Planta (2013) 237:1571–1583 DOI 10.1007/s00425-013-1866-4
ORIGINAL ARTICLE
ATP produced by oxidative phosphorylation is channeled toward hexokinase bound to mitochondrial porin (VDAC) in beetroots (Beta vulgaris) Flor C. Alca´ntar-Aguirre • Alicia Chagolla Axel Tiessen • John Paul De´lano • Luis Eugenio Gonza´lez de la Vara
•
Received: 17 December 2012 / Accepted: 26 February 2013 / Published online: 17 March 2013 Ó Springer-Verlag Berlin Heidelberg 2013
Abstract Mitochondrial porins or voltage-dependent anion channels (VDAC) are the main route for solute transport through outer mitochondrial membranes (OMM). In mammals, hexokinase (HK) binds to VDAC, which allows the channeling of ATP synthesized by oxidative phosphorylation toward HK. In plants, although HK has been found associated with OMM, evidence for an interaction with VDAC is scarce. Thus, in this work, we studied the physical and functional interaction between these proteins in beetroot mitochondria. To observe a physical interaction between HK and VDAC, OMM presenting HK activity were prepared from purified mitochondria. Protein complexes were solubilized from OMM with mild detergents and separated by centrifugation in glycerol gradients. Both HK activity and immunodetected VDAC were found in small (9S–13S) and large ([40S) complexes. OMM proteins were also separated according to their hydropathy by serial phase partitioning with Triton X-114. Most of HK activity was found in hydrophobic fractions where VDAC was also present. These results indicated that HK could be bound to VDAC in beetroot mitochondria. The functional interaction of HK with VDAC was demonstrated by
Electronic supplementary material The online version of this article (doi:10.1007/s00425-013-1866-4) contains supplementary material, which is available to authorized users. F. C. Alca´ntar-Aguirre A. Chagolla J. P. De´lano L. E. Gonza´lez de la Vara (&) Departamento de Biotecnologı´a y Bioquı´mica, Cinvestav, Unidad Irapuato, Km 9.6 Libramiento Norte, C.P. 36821 Irapuato, Guanajuato, Mexico e-mail:
[email protected] A. Chagolla A. Tiessen Departamento de Ingenierı´a Gene´tica, Cinvestav, Unidad Irapuato, Irapuato, Mexico
observing the effect of apyrase on HK-catalyzed glucose phosphorylation in intact mitochondria. Apyrase, which hydrolyzes freely soluble ATP, competed efficiently with hexokinase for ATP when it was produced outside mitochondria (with PEP and pyruvate kinase), but not when it was produced inside mitochondria by oxidative phosphorylation. These results suggest that HK closely interacts with VDAC in beetroot mitochondria, and that this interaction allows the channeling of respiratory ATP toward HK through VDAC. Keywords Metabolic channeling Energy metabolism Outer mitochondrial membrane proteins Plant mitochondria Abbreviations DDM Dodecylmaltoside HK Hexokinase LC–MS/MS Liquid chromatography coupled to tandem mass spectrometry MS Mass spectrometry OMM Outer mitochondrial membrane(s) PK Pyruvate kinase PMSF Phenylmethylsulfonyl fluoride SUS Sucrose synthase VDAC Voltage-dependent anion channel (mitochondrial porin)
Introduction Plant metabolism is complex, redundant, flexible and more compartmented than the metabolism in animal cells (Heldt 2005; Lunn 2007). In non-photosynthetic tissues, energy is
123
1572
obtained through the degradation of organic molecules, mainly carbohydrates. Sucrose, provided by photosynthesis, is cleaved by the action of reversible sucrose synthases (SUS) or of invertases (Koch 2004). Glucose produced by these latter enzymes is then phosphorylated by hexokinase (HK). This enzyme phosphorylates glucose to produce glucose-6-phosphate (glucose-6P), using ATP as phosphate-group donor. Glucose-6P can then enter several metabolic pathways such as glycolysis, the pentose phosphate pathway (to produce pentoses and/or NADPH) or sugar nucleotide biosynthesis (Galina and da Silva 2000; Wilson 2003). In plants, metabolism is often regulated by compartmentation. Many reactions can occur in parallel and, in certain cases, the mass action ratios of substrates and products are specific for each subcellular compartment (Tiessen et al. 2002, 2012). The subcellular location and the enzymatic source of nucleotides can also be relevant for metabolic regulation. For example, in Arabidopsis seeds it has been shown that glucose nucleotides produced by SUS favor starch synthesis in the amyloplast but not so much lipid or oil synthesis (Angeles-Nunez and Tiessen 2010). Adenine or uridine feeding also had differential effects on primary metabolism (Loef et al. 1999; Florchinger et al. 2006). Plant cell metabolism can also be regulated through the physical coupling of subsequent reactions: a mechanism known as metabolic channeling. Through it, the product of one reaction is directly used as substrate by the next enzyme. For instance, it is known that SUS binds to the cellulose synthasome to obtain UDP and supply UDPglucose (Delmer 1999; Koch 2004). Metabolic channeling also occurs within enzymes, where a reaction intermediate migrates from the site of its formation to that of its utilization through an intra-molecular tunnel. Typical examples are the enzymes that use glutamine as ammonia donor, such as glutamate synthases (Vanoni and Curti 2008). A special case of metabolic channeling has been observed in mammalian cells. Here, HK binds to mitochondria through an interaction with the channel its substrate (ATP) flows through (Fiek et al. 1982; Linden et al. 1982; Nakashima et al. 1986). This channel, named mitochondrial porin or voltage-dependent anion channel (VDAC), is the most abundant protein in the outer mitochondrial membrane (OMM). The binding of HK to VDAC involves mainly the hydrophobic amino terminal end of HK, which can form an a-helix that has been proposed to be inserted into the membrane (Xie and Wilson 1988), or inside the VDAC pore (Rosano 2011). Of special interest is the interaction of VDAC with HK-II in cancer cells. Here, the ATP produced by oxidative phosphorylation inside mitochondria is preferentially channeled toward the HK-II bound to VDAC. This produces glucose-6P which is used
123
Planta (2013) 237:1571–1583
mainly to form lactate under aerobic conditions (‘‘Warburg effect’’; Pedersen 2008). Because of this, chemicals that disrupt the VDAC-HK interaction, like methyl-jasmonate, have been proposed as drugs for cancer therapy (Goldin et al. 2008). In higher plant cells, there are two main types of HKs: type-A, which are located in chloroplast’s stroma, and type-B, which are mainly bound to mitochondria (Claeyssen and Rivoal 2007; Granot 2008). In addition, there are soluble cytosolic HKs with biochemical properties different from either plastidic or mitochondria-bound ones (Miernyk and Dennis 1983; Galina et al. 1995; da-Silva et al. 2001). The function of these HK types is also different: HKs bound to mitochondria appear to be involved in sugar nucleotide synthesis, while cytosolic HK produce mainly glucose-6P for glycolysis in maize root cells (Galina and da Silva 2000; da-Silva et al. 2001). Most type-B HKs have a hydrophobic segment at their N-termini (Karve et al. 2010), which has led to presume that the physical interaction of HK with OMMs proceeds through VDAC as in animal cells. However, the amino-acid sequences of these segments, although conserved among plant HKs (Claeyssen and Rivoal 2007), are different from N-terminal sequences in animal HKs. This fact has led to the proposal that, in plants, HK binding to mitochondria does not involve VDAC (Rezende et al. 2006). In addition, the information about a possible physical or functional interaction of plant HKs with VDAC is scarce (Miernyk and Dennis 1983; Balasubramanian et al. 2007, Camacho-Pereira et al. 2009). In non-photosynthetic plant tissues, ATP is produced either in the cytosol by substrate-level phosphorylation in glycolysis, or in the mitochondria, by oxidative phosphorylation. Since these tissues receive free glucose and fructose resulting from invertase activity, cytosolic HKs (fructo- or glucokinases) may consume large amounts of ATP (da-Silva et al. 2001). It is thus interesting to know the source of ATP used by mitochondria-bound HKs in plant non-photosynthetic cells. To address this problem, we chose the beetroot (Beta vulgaris L.) as our model system, since it is a heterotrophic organ that accumulates sucrose and hexoses in the cytosol and vacuole. Moreover, beetroot cell metabolism is dependent on respiration, since beetroots are particularly sensitive to hypoxia (Petraglia and Poole 1980). In addition, we can have access to large amounts of fresh tissue at all seasons. We therefore decided to study the possible physical interaction between VDAC and hexokinase in beetroot OMM. We found evidence suggesting the existence protein complexes in OMM containing both VDAC and HK. In addition, our results support the functional importance of the interaction between these proteins, since it allows the metabolic channeling of ATP toward HK in this crop species.
