity. The image shown in Fig. 6B indicates the positions of the NAD+-binding site and the active site, both of which lie close to a groove that can accommodate the formate molecule. Thr76 and Thr333 are both on the outer surface of the protein and thus easily accessible to kinases and phosphatases.



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  		FIG. 6.
Structural homology model of S. tuberosum FDH. S. tuberosum FDH (NCBI-CAA79702) shares high sequence and structural similarity to the bacterial FDH from Pseudomonas sp. 101 (NCBI-999845). The template and target sequences have 51.8% identity; NAD+-binding domains have 50% identity, and the substrate-binding site (Arg residue) is conserved. The three-dimensional structure of mitochondrial FDH was constructed by homology modeling using Swiss model 3.5 protein modeling server and the Swiss-PdbViewer version 3.7b2 (30, 31), based on the x-ray structure of bacterial FDH (Molecular Modeling Database): 2827; Protein Data Bank code 2NAC [PDB] ) (47, 48); sequence alignment was done in LALIGN (49). A, backbone alignment of the mitochondrial FDH (red) and bacterial FDH (blue). The quality of the model was evaluated by WHAT IF program version 19970813-1517 (50, 51). The root mean square Z scores for bond lengths and for bond angles were 0.758 and 1.283, respectively, and displayed normal variability. B, the model of potato FDH. The image was generated in Swiss-PdbViewer version 3.7b2 (30). Active site, NAD+-binding domain, and the two phosphorylation sites are indicated.

 

Regulation of FDH (and PDH) Phosphorylation—To investigate the regulation of protein phosphorylation, intact mitochondria or matrix fraction was incubated with [32P]ATP and various compounds either alone or in combination. In Fig. 7 the proteins were separated by SDS-PAGE, and phosphorylation was quantified by PhosphorImager scanning. Fig. 7C shows the protein bands that were cut out and identified by ESI-MS/MS (Table I).

In intact mitochondria the labeled band 20 contained both PDH and FDH. In the matrix fraction, band 21 contained PDH + FDH, whereas the lower band 22 contained only FDH (and isocitrate dehydrogenase, not shown) (Fig. 7B and Table I). The resolution on the PhosphorImager did not allow us to exclude any of these proteins from contributing to the labeling pattern. However, when bands 21 and 22 were cut out of the gel and read by PhosphorImaging separately, they both displayed 32P incorporation, and the labeling intensity in spot 21 was about 7 times higher than that in spot 22 (results not shown). Formate had almost no effect on the amount of 32P incorporation in intact mitochondria but clearly depressed phosphorylation in the matrix fraction. Formate and NAD+ inhibited phosphorylation in the matrix fraction in an additive manner by reducing labeling by 36 and 60% when added separately and by 90% when added together. Pyruvate also inhibited phosphorylation very strongly both in intact mitochondria (Fig. 7, A and B) and in the matrix fraction (not shown), whereas NADH only inhibited labeling in the matrix fraction by about 40% (Fig. 7).

A matrix fraction was also labeled either without additions (control) or in the presence of the FDH substrates formate + NAD+, and these two fractions were separated on IEF-PAGE two-dimensional gels. In the presence of formate + NAD+, two protein spots disappeared (Fig. 8, A and C), spot 12 and spot 13 containing both PDH and FDH (Table I), and labeling decreased markedly in that area (Fig. 8, B and D). This indicates that these spots contained phosphorylated FDH and PDH formed during the labeling incubation. The Coomassie staining of spot 16 containing only FDH did not appear to alter when labeling was inhibited, whereas spots 14 and 15 containing both enzymes increased in size (Fig. 8, A and C; Table I); but the labeling of all three spots almost completely disappeared (Fig. 8, B and D). The amount of protein in the unlabeled spots 17–19 was apparently unaffected by formate + NAD+. The results indicate clearly that formate + NAD+ inhibited the labeling of both PDH and FDH.



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  		FIG. 8.
Regulation of phosphorylation of FDH and PDH in the mitochondrial matrix fraction as analyzed by isoelectric focusing/Tricine SDS two-dimensional gel electrophoresis and PhosphorImaging. A, image of the Coomassie-stained gel for control experiment; B, PhosphorImager of control experiment; C and D, Coomassie staining and corresponding PhosphorImager, respectively, for the experiment in the presence of 15 mM formate and 1 mM NAD+. The concentrated matrix proteins (1 mg) were phosphorylated in the presence of [32P]ATP for 10 min at room temperature. The reaction was terminated by precipitation with 80% ice-cold acetone containing 35 mM mercaptoethanol. The proteins were resolubilized by addition of rehydration-solubilization buffer and separated by IEF/SDS two-dimensional PAGE. The numbers on the gels refer to the protein spots investigated by nano-ESI-MS/MS or nano-flow LC-MS/MS analysis shown in Table I.

