Báo cáo khoa học: L-Lactate metabolism in potato tuber mitochondria docx

L-Lactate metabolism in potato tuber mitochondria Gianluca Paventi, Roberto Pizzuto, Gabriella Chieppa and Salvatore Passarella Dipartimento di Scienze per la Salute, Universita ` del Molise, Campobasso, Italy According to the Davies–Roberts hypothesis, plants primarily respond to oxygen limitation by a burst of l-lactate production ([1] and refs there in). The acidifica- tion of the cytoplasm during the first phase of anaerobi- osis arising from lactic fermentation results in inhibition of lactate dehydrogenase (LDH) and activation of pyruvate decarboxylase [2]. As a result, a switch from lactic to ethanolic fermentation occurs. In those organ- isms that cannot switch to ethanolic fermentation, when oxygen falls below 1%, glycolysis is stimulated and l-lactate accumulates [3], leading to decreased cytoplasmic pH and cell death [4,5]. Thus, according to the Davies– Roberts concept, cytoplasmic acidification potentially induces damage and death of intolerant plants. Because of the damage that can arise from l-lactate accumulation, a cellular safety valve to minimize that damage is to be expected. It has been consistently repor- ted that metabolism of l-lactate in potato after a period of anoxia is accompanied by a two-fold increase in LDH activity and by the induction of two LDH iso- zymes [6]. These observations related to l-lactate meta- bolism occurring in the cytoplasm involved pyruvate formation via LDH, and further pyruvate metabolism, both in mitochondria and in the cytoplasm. There is rea- son to suspect, however, that mitochondria themselves may be involved in l-lactate metabolism. This is based on our previous work, which has shown that l-lactate is transported into the organelles isolated from both rat Keywords L-lactate; L-lactate dehydrogenase; mitochondrial transport; plant mitochondria; shuttle Correspondence S. Passarella, Dipartimento di Scienze per la Salute, Universita ` del Molise, Via De Sanctis, 86100 Campobasso, Italy Fax: +39 0 874 404778 Tel: +39 0 874 404868 E-mail: passarel@unimol.it (Received 2 August 2006, revised 20 December 2006, accepted 10 January 2007) doi:10.1111/j.1742-4658.2007.05687.x We investigated the metabolism of l-lactate in mitochondria isolated from potato tubers grown and saved after harvest in the absence of any chemical agents. Immunologic analysis by western blot using goat polyclonal anti- lactate dehydrogenase showed the existence of a mitochondrial lactate dehydrogenase, the activity of which could be measured photometrically only in mitochondria solubilized with Triton X-100. The addition of l-lac- tate to potato tuber mitochondria caused: (a) a minor reduction of intra- mitochondrial pyridine nucleotides, whose measured rate of change increased in the presence of the inhibitor of the alternative oxidase salicyl hydroxamic acid; (b) oxygen consumption not stimulated by ADP, but inhibited by salicyl hydroxamic acid; and (c) activation of the alternative oxidase as polarographically monitored in a manner prevented by oxamate, an l-lactate dehydrogenase inhibitor. Potato tuber mitochondria were shown to swell in isosmotic solutions of ammonium l-lactate in a stereo- specific manner, thus showing that l-lactate enters mitochondria by a pro- ton-compensated process. Externally added l-lactate caused the appearance of pyruvate outside mitochondria, thus contributing to the oxidation of extramitochondrial NADH. The rate of pyruvate efflux showed a sigmoidal dependence on l-lactate concentration and was inhibited by phenylsucci- nate. Hence, potato tuber mitochondria possess a non-energy-competent l-lactate ⁄ pyruvate shuttle. We maintain, therefore, that mitochondrial metabolism of l-lactate plays a previously unsuspected role in the response of potato to hypoxic stress. Abbreviations AOX, alternative oxidase; COX IV, subunit IV of cytochrome oxidase; FCCP, carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone; LDH, L-lactate dehydrogenase; PTM, potato tuber mitochondria; SHAM, salicyl hydroxamic acid. FEBS Journal 274 (2007) 1459–1469 ª 2007 The Authors Journal compilation ª 2007 FEBS 1459 heart [7] and liver [8] and metabolized there. Moreover, a major role for the mitochondrial LDHs in the transfer of reducing equivalents from the cytosol to the respirat- ory chain (lactate shuttle) was also proposed [7]. In order to ascertain whether and how energy meta- bolism, and in particular l-lactate metabolism, can change as a result of spontaneous hypoxia in plants, we used potato, which is an important crop whose tubers show a high sensitivity to O 2 deprivation [3]. We show here for the first time the existence of LDH in isolated potato tuber mitochondria (PTM). This enzyme is localized in the inner mitochondrial compartments and uses NADP + as a cofactor, the product, NADPH, being oxidized essentially by the alternative oxidase (AOX), which is activated by pyru- vate. The latter can also exit from the mitochondria in a novel l-lactate ⁄ pyruvate shuttle operating in a non- energy-competent manner. Results The