Planta (2013) 237:1571–1583
1573
Materials and methods Plant material and chemicals Beetroots were obtained fresh at a local market and used immediately or kept on moist towels at 4 °C used, usually 2 days later. Chemicals and enzymes obtained from Sigma (St Louis, MO, USA) unless cated otherwise.
were until were indi-
Preparation of mitochondria and outer mitochondrial membranes Beetroot mitochondria were prepared by homogenizing 720 g of beetroots with 1,080 ml of a medium containing 300 mM sucrose, 4 mM dithiothreitol (DTT), 3 mM ethylene-diamine tetraacetic acid (EDTA), 0.1 % bovine serum albumin (BSA), 0.5 % polyvinyl-polypyrrolidone (insoluble PVP), 0.2 mM phenylmethylsulfonyl fluoride (PMSF) and 70 mM Tris–HCl (pH 8.0), in a blender for 10 s. This homogenate was filtered through eight layers of cheesecloth and centrifuged at 3,000 rpm (1,4009g) for 20 min in a JA-14 rotor (Beckman Coulter, Inc., Indianapolis IN, USA). The supernatant was then collected and centrifuged for 20 min at 9,000 rpm (12,4009g). Each pellet obtained was suspended in 2 ml of washing medium [300 mM mannitol, 10 mM MOPS–KOH buffer (pH 7.4), 1 mM EDTA and 0.1 % BSA]. These mitochondria were centrifuged at 3,500 rpm (1,5009g) for 10 min in a Beckman JA-20 rotor. The pellet was discarded and the supernatant was centrifuged at 9,000 rpm (9,8009g) for 10 min in the same rotor. The pellet was resuspended in 4 ml of washing medium. This mitochondrial suspension was mixed with 36 ml of 28 % (w/v) Percoll in 300 mM sucrose, 10 mM potassium phosphate buffer (pH 7.2), 1 mM EDTA and 0.1 % BSA, and centrifuged at 18,000 rpm (39,0009g) for 1 h at 4 °C in a Beckman JA-20 rotor. Purified mitochondria were collected from the light-brown broad band near the top of the Percoll gradient formed during the centrifugation. This fraction was well mixed with 15 times its volume of washing medium and centrifuged at 9,000 rpm (9,8009g) for 15 min (Douce et al. 1987; Camacho-Pereira et al. 2009). The pellets contained purified mitochondria. Outer mitochondrial membranes were prepared from purified mitochondria essentially as described by Mannella (1987). Purified mitochondria-containing pellets were suspended in 16 ml of a hypotonic solution (lysis medium: 0.25 mM EDTA, 0.25 mM EGTA, pH 7.0 set with NaOH). Enough mannitol was added to this suspension to give a 5 % solution, which was then placed on ice and submitted to five 30-s pulses of sonication. A 17/27 % (w/w; 10 ml of each) step sucrose gradient was prepared in 10 mM
potassium phosphate buffer (pH 7.0), 0.25 mM EDTA and 0.25 mM EGTA. The lysed mitochondria suspension was applied on top of this gradient before centrifuging it at 23,000 rpm (95,0009g) for 1 h in a Beckman SW28 rotor. OMMs were collected separately from both interphases, diluted to 36 ml with 5 mM Tris–Mes buffer (pH 6.5) containing 2 mM DTT and centrifuged at 23,000 rpm for 15 min in the same rotor. The pellet was suspended in a small volume of 25 mM Tris–Mes (pH 7.5), 2 mM EDTA, 1 mM DTT, and 45 % (v/v) glycerol, and kept at -70 °C for a few days, until used. Hexokinase activity and protein content of each OMM preparation were determined. Preparations presenting highest HK-specific activity, usually those from the top (lysed mitochondria suspension sample/17 % sucrose) interphases, were selected for subsequent work. Hexokinase activity assay Hexokinase activity was determined in a medium containing 50 mM Tris–HCl (pH 7.4), 6 mM MgCl2, 1 mM glucose, 1 mM ATP, 2 mM PEP, 0.1 % Triton X-100, 0.3 mM b-NADP?, 6 U ml-1 pyruvate kinase (PK) and 1 U ml-1 glucose-6P dehydrogenase (NADP-dependent, from Saccharomyces cerevisiae). The reaction was started by adding mitochondria, OMMs, or protein fractions obtained from them. Triton X-100 was omitted in fractions already containing detergents (Zwittergent 3-14, dodecyl maltoside or Triton X-114). Glucose-6P production was determined by measuring the NADPH formation through its absorption at 340 nm, using an extinction coefficient of 6.22 mM-1. Electrophoresis and immunodetection The electrophoretic separation of proteins was performed in tricine-SDS gels as described by Scha¨gger and von Jagow (1987). When fractions from serial phase partitions with Triton X-114 were loaded into gels, this detergent was replaced with SDS as described (Gonza´lez de la Vara and Lino Alfaro 2009). After electrophoresis, proteins in gels were either fixed and stained with Coomassie Blue (Scha¨gger and von Jagow 1987) or blotted onto polyvinylidene difluoride (PVDF) membranes. VDAC was immunodetected in these membranes as described (Gonza´lez de la Vara and Lino Alfaro 2009). The primary antibody was anti-Arabidopsis thaliana VDAC (from Agrisera, Va¨nna¨s, Sweden) used at a dilution of 1:5,000. Blots were revealed with both 5-bromo-4-chloro3-indolyl phosphate and nitro blue tetrazolium (Gonza´lez de la Vara and Medina 1990) or with the Pierce ECL system (Thermo Fisher Scientific, Inc., Rockford, IL, USA) following the instructions of the manufacturer.