 

FDH Activity and the Oxygen Concentration Inside Sprouting Potato Tubers Are Negatively Correlated—FDH is induced by a variety of stresses including hypoxia and anoxia (20, 36). The oxygen concentration inside actively growing potato tubers is so low as to limit respiration (37). For this reason we measured the activity of FDH in extracts from whole sprouting potato tubers and compared that to the activity of CCO, as an indicator of aerobic respiratory capacity, measured in the same extracts. These values were correlated with the concentration of oxygen measured in the same tubers before making the extracts. The oxygen concentration in the tubers was 30–75% that in air-saturated water or 80–200 µM similar to what was reported for growing tubers (37). However, unlike Geigenberger et al. (37), we did not observe any oxygen gradients in the tubers, the oxygen concentration was very similar whether measured immediately below the cork layer or at depths of 5–25 mm (the diameter of the tubers being 4–5 cm).

FDH activity increased and CCO activity decreased in the tuber extracts with decreasing oxygen concentration in the intact tubers (Fig. 9, A and B). As a result, FDH activity increased 4-fold relative to CCO activity with decreasing oxygen concentration (Fig. 9C). For a comparison, tubers stored at 4 °C with a low metabolic activity had an oxygen concentration above 300 µM, FDH and CCO activities of 6.8 ± 1.1 and 287 ± 49 nmol min–1 g fresh weight–1, and an FDH/CCO ratio of 0.025 ± 0.006 (n = 9) or very similar to that observed for sprouting tubers with oxygen concentrations above 200 µM (Fig. 9).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we have shown that phosphorylated and unphosphorylated PDH and FDH can be separated by IEF/SDS-PAGE two-dimensional gel electrophoresis of intact potato tuber mitochondria. The sites of phosphorylation in both proteins have been identified for the first time, and it has been shown that in FDH these sites are found on the outer, accessible surface of the enzyme. Finally, we have found that phosphorylation of FDH is strongly regulated by NAD+, formate, and pyruvate and that FDH activity is induced at low oxygen concentrations in the tubers.

Phosphorylation of PDH—No PDH phosphopeptide was found in spots 15 and 17 (Fig. 8, A and C) indicating that potato tuber mitochondria contain two unphosphorylated forms of the E1{alpha}-subunit of PDH. Whether these are products of different genes or caused by post-translational modification, we do not know. We identified Ser294 as a site of phosphorylation in plant PDH (Fig. 3) consistent with the fact that it is the only one of three serines phosphorylated in mammalian PDH that is conserved in plants (2). However, the phosphopeptide was found in three protein spots (spots 12–14; Fig. 8, A and B), two of which disappeared when phosphorylation was inhibited (spots 12 and 13, Fig. 8, A and C). It is therefore possible that both PDH forms are phosphorylated on Ser294, but we cannot exclude that there are more, as yet undetected and unidentified, sites of phosphorylation in PDH.

Phosphorylation of FDH—Previously it was assumed that 32P labeling was observed around 40–42 kDa for potato tuber mitochondria separated on one-dimensional gels derived from PDH (2, 3, 38). We now show that FDH is phosphorylated and that FDH phosphorylation is also complex.

Spots 18 and 19 contained only unlabeled FDH, and unlabeled FDH could have been present in other spots (Fig. 8, A and B). Because only one FDH gene product was detected in potato callus (19), it seems likely that either spot 18 or 19 contains post-translationally modified FDH. We did detect several post-translational modifications in the form of deamidations and methionine oxidation in one of the identified phosphopeptides from FDH (Fig. 5). A protein with these deamidations would have a lower pI than the unmodified protein, and this could be one of the reasons for finding FDH in a number of spots with pI ranging from pH 6.74 to 7.19 (Fig. 8 and Table I).

We identified both Thr76 and Thr333 as sites of phosphorylation on FDH (Figs. 4 and 5). We cannot at the moment exclude the possibility that there are more phosphorylation sites. A structural homology three-dimensional model of the enzyme indicates that the two phosphorylated residues are found on the outer surface of the protein (Fig. 6). The residues are therefore conveniently located to be reached by kinases and phosphatases.

Regulation of FDH Phosphorylation—Pyruvate, formate, and NAD+, the substrates of FDH and PDH, all reduced the phosphorylation level of FDH + PDH strongly (Fig. 7). PDH kinase is inhibited by pyruvate (39), so a pyruvate inhibition of PDH labeling was expected. However, it was unexpected that pyruvate completely abolished all phosphorylation in the 40– 43-kDa region because this implies that pyruvate also inhibits the kinase responsible for FDH labeling (Figs. 7, A and B, and 8). By analogy with PDH kinase, the inhibition of FDH phosphorylation by formate and by formate + NAD+ was expected. However, the complete inhibition of all phosphorylation in the 41–43-kDa region by formate + NAD+ (Figs. 7 and 8) was also unexpected because it implies that PDH kinase is inhibited by formate + NAD+.

The results indicate that FDH phosphorylation and PDH phosphorylation are regulated in the same way, which can only be interpreted after we have considered the physiological role of FDH. It appears clear, however, that PDH kinase can not be responsible for FDH phosphorylation on Thr76 or Thr333, because the PDH kinase is specific for serine residues (2).