existence of LDH in mitochondria isolated from potato tubers In order to verify the occurrence of LDH in PTM, use was made of goat polyclonal antibodies raised against LDH, which have already been shown to cross-react with LDHs from different species [9–11]. Solubilized mitochondrial proteins were analyzed by SDS ⁄ PAGE, blotted onto poly(vinylidene difluoride) membrane, and then probed with the antibody to LDH. In agreement with Hondred & Hanson [12], LDH protein was visual- ized as a single band with a molecular mass of about 39 kDa. A typical experiment is reported in Fig. 1, which shows clearly the presence of LDH in the mitochondrial fraction. Confirmation of this site of ori- gin was provided by use of a specific antibody against subunit IV of the cytochrome c oxidase (COX IV). A band corresponding to a protein of molecular mass 35 kDa was observed; this is likely to arise from an aggregate of COX IV (13 kDa [13]) and other unidenti- fied protein ⁄ s, as already shown in pea mitochondria [14]. The occurrence of respirasomes in potato mito- chondria has been recently reported [15], making poss- ible the occurrence of aggregates not separated in the SDS ⁄ PAGE procedure. Whatever its origins, the lack of this band in the cytosolic fraction showed that the 35 kDa band is specific for PTM and not a technical artefact. In the same experiment, it was shown that the PTM fraction did not contain b-tubulin, a protein restricted to the cytoplasm, thus ruling out the possibil- ity that the LDH detected arose from cytosolic contam- ination. Contamination by other particulate ⁄ membrane fractions was also ruled out, as we used purified mito- chondria free of subcellular contamination (see Experi- mental procedures). The cytosolic fraction was free of mitochondrial COX IV, showing that minimal rupture of PTM had occurred during isolation. The intactness of the mit- ochondrial outer membrane was measured as in Douce et al. [16], and found to be 95%. In addition, we found negligible fumarase activity, a plant mitochondrial marker [17], in suspensions of mitochondria, thus fur- ther confirming the intactness of the inner membrane. To establish where LDH is localized within the mitochondria and whether it is active, LDH was assayed photometrically by measuring the absorbance decrease of NADH [18] in the presence of pyruvate in isolated PTM. When PTM (0.1 mg protein) were incu- bated in the presence of NADH (0.2 mm), oxidation occurred, catalyzed by external NADPH dehydro- genases (Fig. 2A). The constant rate of decrease in absorbance (about 130 nmolÆmin )1 Æmg protein) remained unchanged when pyruvate (10 mm) was added; that is, the LDH was not accessible to sub- strates. Consistently, no NADH formation was found in the presence of 10 mml-lactate (not shown). In order to rule out the possibility that l-lactate is oxidized on the external face of the inner membrane, with electrons transferred to the inner surface, intact PTM were assayed for LDH activity by using phena- zine methosulfate and dichloroindophenol (Fig. 2B), as in Atlante et al. [19]. A negligible decrease in dichloro- indophenol absorbance at 600 nm was found when l-lactate (10 mm) was added to the PTM, either in the absence or in the presence of 1 mm NAD + , confirming the absence of LDH activity in the outer membrane, in Fig. 1. Immunodetection of mitochondrial LDH. Solubilized protein (30 and 40 lg) from both mitochondrial and cytosolic fractions was analyzed by western blot as described in Experimental procedures. Membrane blots were incubated with polyclonal anti-LDH, anti- COX IV and anti-b-tubulin. COX IV and b-tubulin were used as mit- ochondrial and cytosolic markers, respectively. L-Lactate metabolism in PTM G. Paventi et al. 1460 FEBS Journal 274 (2007) 1459–1469 ª 2007 The Authors Journal compilation ª 2007 FEBS the intermembrane space or on the outer side of the mitochondrial inner membrane, or in any contamin- ation of the mitochondrial suspension. Addition of LDH externally produced a rapid decrease in absorp- tion by dichloroindophenol. To validate the experi- mental protocol that we had used, we confirmed that addition of 0.3 mm glycerol 3-phosphate to PTM in the presence of phenazine methosulfate and dichloroin- dophenol resulted in a decrease of dichloroindophenol absorbance with a rate of about 22 nmolÆmin )1 Æmg )1 protein, arising from the activity of glycerol 3-phos- phate dehydrogenase (EC 1.1.1.8), which is located on the outer side of the mitochondrial inner membrane (Fig. 2B,a). On the other hand, no oxidation of succi- nate by succinate dehydrogenase (which is located on the matrix side of the inner mitochondrial membrane) occurred with intact PTM. Oxidation did occur after the addition of 0.1% Triton X-100, which solubilized the mitochondrial membranes and allowed the interac- tion between dichloroindophenol and the succinate dehydrogenase complex (Fig. 2B,b). To confirm that LDH is located in the internal mit- ochondrial compartments, i.e. in the inner face of the mitochondrial membrane or in the matrix, PTM were solubilized with Triton X-100 (0.2%). Added