123
1574
Planta (2013) 237:1571–1583
Centrifugation in glycerol gradients
Analysis of proteins by mass spectrometry
To separate protein complexes according to their masses, OMM were suspended at a final concentration of 0.32 mg of protein per ml in 0.5 ml of a medium containing 30 mM Hepes–Tris buffer (pH 7.0), 2 mM EDTA and 1 mM DTT. To this suspension, 20 % solution of the detergents Zwittergent 3-14 (Calbiochem Merck KGaA, Darmstadt, Germany) or dodecylmaltoside (DDM) was added slowly to get a protein: detergent relation of 1:1. These mixtures were applied onto 20–42 % (w/w) 4.5-ml linear glycerol gradients prepared in the medium mentioned above. These gradients were centrifuged at 52,000 rpm for 4.7 h in a Beckman SW55Ti rotor in x2t mode (x2t = 5 9 1011 s-1). After centrifugation, gradients were collected in 0.5-ml fractions. In each fraction, glycerol concentration (through its refraction index), protein content and hexokinase activity were measured. Proteins in these fractions were analyzed by SDS-PAGE and VDAC immunodetection. Sedimentation coefficients corresponding to protein complexes in each fraction were calculated from glycerol concentrations and x2t values as described (Young 1984).
Sample preparation and in-gel digestion of proteins
Serial phase partitioning with Triton X-114 To separate membrane proteins according to their hydropathy, these were solubilized with Triton X-114 and subjected to nine-step serial phase partitionings as described (Gonza´lez de la Vara and Lino Alfaro 2009). Briefly, OMM were diluted to 1 mg of protein per ml in 25 mM potassium phosphate buffer (pH 7.8), 2 mM EDTA, 0.2 mM PMSF and 3 % (v/v) Triton X-114. This mixture was placed in an ice bath and stirred gently for 25 min, before raising the temperature of the sample to 25 °C in a water bath until the suspension became cloudy. It was then centrifuged at 15,000 rpm (21,0009g) for 10 min at 25 °C (in an IEC Micromax RF tabletop centrifuge) to get two phases. The upper phase was removed and placed in a tube with ‘‘clean’’ lower phase (without membrane proteins) and ‘‘clean’’ upper phase was added to the lower phase with membrane proteins. These new mixtures were stirred at 0 °C for 15 min before raising the temperature to 25 °C and centrifuging them as before. After this centrifugation, the upper phase of the second tube was placed in a third tube with clean lower phase, the upper phase of the first tube was transferred onto the lower phase left in the second tube, and clean upper phase was added to the first tube. These three new mixtures were gently stirred in an ice bath for 10 min, incubated at 25 °C until they became cloudy, and centrifuged as described. This procedure was repeated until ten fractions (numbered from 0 to 9) were obtained. Final fractions were mixed at 0 °C and stored at -70 °C until used.
123
Phase-partitioning fractions with proteins to be analyzed by mass spectrometry were concentrated by precipitation. Fifty- or 100-ll aliquots were treated with the Pierce CleanUp kit (Thermo Fisher Scientific, Inc.) following the manufacturer instructions. Pellets were dissolved in electrophoresis sample buffer. Proteins were then separated by tricine-SDS-PAGE, and the gel was stained with Coomassie Blue. Protein bands of interest were cut from this gel, and gel fragments were washed with water, then with 50 % (v/v) acetonitrile in water, with acetonitrile mixed with 100 mM ammonium bicarbonate, and finally with 100 % acetonitrile as described (Lino et al. 2006). Proteins in gel fragments were then reduced, carbamidomethylated and digested with trypsin. The resulting peptides were extracted from the gel as described by Shevchenko et al. (1996). Mass spectrometric analysis of peptides Selected proteins were identified by sequencing their peptides. These were separated by HPLC in an Accela (Thermo Fisher Scientific, Inc.) instrument with a Picofrit C18 column, run with a linear 3–70 % acetonitrile gradient in 0.1 % formic acid (over 15 min; flow rate 600 nl min-1). Resolved peptides were analyzed in an LTQ-Velos ion-trap mass spectrometer (Thermo Fisher Scientific, Inc.) with a nanospray ion source. The most abundant peptides were fragmented by collision-induced dissociation (CID) and pulsed CID (PQD) in a cycle producing approximately one fragmentation spectrum per second. The spectrometer was controlled by Xcalibur 2.1 software. Data treatment The sequences of analyzed peptides were obtained manually from their fragmentation spectra with Xcalibur 2.1 software and compared with those in databases using the MS-Blast search program at http://genetics.bwh.harvard. edu/msblast/ (Shevchenko et al. 2001). Assessment of channeling of ATP to hexokinase To assess if there is a functional interaction between VDAC and HK, the channeling of ATP toward this enzyme was estimated by comparing the effects of apyrase on glucose-6P production, with ATP formed either by the ATP synthase inside beetroot mitochondria or outside they with added PK, phospho-enol-pyruvate (PEP) and ADP. These assays were performed in a enzymatic 0.5-ml coupled assay system containing 0.3 M mannitol, 3 mM MgSO2, 10 mM NaCl, 0.1 % (w/v) fatty-acid-free BSA,
Planta (2013) 237:1571–1583
0.15 mM ADP, 1 mM glucose, 0.3 mM b-NADP and 0.5 U ml-1 S. cerevisiae glucose-6P dehydrogenase in 50 mM Tris–HCl buffer (pH 7.4). Percoll gradient-purified functional mitochondria prepared just before the experiments, as described above, were added to this mixture at a final 0.1 mg ml-1 protein concentration. When ATP was produced outside mitochondria (to simulate a no-channeling scenario), the reaction mixture additionally contained 2 mM PEP. In those cases, glucose-6P production was started by adding 0.6 U ml-1 of PK, and changes in absorbance at 340 nm due to the production of NADPH were recorded every 15 s in a Beckman DU640 spectrophotometer. When a constant glucose-6P production rate was attained, few microliters of a 4.5 U ml-1 apyrase (Sigma, Grade VI, from white potato. Dissolved at 450 U ml-1 in 50 % glycerol buffered with 10 mM Tris– HCl, pH 7.4 and kept at 4 °C) solution were successively added, and the rates after each addition were recorded (Fig. 3c). When ATP was produced inside mitochondria by oxidative phosphorylation (to allow and observe a possible channeling of ATP toward VDAC-bound HK), 5 mM potassium phosphate was included in the medium. Glucose6P production was started by adding 10 mM potassium succinate. Then, small successive apyrase amounts were added, and glucose-6P production rates recorded, as described above and in Fig. 3d. The effect of apyrase on glucose-6P rates was also observed in microplate assays. Glucose 6P production with ATP produced outside mitochondria was observed in reaction mixtures (200 ll) containing 0.3 M mannitol, 20 mM Hepes–Tris buffer (pH 7.4), 6 mM MgSO4, 10 mM NaCl, 5 mM KH2PO4, 0.1 % fatty-acid-free BSA, 0.6 U ml-1 PK, 3 mM PEP, 0.2 mM ADP, 1 mM glucose, 0.3 mM b-NADP?, 0.5 U ml-1 glucose-6P dehydrogenase and the amounts of apyrase indicated in Fig. 4b. When ATP had to be produced inside mitochondria, 10 mM potassium succinate was added instead of PK and PEP. Reactions were started by adding recently prepared mitochondria (at a protein concentration of 0.1 mg ml-1) to each well. Glucose-6P relative production rates were calculated from A340 readings (taken every 30 s) obtained in a Synergy 2 microplate reader with Gen5 analysis software (Bio Tek Instruments, Inc., Winooski, VT, USA). Protein determination Protein concentration was determined according to Stoschek (1990) with BSA as standard. Reproducibility of the results All the experiments described in the figures were repeated at least three times with different preparations. Results
1575
were very similar, and those from a typical experiment are shown in each figure.