Other Post-translational Modifications of FDH—The identified phosphopeptide Tyr326–Arg346 in FDH contained several other modifications, oxidation of methionine and deamidation of asparagine and glutamine (Fig. 5). The oxidized methionines were probably, but not certainly, artifacts. Amino acid residues containing thioethers are easily oxidized during protein purification, derivatization, and/or digestion (40, 41).

However, the two deamidations in the phosphopeptide Tyr326–Arg346 could be biologically relevant. Deamidation is a modification formed both naturally and artificially. As a natural post-translational modification, it has been described in the context of protein aging and cataract formation (42, 43) and G-protein regulation (44). A deamidating enzyme has been described in Escherichia coli (45). Artificial deamidation can occur spontaneously under both acidic and alkaline conditions (46). We attempted to test the biological relevance by comparing the prediction score for threonine phosphorylation for the predicted peptide with Asn and Gln and the modified peptide containing Asp and Glu (Table II). The modified peptide with two extra negative charges gave a much higher prediction score than the encoded peptide implying that the modification in vivo could facilitate the recognition by a specific protein kinase.

The Physiological Role of FDH—We found a marked increase of FDH activity relative to aerobic respiration (monitored as CCO activity) at lower oxygen concentrations in the potato tubers (Fig. 9). This implies that FDH has a role in hypoxic metabolism, which is consistent with the observation that FDH synthesis is induced under hypoxic (36) or anoxic (20) conditions.

The physiological role of FDH in plants is uncertain as discussed by Hourton-Cabassa et al. (36) mainly because the source of formate is uncertain (21). One possible source of formate is pyruvate provided that the enzyme formate C-acetyltransferase (EC 2.3.1.54 [EC] ), also called pyruvate formate-lyase (PFL), is present. In microorganisms and mitochondria from unicellular algae, the enzyme is strictly anaerobic, but although fairly strongly hypoxic conditions are observed inside potato tubers (Fig. 9) (37), the conditions in the tubers are far from anoxic. Still, we searched the Arabidopsis data base for genes that might encode PFL. That did not give significant hits so instead we searched for the conserved local sequence motif in PFL and found a gene containing it in Arabidopsis (Fig. 10).



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  		FIG. 10.
Tentative identification of PFL in plants by in silico search for homologues to the conserved local sequence motif in PFL (52). Results of NCBI Conserved Domain Database search (53) with reverse position-specific BLAST (54) are shown. NCBI accession numbers of the aligned PFL sequences from different organisms are indicated. The consensus sequence was computed from nine imported alignments processed at NCBI (four of them are displayed and also Chlamydomonas reinhardtii PFL sequence was added) and represents the most frequently occurring residue in each column. Aligned and unaligned residues are shown as uppercase and lowercase letters, respectively. The conserved identical amino acid residues are shown in boldface, and similar residues are underlined.

 

If PFL is present in potato tubers, the combined action of PFL and FDH gives the same net result as that of PDH alone as shown in Reactions 1, 2, 3,

(1)

(2)

(3)
where Reaction 1 is catalyzed by PFL; Reaction 2 is catalyzed by FDH, and Reaction 3 is catalyzed by PDH. This means that PFL + FDH can perform the same function as PDH and produce acetyl-CoA that is used further in the tricarboxylic acid cycle. It would therefore make sense to have them regulated in the same way (see above). This additional PFL pathway may contribute to the total glycolytic flux especially under stress conditions.


   FOOTNOTES
 
* This work was supported by grants from the Danish Agricultural and Veterinary Research Council (to N. V. B. and I. M. M.) and from the Danish Natural Science Research Council (to O. N. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Present address: Dept. of Physics and Astronomy, University of Manitoba, 301 Allen Bldg., Winnipeg, Manitoba R3T 2N2, Canada. Back

|| To whom correspondence should be addressed: Plant Research Dept., Risø National Laboratory, Bldg. 301, P. O. Box 49, DK-4000 Roskilde, Denmark. Tel.: 45-46-77-42-13; Fax: 45-46-77-41-22; E-mail: ian.max.moller{at}risoe.dk.

1 The abbreviations used are: PDH, pyruvate dehydrogenase; BN, Blue Native; CCO, cytochrome c oxidase; FDH, formate dehydrogenase; IEF, isoelectric focusing; LC, liquid chromatography; MOPS, 4-morpholinopropanesulfonic acid; PFL, pyruvate formate-lyase; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; LC/MS, liquid chromatography/mass spectrometry; ESI, electrospray ionization; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; IMAC, immobilized metal affinity chromatography. Back


   ACKNOWLEDGMENTS
 
We are grateful to Ina Blom Hansen and Søren Andersen for excellent technical assistance, to Unisense, Århus, Denmark for the generous loan of oxygen-monitoring equipment, and to The Danish Biotechnology Instrument Center for supporting protein mass spectrometry research at the University of Southern Denmark.



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