Results Beetroots, being heterotrophic plant organs very sensitive to anoxia, are dependent on mitochondrial respiration for their energy needs. We therefore chose beetroots as a source of active mitochondria. These mitochondria were also useful to prepare partially purified OMM. HK activity was present in all mitochondria and OMM preparations obtained. This enzyme, unlike HK bound to mammalian mitochondria, could not be detached from beetroot mitochondria with clotrimazole (up to 50 lM) or with glucose-6P (up to 5 mM; data not shown). Protein complexes containing VDAC and HK in outer mitochondrial membranes To know if HK is bound to VDAC in plant OMM, the latter were prepared from beetroot mitochondria and protein complexes were then extracted using mild detergents. These complexes were subsequently separated, either according to their size, by centrifugation in glycerol gradients, or according to their hydropathy, by serial phase partitioning with Triton-X114. Two different detergents were used to extract protein complexes from OMM: Zwittergent 3:14 and dodecylmaltoside (DDM). Proteins extracted with them were separated according to their complex sizes by centrifugation in glycerol gradients. Here, a value for the sedimentation coefficient was calculated for each fraction collected. In addition, HK activity was measured and VDAC was immunodetected in each fraction. In Fig. 1a, it is shown that, when Zwittergent 3-14 was used to extract proteins from OMM, most of HK activity is recovered in fractions with protein complexes with sedimentation coefficients about 12S. These values correspond to proteins with molecular masses near 200 kDa; HK (with a monomer mass of about 50 kDa) was thus forming mainly small complexes. In addition, a smaller HK peak appeared at 43S (approximate estimated mass 2,000 kDa). HK monomers, appearing in 4S fractions, were also observed. VDAC, on the other hand, was immunodetected in all fractions in proteins extracted with Zwittergent 3-14, but higher amounts were observed in fractions with 12S and heavier protein complexes (Fig. 1c). When DDM was employed to extract protein complexes, the highest HK activity peak appeared centered at 11S (Fig. 1b). A peak at 42S was also observed, but very little HK activity was found in the 3S and 7S fractions, where HK monomers were expected. VDAC was also barely observed in fractions with 7S and lighter proteins. On the contrary, in the 11S fraction and in fractions with
123
1576
Planta (2013) 237:1571–1583
Fig. 1 Hexokinase activity is found in VDAC-containing multimeric complexes. Beetroot mitochondria were isolated and outer mitochondrial membranes (OMM) prepared as described in ‘‘Materials and methods’’. OMM protein complexes were solubilized with Zwittergent 3-14 (1:1 protein:detergent, a, c) or dodecylmaltoside (1:1 protein:detergent, b, d) and centrifuged in a 20–42 % glycerol
gradient at 52,000 rpm for 4.7 h. The hexokinase activity of each 0.5-ml fraction was measured and shown in a and b. Proteins in a 10-ll aliquot of each fraction were separated by SDS-PAGE, blotted and immunodetected with anti-A. thaliana VDAC antibodies (c, d). Sedimentation coefficients are given in Svedberg (S) units
heavier protein complexes, VDAC was conspicuously immunodetected (Fig. 1d). These results indicate that both VDAC and HK are located at OMM mainly forming complexes. The observation that there was always large VDAC amounts in fractions with peak HK activity suggests that these two proteins could be bound together in beetroot OMM. In agreement with these results, when OMM protein complexes were extracted with DDM and separated by blue-native PAGE (Wittig et al. 2006), VDAC was found, by both immunodetection and MS, in complexes with masses of 200- and 600 kDa. In the 200-kDa complex, HK peptides were also found by MS (data not shown). Protein complexes in OMM were also separated according to their hydropathy by serial phase partitioning with the detergent Triton X-114. Micelle solutions of this detergent have the property of separating in two phases above a temperature threshold (22 °C). Membrane proteins extracted with this detergent at a low temperature distribute themselves between the phases formed when the temperature is raised above the threshold. Hydrophobic proteins are concentrated in the lower (detergent-rich) phase, whereas hydrophilic ones are found mainly in the upper phase. If this partition is repeated as described above, a series of fractions with increasingly hydrophilic proteins is obtained. Each protein, or protein complex, distributes itself in several fractions according to its hydropathy following the binomial distribution law (Gonza´lez de la Vara and Lino Alfaro 2009).
Beetroot OMM proteins were analyzed using this method. In Fig. 2a, an SDS-PAGE gel image is shown with the proteins in each fraction of a nine-step serial partition. In fraction 0, having the most hydrophobic proteins, a protein band with a molecular mass of 33 kDa (identified immunologically as VDAC) was most conspicuous. VDAC was also immunodetected, although in lower amounts, in fraction 1 and in trace amounts in the following fractions (Fig. 2c). This behavior was expected for a very hydrophobic protein. HK activity was measured in serial phase partitioning fractions. It was distributed in two peaks, which fitted binomial distribution functions as expected. The first peak, centered about fraction 1, is the expected for a hydrophobic protein complex [with a probability (p) of being at the upper hydrophilic phase of 0.164]. This peak contained 61 % of the HK activity found in all fractions. The remaining 39 % of the HK activity was found in a peak about fraction 6, with a calculated p value of 0.645 (Fig. 2b). This p value indicated that, in this second peak, HK presents a more hydrophilic character. These results can be explained postulating that in the first (hydrophobic) HK activity peak, HK is bound to a hydrophobic membrane protein, probably VDAC, while in the second peak it is bound to OMM through weak hydrophobic interactions. It is also possible that this activity peak could be due to HK detached from VDAC by the detergent Triton X-114, which is used necessarily at high concentrations.
123
Planta (2013) 237:1571–1583
1577
Fig. 2 Hexokinase is found in hydrophobic protein fractions where VDAC is present. A membrane fraction enriched in outer mitochondrial membranes (OMM) was extracted with 3 % (w/w) Triton X-114, and their proteins were separated into different fractions (0–9) according to their hydropathy by serial phase partitioning. Fraction 0 (where p, the probability of finding a protein in the upper hydrophilic phase, is zero) contains the most hydrophobic proteins and fraction 9 (p = 1) the most hydrophilic ones. Proteins in 100-ll aliquots from each fraction were precipitated and separated by SDS-PAGE. a A Coomassie Blue-stained gel is shown. Protein bands in brackets and
numbered are those analyzed by ion-trap MS (see Table 1). Positions of molecular weight markers (in kDa) are at the left margin. b Hexokinase activity in fractions with different hydropathy. The solid line is the best-fitting curve formed by the sum of two binomial distribution curves with p values of 0.164 and 0.645, respectively (broken lines). c Protein immunoblot using an anti-VDAC antibody. Ten microliters of each fraction were applied to an SDS-PAGE gel before proteins were separated, blotted and immunodetected. OM outer (mitochondrial) membrane
Proteins in fractions within the hydrophobic peak of HK activity were identified by LC–MS/MS. As shown in Table 1, the 33-kDa protein in fraction 1 was identified as VDAC. Comparing the sequences of six abundant peptides in this protein band with those in NCBInr database, the best match obtained was with spinach VDAC (with a significant score of 494). Proteins with masses about 50 kDa in fraction 2 appeared to be mainly inner membrane abundant proteins (ATP synthase or respiratory complex III subunits). However, in a *55-kDa protein band (Fig. 2a, band 3), two peptides significantly matching with those of spinach HK were found. These results confirmed the presence of HK in hydrophobic protein complexes; supporting thus the existence of protein complexes containing both VDAC and HK in plant mitochondria.
phosphatase that hydrolyzes ATP, on the glucose-6P production dependent on the ATP formed by oxidative phosphorylation. We expected that apyrase could compete with HK for ATP if it diffused out the mitochondria into the mitochondrial outer medium (i.e. the cytosol, in the cell) before reaching HK. This condition was simulated, as depicted in Fig. 3a, by providing ATP to hexokinase externally with PEP and PK, and observing the HK-catalyzed glucose-6P production rate. Apyrase was then added (in increasing amounts) and its effect on this rate recorded. If, otherwise, the physical proximity of VDAC with HK allowed an efficient channeling of ATP produced by oxidative phosphorylation toward HK (Fig. 3b), we expected no, or little, competition of apyrase for ATP with HK. In this case, succinate was provided as a respiratory substrate to allow ATP synthesis by the ATP synthase in the mitochondrial matrix. Freshly prepared mitochondria that showed an increase in their oxygen consumption rate with ADP (respiratory control), were used for these experiments. An example of a typical experimental trace with an external source of ATP and one with ATP produced by respiration are shown in
Assessment of channeling of ATP toward hexokinase bound to mitochondria To evaluate the physiological importance of the proximity between VDAC and HK, we evaluated the existence of channeling by observing the effects of apyrase, a
123
1578
Planta (2013) 237:1571–1583
Table 1 Ion-trap MS identification of beetroot OMM proteins Protein band
gi ID number
1
2 3
1256259
13959067 11386886 525291
587564
Identified proteins
MS-Blast scorea 494
Voltage-dependent anion channel protein (Spinacia oleracea)
Mitochondrial-processing peptidase (Avicennia marina) Hexokinase 1 (Spinacia oleracea) ATP synthase beta subunit (Triticum aestivum)
Mitochondrial-processing peptidase (Solanum tuberosum)
Peptides m/z 511.01
BGPGlYTDlGK
654.69
BKGElFlADVSTK
679.36
lVYDllYR
819.2
EDlSGEVDTSAlEK
974.84
BFTNTTFTSNGVAlTSTGTK
128
485.6
BVTVlPDGlR
195
708.7 664.19
BlSTDPTTAAQlVAK NlGTGTNAAYVER
772.75
BVTAlVNDTVGTlAGGR
318?
488.62
BlGlFGGAGVGK
517.01
BTVlGSVDDVK
279?
587.56
BVVDllAPYQR
632.1
BTlAMDGTEGVlR
712.76
BVlNTGSPMTYQGR
495.07
BlDAVDASAVK
630.02
BMGDVPVAEDlGR
708.69 1,281.39 4
266567
Mitochondrial-processing peptidase subunit alpha (Solanum tuberosum)
Sequenceb
276?
BlSTDPTTAAQlVAK TSQlVASDPVNFTGSPMR
724.14
EAVGGNVTASASR
883.78
VPEmVEllVDSVR
977.34 1,020.29
YAVATPGPMTQlQlDR ATGNEQFVAENYTAPR
a
Scores obtained with the sequences shown. When the score has a plus sign (?), more sequences were obtained, but not considered
b
Sequences given in one letter aminoacid code. B: K or R, lowercase l: I or L, lowercase m: oxidized M
Fig. 3c and d, respectively. When ATP was provided externally (i.e. after adding PK and PEP to mitochondria), glucose 6-P production (measured as an increase of NADPH absorbance) was clearly observed. This production rate decreased, as expected, when adding increasing amounts of apyrase (Fig. 3c). On the other hand, when ATP was produced by oxidative phosphorylation (with succinate as substrate), much higher apyrase amounts had to be added to respiring mitochondria to get only a small decrease in the glucose-6P production rate (Fig. 3d). Glucose-6P production rates in Fig. 3c and d were calculated and plotted against the apyrase added (Fig. 4a). Here, it is clearly shown that, when ATP was provided externally to HK, \0.07 U of apyrase was enough to bring glucose-6P production rate to almost zero. In contrast, when ATP was produced by oxidative phosphorylation, this same amount of apyrase was unable to decrease glucose-6P production (actually, an increase in its rate was observed). To rule out the possibility that the declining rates of glucose-6P production observed were not due to apyrase, but to the decay of mitochondria or to substrate exhaustion, all necessary reagents and different apyrase amounts were
123
added from the start to reaction media in microplate wells. Glucose-6P production was then measured simultaneously after adding mitochondria. Relative glucose-6P production rates were plotted against the apyrase in the reaction medium (Fig. 4b). In these experiments, very similar results to that in Fig. 4a were obtained: apyrase caused a marked decrease in glucose-6P production rates only when ATP was provided externally. When ATP was produced by oxidative phosphorylation, larger amounts of apyrase were needed to get a significant decrease in glucose-6P production. Finally, we observed that ATP produced in respiration is channeled only to HK bound to mitochondria. When soluble yeast HK was added to respiring mitochondria, it can utilize the ATP produced by them, and an increase in the glucose-6P production rate was observed. This increase was sensitive to apyrase addition, as expected for an enzyme not having preferential access to respiratory ATP (Fig. S1 in Online resource 1). All these results indicate that HK is functionally bound to VDAC in beetroot mitochondria. The proximity of these two proteins allows the channeling of ATP produced inside mitochondria through VDAC directly to HK.
Planta (2013) 237:1571–1583
1579
Fig. 3 Channeling of ATP produced by oxidative phosphorylation toward VDAC-bound hexokinase. To assess if ATP produced by respiration is channeled through the outer membrane porin (VDAC) toward hexokinase (HK), the effect of apyrase on glucose-6P production was observed. When there is no channeling, as depicted in a, ATP goes to the outside of mitochondria before interacting with HK to produce glucose-6P. This condition is simulated here by providing ATP externally through the reaction of PEP with ADP catalyzed by added pyruvate kinase (PK). In this case, it is expected that added apyrase (that hydrolyzes external ATP and ADP) competes with HK for ATP, thus decreasing glucose-6P production. In this condition, both outer membrane (OM) and inner membrane (IM) solute transporters: the succinate/fumarate carrier (SFC), the phosphate carrier (PIC) and the adenine nucleotide translocator (ANT), as well as the electron transport chain (ETC) and the ATP synthase are
inactive. If, otherwise, ATP is directly channeled to HK, as depicted in b, apyrase would be unable to hydrolyze it, thereby permitting glucose-6P production to remain unaffected by the presence of this hydrolase. c, d Glucose-6P production was measured using an enzymatic coupled assay system with glucose-6P dehydrogenase and NADP where the amount of NADPH produced was plotted against time. When indicated, apyrase (Ap) was added to the reaction mixture at the amounts pointed out in parentheses (microliters of a solution containing 4.5 U ml-1). In c, PK was added to a reaction mixture containing PEP, ADP and mitochondria (Mit) at the times indicated, to provide an external source of ATP; simulating a no-channeling condition. d Succinate (Succ) was added as a respiratory substrate (to a reaction mixture containing ADP and Pi) to fuel respiratory production of ATP. Here, the occurrence of channeling of ATP to HK was inferred since apyrase did not affect glucose-6P production as much as in c
Discussion
In plant cells, metabolic compartmentation becomes more important because these cells possess unique organelles (i.e. plastids). However, metabolic pathways in every compartment depend on other parts of the cell for supplies of energy (ATP) or metabolic precursors, and some pathways actually involve the activity of enzymes in different compartments (Lunn 2007).
Hexokinase is associated with outer mitochondrial membranes in several plant species Intracellular compartmentation of different metabolic pathways is a characteristic feature of eukaryotic cells.
123
1580
Fig. 4 ATP produced by oxidative phosphorylation is channeled toward VDAC-bound hexokinase. Glucose-6P production was measured by including glucose-6P dehydrogenase and NADP in reaction media and monitoring absorbance changes at 340 nm. All reaction media also included glucose (1 mM) and ADP (0.15 mM). When an external source of ATP was provided to simulate a no-channeling condition (open circles), PEP was also included. To produce ATP by oxidative phosphorylation inside mitochondria, Pi was included (solid circles). a Glucose-6P production rates of the experiment shown in Fig. 3c and d are plotted against total apyrase added. In a nochanneling condition, PK was added to start the production of ATP outside mitochondria. In a channeling condition, respiration was started with succinate to produce ATP inside mitochondria. b Complete reaction mixtures (with all appropriate substrates and the indicated amounts of apyrase, in 0.18 ml) were placed in 96-well microplates. Reactions were started by adding mitochondria (20 ll) and recording the reduction of NADP (i.e. the production of glucose-6P). Controls run in these experiments (and results observed) were: no mitochondria (no NADP reduction); mitochondria substituted with yeast hexokinase and ATP produced with PEP ? PK (NADP reduction was inhibited by apyrase); no glucose (no NADP reduction). 5 lM CCCP in reaction mixtures (when ATP was produced with PEP ? PK, NADP reduction was inhibited by apyrase; when ATP was produced by respiration, NADP reduction without apyrase decreased more than 70 %). Finally, soluble yeast HK added to respiring ATP-producing mitochondria (NADP reduction rate was increased; this increase was sensitive to apyrase)
Metabolic compartmentation is maintained mainly by the enclosure of (mostly soluble) enzymes inside membrane-bound organelles. However, a different kind of
123
Planta (2013) 237:1571–1583
compartmentation arises when the enzymes involved in a pathway physically associate themselves to allow metabolic channeling of substrates and products. These physical associations can be facilitated by the binding of enzymes to membranes. For instance, it has been proposed that all glycolytic enzymes are bound to mitochondria in Arabidopsis cells so that pyruvate, the product of this pathway, can be directly channeled into mitochondria as a respiratory substrate (Giege´ et al. 2003). The association of HK with mitochondria has been observed in several animal cells. Only HKs possessing a conserved hydrophobic segment at their N-termini (type Ior type II-HKs) bind to mitochondria, whereas type III-HK, not having this segment, do not (Wilson 2003). Most probably, the interaction of HK with mammalian mitochondria in vivo is transient, since it can be disrupted by metabolites like ATP or glucose-6P at physiological concentrations (Arora and Pedersen 1988; Rezende et al. 2006), and regulated by phosphorylation and OMM lipid composition (Pastorino and Hoek 2008). In plants, the association of HK with mitochondria has been observed in many plant species and organs (Giege´ et al. 2003; Claeyssen and Rivoal 2007; Damari-Weissler et al. 2007). This association appears to be more permanent in plant than in animal mitochondria. For instance, it has been observed that, in maize and rice root mitochondria, HK is bound to mitochondria, and that this union cannot be disrupted either with glucose-6P or with clotrimazole or thiopental, which are pharmacological agents that detach HK from rat brain mitochondria (Rezende et al. 2006). Similarly, HK could not be detached from beetroot mitochondria with glucose-6P or clotrimazole (data not shown). It has also been proposed that, in plant cells, non-plastidic HK is bound to mitochondria, with little or no HK left soluble in the cytosol (Granot 2008). In agreement with this, it has been reported that two (AtHXK1 and AtHXK2) out of the four presumably active hexokinases encoded in the A. thaliana genome are bound to mitochondria (Karve et al. 2008). However, soluble cytosolic HKs with biochemical properties different from those of mitochondriabound HKs have been found in Ricinus seeds and maize roots (Miernyk and Dennis 1983; Galina et al. 1995). In addition, genes encoding HKs lacking an N-terminal hydrophobic segment, thus presumably cytosolic, have been found in cereal and moss genomes (Karve et al. 2010, Nilsson et al. 2011). Our results are in agreement with reported experimental results indicating that mitochondria-bound HK is located at the outer membrane. However, obtaining very pure OMM is difficult because contaminating proteins from other cell compartments, especially from the protein-rich inner mitochondrial membranes, are hard to remove. In this article, for instance, inner mitochondrial membrane
Planta (2013) 237:1571–1583
proteins were always present in OMM preparations (Table 1), even though all OMM preparations we obtained were enriched in HK activity and VDAC content. To overcome these restrictions, a careful study was carried out in Arabidopsis by Duncan et al. (2011). In it, several lines of evidence were considered to distinguish true OMMresident proteins from contaminants. They identified 42 bona fide OMM proteins. Among these were the products of four VDAC genes, as well as three active HK isoforms and the inactive one HXKL-1. Hexokinase forms membrane protein complexes with VDAC in beetroot mitochondria It is generally accepted that HK binds to mammalian mitochondria because of its high affinity for VDAC (Nakashima et al. 1986). In contrast, the evidence favoring the binding of HK to VDAC in plant mitochondria is scarce or even contradictory. On one side, taking into account that the hydrophobic N-terminus of plant and mammalian HKs are different (Claeyssen and Rivoal 2007) and that plant HKs behave differently than animal HK with chemicals affecting its binding to mitochondria, it has been proposed that the binding mechanism of plant HKs to OMM is different from that of animal HKs (i.e. it could not involve VDAC; Rezende et al. 2006). On the other side, HK could be removed from Ricinus communis mitochondria with 2 mM glucose-6P (Miernyk and Dennis 1983); as observed in mammalian mitochondria. In addition, the N-terminus of A. thaliana HXK1 has been found to be necessary and sufficient for the binding of HK to mitochondria. Finally, VDAC1 and VDAC3 interact with HXK1 in immunocapture and co-immunoprecipitation experiments (Balasubramanian et al. 2007). To find a protein complex in beetroot OMM containing both VDAC and HK, we extracted OMM proteins with two mild detergents and separated the extracted proteins by centrifugation in glycerol gradients. We observed that VDAC was found in all fractions with protein complexes having sedimentation coefficients larger than 8S, whereas HK was found mainly in a peak with proteins with sedimentation coefficients about 11–12S (Fig. 1). These results agree with those of Hoogenboom et al. (2007), who found, by atomic-force microscopy, that VDAC could form aggregates of several different sizes (monomers, tetramers, hexamers and higher oligomers) in potato tuber OMM. VDAC complexes of different sizes have also been observed in Arabidopsis mitochondria by 2D blue-native electrophoresis (Klodmann et al. 2011). In that study, 471 mitochondrial proteins were identified by mass spectrometry and displayed in a ‘‘map’’ available online (http:// www.gelmap.de/arabidopsis_mito). VDAC was identified in 15 complexes, with highest scores in those with
1581
molecular masses of about 180- and 110 kDa. In agreement with our results, HK (mainly HXK1) appeared forming part of small complexes with molecular masses of 77-, 115- and 163 kDa. All these results suggest that the existence of complexes containing both VDAC and HK in plant OMMs is possible. This possible association of VDAC with HK was also studied by separating OMM proteins according to their hydropathy. This was achieved by serial phase partitioning with Triton X-114. This method has been used to separate beetroot plasma membrane proteins, and to analyze the hydropathy of the calcium-dependent protein kinases present in them (Gonza´lez de la Vara and Lino Alfaro 2009). Using this method to analyze beetroot OMMs, VDAC was found, both by immunodetection and LC–MS/MS, in the first hydrophobic fractions; as expected for an intrinsic membrane protein. HK activity, on the other hand, appeared in two peaks: the larger one in hydrophobic fractions and the other in middle (amphipathic) ones (Fig. 2). HK was expected to be an amphipathic protein due to its N-terminal hydrophobic domain. Its presence in hydrophobic fractions can thus be explained if it interacts with a hydrophobic protein, probably VDAC. These results then add to the evidence suggesting that HK binds to mitochondria through VDAC. Membrane proteins prepared by serial phase partitioning with Triton X-114 can be identified by mass spectrometry (Gonza´lez de la Vara and Lino Alfaro 2009 and unpublished results). In this work, VDAC was identified unambiguously as a *30-kDa hydrophobic protein. HK peptides were found in a hydrophobic *55-kDa protein band, which confirm the results of HK activity measurements suggesting the existence of HK in hydrophobic protein complexes in beetroot OMM. Channeling of ATP produced by oxidative phosphorylation toward hexokinase is allowed by its proximity with VDAC Many methods used to observe the association of membrane proteins involve extracting protein complexes with mild detergents. However, they could alter the aggregation status of membrane proteins during their extraction. Because of this, methods for observing interactions of membrane proteins without using detergents are preferred, especially to assess the physiological relevance of these interactions. In mammalian mitochondria, many experimental results indicate that there is a channeling of ATP transported through VDAC toward the HK bound to it. In hepatoma cells, which contained a high amount of HK bound to mitochondria, it was observed that glucose-6P formation was higher when ATP was produced inside mitochondria
123
1582
than when produced in the cytosol. In addition, with [c-32P]ATP added to mitochondria, the specific radioactivity of the glucose-6P produced was lower than that of the ATP in the medium under phosphorylating conditions (where unlabeled ATP was produced from 31Pi inside mitochondria by oxidative phosphorylation), whereas in non-phosphorylating mitochondria, the specific radioactivity of glucose-6P and ATP was the same (Arora and Pedersen 1988). These results were expected if HK bound to mitochondria preferentially consumed ATP produced in the mitochondrial matrix by oxidative phosphorylation. In agreement with these results, it was observed that respiring brain mitochondria phosphorylated glucose at a rate that was independent of the external (added) ATP concentration (BeltrandelRio and Wilson 1992; Wilson 2003). Moreover, this rate was not altered when phosphorylating liver mitochondria (which had little or no bound HK, and delivered the ATP they produced to the medium) were mixed with phosphorylating brain mitochondria (reviewed in Wilson 2003). In contrast with all the above results, there are no experiments designed to observe the possible channeling of ATP produced by respiration toward the HK bound to plant mitochondria. We thus devised an experiment in which the ATP released at the outside of mitochondria was distinguished from that channeled to HK through VDAC, by adding apyrase. It was expected that ATP in the medium would be hydrolyzed by apyrase, whereas that produced in the mitochondrial matrix and channeled to VDAC-bound HK would be not. Our results indicated that most of the glucose-6P produced by HK bound to respiring beetroot mitochondria utilized mainly ATP produced by oxidative phosphorylation, since apyrase lowered the rate of glucose-6P production only when added at high amounts. In contrast, the glucose-6P production rate was greatly decreased by small amounts of apyrase when ATP was delivered outside mitochondria; or when glucose-6P production was catalyzed by added yeast HK, which does not attach to mitochondria. A close functional interaction between VDAC and HK in plant mitochondria could allow, besides a direct provision of ATP to HK, the removal of the ADP produced by it to be phosphorylated again by the ATP synthase. In agreement with this, it was observed in potato tuber mitochondria that glucose increased their respiratory rate, the same way as ADP did. This increase was sensitive to atractyloside, oligomycin and mannoheptulose, inhibitors of the adenine nucleotide translocator (ANT), the ATP synthase and HK, respectively. This suggested that active HK provided the ADP needed to accelerate the respiration. In addition, it was observed that HK bound to mitochondria was much more effective in increasing the respiratory rate than unbound HK, since yeast HK had to be added at higher concentrations than naturally bound HK to attain the
123
Planta (2013) 237:1571–1583
same effect on the respiratory rate (Camacho-Pereira et al. 2009). In conclusion, the results in this article provide evidence suggesting that HK is physically and functionally bound to VDAC in beetroot OMM. This formation of a multi-heteromeric protein complex could allow the linkage of the respiratory activity to the activation of glucose by HK. Acknowledgments We thank Ba´rbara Lino, Marisol Piceno and Rocı´o Medina for their help. This work was supported by Consejo Nacional de Ciencia y Tecnologı´a (Conacyt, Mexico) by granting a postgraduate fellowship to FC Alca´ntar and funding basic-science projects conducted by L. Gonza´lez, J.P. Delano and A. Tiessen.
References Angeles-Nunez JG, Tiessen A (2010) Arabidopsis sucrose synthase 2 and 3 modulate metabolic homeostasis and direct carbon towards starch synthesis in developing seeds. Planta 232:701–718 Arora KK, Pedersen PL (1988) Functional significance of mitochondrial bound hexokinase in tumor cell metabolism. Evidence for preferential phosphorylation of glucose by intramitochondrially generated ATP. J Biol Chem 263:17422–17428 Balasubramanian R, Karve A, Kandasamy M, Meagher RB, Moore Bd (2007) A role for F-actin in hexokinase-mediated glucose signaling. Plant Physiol 145:1423–1434 BeltrandelRio H, Wilson JE (1992) Interaction of mitochondrially bound rat brain hexokinase with intramitochondrial compartments of ATP generated by oxidative phosphorylation and creatine kinase. Arch Biochem Biophys 299:116–124 Camacho-Pereira J, Meyer LE, Bender ML, Fernandes OM, Galina A (2009) Reactive oxygen species production by potato tuber mitochondria is modulated by mitochondrially bound hexokinase activity. Plant Physiol 149:1099–1110 Claeyssen E, Rivoal J (2007) Isozymes of plant hexokinase: occurrence, properties and functions. Phytochemistry 68:709– 731 Damari-Weissler H, Ginzburg A, Gidoni D, Mett A, Krassovskaya I, Weber APM, Belausov E, Granot D (2007) Spinach SoHXK1 is a mitochondria-associated hexokinase. Planta 226:1053–1058 da-Silva WS, Rezende GL, Galina A (2001) Subcellular distribution and kinetic properties of cytosolic and non-cytosolic hexokinases in maize seedling roots: implications for hexose phosphorylation. J Exp Bot 52:1191–1201 Delmer DP (1999) Cellulose biosynthesis: exciting times for a difficult field of study. Annu Rev Plant Physiol Plant Mol Biol 50:245–276 Douce R, Bourguignon J, Brouquisse R, Neuburger M (1987) Isolation of plant mitochondria: general principles and criteria of integrity. Methods Enzymol 148:403–415 Duncan O, Taylor NL, Carrie C, Eubel H, Kubiszewski-Jakubiak S, Zhang B, Narsai AR, Millar H, Whelan J (2011) Multiple lines of evidence localize signaling, morphology, and lipid biosynthesis machinery to the mitochondrial outer membrane of Arabidopsis. Plant Physiol 157:1093–1113 Fiek C, Benz R, Roos N, Brdiczka D (1982) Evidence for identity between the hexokinase-binding protein and the mitochondrial porin in the outer membrane of rat liver mitochondria. Biochim Biophys Acta 688:429–440 Florchinger M, Zimmermann M, Traub M, Neuhaus HE, Mohlmann T (2006) Adenosine stimulates anabolic metabolism in
Planta (2013) 237:1571–1583 developing castor bean (Ricinus communis L.) cotyledons. Planta 223:340–348 Galina A, da Silva WS (2000) Hexokinase activity alters sugarnucleotide formation in maize root homogenates. Phytochemistry 53:29–37 Galina A, Reis M, Albuquerque MC, Go´mez Puyou A, Gomez Puyou MT, de Meis L (1995) Different properties of the mitochondrial and cytosolic hexokinases in maize roots. Biochem J 309: 105–112 Giege´ P, Heazlewood JL, Ressner-Tunali U, Millar H, Fernie AR, Leaver CJ, Sweetlove LJ (2003) Enzymes of glycolysis are functionally associated with the mitochondrion in Arabidopsis cells. Plant Cell 15:2140–2151 Goldin N, Arzoine L, Heyfets A, Israelson A, Zaslavsky Z, Bravman T, Bronner V, Notcovich A, Shoshan-Barmatz V, Flescher E (2008) Methyl jasmonate binds to and detaches mitochondriabound hexokinase. Oncogene 27:4636–4643 Gonza´lez de la Vara LE, Lino Alfaro M (2009) Separation of membrane proteins according to their hydropathy by serial phase partitioning with Triton X-114. Anal Biochem 387:280–286 Gonza´lez de la Vara LE, Medina G (1990) Phosphorylation by inorganic phosphate of the plasma membrane H?-ATPase from red beet (Beta vulgaris L.). Plant Physiol 94:1522–1527 Granot D (2008) Putting plant hexokinases in their proper place. Phytochemistry 69:2649–2654 Heldt HW (2005) Plant biochemistry, 3rd edn. Elsevier Academic Press, London. ISBN: 0-12-088391-033 Hoogenboom BW, Suda K, Engel A, Fotiadis D (2007) The supramolecular assemblies of voltage-dependent anion channels in the native membrane. J Mol Biol 370:246–255 Karve A, Rauh BL, Xia X, Kandasamy M, Meagher RB, Sheen J, Moore Bd (2008) Expression and evolutionary features of the hexokinase gene family in Arabidopsis. Planta 228:411–425 Karve R, Lauria M, Virnig A, Xia X, Rauh BL, Moore Bd (2010) Evolutionary lineages and functional diversification of plant hexokinases. Mol Plant 3:334–346 Klodmann J, Senkler M, Rode C, Braun HP (2011) Defining the protein complex proteome of plant mitochondria. Plant Physiol 157:587–598 Koch K (2004) Sucrose metabolism: regulatory mechanisms and pivotal roles in sugar sensing and plant development. Curr Opin Plant Biol 7:235–246 Linden M, Gellerfors P, Nelson BD (1982) Pore protein and the hexokinase-binding protein from the outer membrane of rat liver mitochondria are identical. FEBS Lett 141:189–192 Lino B, Carrillo-Rayas MT, Chagolla A, Gonza´lez de la Vara LE (2006) Purification and characterization of a calcium-dependent protein kinase from beetroot plasma membranes. Planta 225: 255–268 Loef I, Stitt M, Geigenberger P (1999) Orotate leads to a specific increase in uridine nucleotide levels and a stimulation of sucrose degradation and starch synthesis in discs from growing potato tubers. Planta 209:314–323 Lunn JE (2007) Compartmentation in plant metabolism. J Exp Bot 58:35–47 Mannella CA (1987) Isolation of the outer membrane of plant mitochondria. Methods Enzymol 148:453–464 Miernyk JA, Dennis DT (1983) Mitochondrial, plastid, and cytosolic isozymes of hexokinase from developing endosperm of Ricinus communis. Arch Biochem Biophys 226:458–468 Nakashima RA, Mangan PS, Colombini M, Pedersen PL (1986) Hexokinase receptor complex in hepatoma mitochondria:
1583 evidence from N,N0 -dicyclohexylcarbodiimide-labeling studies for the involvement of the pore-forming protein VDAC. Biochemistry 25:1015–1021 Nilsson A, Olsson T, Ulfstedt M, Thelander M, Ronne H (2011) Two novel types of hexokinases in the moss Physcomitrella patens. BMC Plant Biol 11:32 Pastorino JG, Hoek JB (2008) Regulation of hexokinase binding to VDAC. J Bioenerg Biomembr 40:171–182 Pedersen PL (2008) Voltage dependent anion channels (VDACs): a brief introduction with a focus on the outer mitochondrial compartment’s roles together with hexokinase-2 in the ‘‘Warburg effect’’ in cancer. J Bioenerg Biomembr 40:123–126 Petraglia T, Poole RJ (1980) Effect of anoxia on ATP levels and ion transport rates in red beet. Plant Physiol 65:973–974 Rezende GL, Logullo C, Meyer L, Machado LB, Oliveira-Carvalho AL, Zingali RB, Cifuentes D, Galina A (2006) Partial purification of tightly bound mitochondrial hexokinase from maize (Zea mays L.) root membranes. Braz J Med Biol Res 39:1159–1169 Rosano C (2011) Molecular model of hexokinase binding to the outer mitochondrial membrane porin (VDAC1): implication for the design of new cancer therapies. Mitochondrion 11:513–519 Scha¨gger H, von Jagow G (1987) Tricine-sodium dodecyl sulfate polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368–379 Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal Chem 68:850–858 Shevchenko A, Sunyaev S, Loboda A, Shevchenko A, Bork P, Ens W, Standing KG (2001) Charting the proteomes of organisms with unsequenced genomes by MALDI-quadrupole time-of-flight mass spectrometry and BLAST homology searching. Anal Chem 73:1917–1926 Stoschek CM (1990) Increased uniformity in the response of the coomassie blue G protein assay to different proteins. Anal Biochem 184:111–116 Tiessen A, Hendriks JHM, Stitt M, Branscheid A, Gibon Y, Farre EM, Geigernberger P (2002) Starch synthesis in potato tubers is regulated by post-translational redox modification of ADPglucose pyrophosphorylase: a novel regulatory mechanism linking starch synthesis to the sucrose supply. Plant Cell 14: 2191–2213 Tiessen A, Nerlich A, Faix B, Hu¨mmer C, Fox S, Trafford K, Weber H, Weschke W, Geigernberger P (2012) Subcellular analysis of starch metabolism in developing barley seeds using a nonaqueous fractionation method. J Exp Bot 63:2071–2087 Vanoni MA, Curti B (2008) Structure–function studies of glutamate synthases: a class of self-regulated iron–sulfur flavoenzymes essential for nitrogen assimilation. IUBMB Life 60:287–300 Wilson JE (2003) Isozymes of mammalian hexokinase: structure, subcellular localization and metabolic function. J Exp Biol 206:2049–2057 Wittig I, Braun H-P, Scha¨gger H (2006) Blue native PAGE. Nat Protoc 1:418–428 Xie GC, Wilson JE (1988) Rat brain hexokinase: the hydrophobic N-terminus of the mitochondrially bound enzyme is inserted in the lipid bilayer. Arch Biochem Biophys 267:803–810 Young BD (1984) Measurement of sedimentation coefficients and computer simulation of rate-zonal separations. In: Rickwood D (ed) Centrifugation. A practical approach, 2nd edn. IRL Press, Oxford, pp 127–155. ISBN: 0-9004147-55X
123