NADH (0.2 mm) was oxidized at a rate of about 105 nmolÆ min )1 Æmg )1 protein, but when pyruvate was added, this rate increased to about 170 nmolÆmin )1 Æmg )1 protein (Fig. 2C), showing that LDH is present in the inner mitochondrial compartments. The kinetic characteristics of the LDH reaction were studied by determining the dependence of the rate of oxidation of NADH on increasing concentrations of externally added pyruvate in solubilized mitochondria Fig. 2. Mitochondrial LDH activity assay in PTM. (A) PTM (0.1 mg) were incubated in 2 mL of the standard medium (see Experi- mental procedures) containing 200 l M NADH, and the absorbance (A 340 ) was con- tinuously monitored. Pyruvate (PYR, 10 m M) was added at the time indicated by the arrow. The numbers alongside the traces refer to the rate of oxidation of NADH in nmolÆmin )1 Æmg )1 protein. (B) PTM (0.2 mg) were incubated in 2 mL of standard medium in the presence of phenazine methosulfate (PMS) (30 l M) plus dichloroindophenol (50 l M), either in the presence or in the absence of NAD + , and the absorbance (A 600 ) was continuously monitored. At the times indicated by the arrows, L-lactate ( L-LAC, 10 mM) and LDH (0.1 eu) were added. The insets show control experi- ments: at the times indicated by the arrows, glycerol 3-phosphate (G3P, 0.3 m M) (a) and succinate (SUCC, 5 m M) and Triton X-100 (0.2%) (b) were added to mitochondria trea- ted with phenazine methosulfate and dichlo- roindophenol. Numbers along the curves are rates of L-lactate, succinate or glycerol 3-phosphate oxidation expressed as nmol dichloroindophenol reducedÆmin )1 Æmg )1 pro- tein. (C) PTM solubilized with Triton X-100 (0.2%) were incubated in 2 mL of the stand- ard medium, containing 200 l M NADH, and the absorbance (A 340 ) was continuously monitored. Pyruvate (1.5 m M) was added at the time indicated by the arrow. The num- bers alongside the traces refer to the rate of oxidation of NADH in nmolÆmin )1 Æmg )1 protein. G. Paventi et al. L-Lactate metabolism in PTM FEBS Journal 274 (2007) 1459–1469 ª 2007 The Authors Journal compilation ª 2007 FEBS 1461 (Fig. 3). Saturation kinetics were found with a K m value of 0.63 ± 0.14 mm; the V max value was 85 ± 7 nmolÆ min )1 Æmg )1 sample protein. Unfortunately, spontaneous oxidation of the NADH formed during the oxidation of l-lactate prevented assay with l-lactate and NAD + as the substrate pair. L-Lactate metabolism in mitochondria Uptake and metabolism of l-lactate was further inves- tigated in a set of experiments carried out with isolated coupled PTM. The assumption here is that the mitoch- ondrial LDH is devoted to oxidation of l-lactate rather than reduction of pyruvate, as the latter would be immediately oxidized by the pyruvate dehydroge- nase complex (K m ¼ 0.06 mm [20]). l-Lactate metabo- lism was monitored by determining the ability of externally added l-lactate to reduce intramitochondrial dehydrogenase cofactors. In this case, we resorted to fluorimetric techniques that have previously been used to monitor changes in the redox state of pyridine nu- cleotides [21]. Reduction of mitochondrial NAD(P) + was found to occur at a rate of 0.19 nmolÆmin )1 Æmg )1 protein when l-lactate was added to PTM previously incubated with or without the uncoupler carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone (FCCP) and then treated with cyanide (CN – ) (not shown). The observed rate of reduction was, however, likely to be underestimated, as the newly formed NAD(P)H would be rapidly oxidized by the mitochondrial AOX, which is usually activated by pyruvate, the product of l-lac- tate metabolism. Hence, we checked whether inhibition of AOX would cause an increase in the measured rate of pyridine nucleotide reduction. To achieve this, use was made of salicyl hydroxamic acid (SHAM, 1 mm), an AOX inhibitor [22]. Addition of SHAM resulted in a 150% increase in the measured rate of NAD(P)H formation (Fig. 4A,a). Consistently, addition of l-lac- tate to PTM previously incubated with SHAM caused an increase in the rate of NAD(P)H formation (Fig. 4A,b). In both cases, the addition of oxamate (10 mm), an inhibitor of LDHs [23], completely blocked the increase in fluorescence. The failure of NADH, newly synthesized during l-lactate oxidation, to be oxidized in the cytochrome pathways was confirmed in another experiment (inset to Fig. 4), in which we checked whether addition of l-lactate to PTM could produce an increase in the membrane potential as measured by using safranine O as a fluorimetric probe. In contrast to succinate (5 mm) and d-lactate (10 mm), l-lactate (10 mm) failed to generate a change in electrical membrane potential, DY. As expected, externally added FCCP (1 lm) caused membrane potential collapse. In the same experiment, we investigated, as in Pastore et al. [24], whether l-lactate itself could activate AOX, and obtained the results shown in Fig. 4B. In this case, succinate was added to the mitochondria, fol- lowed by ADP. Oxygen consumption via the electron transfer chain was then blocked with CN – , and finally l-lactate was added either in the absence (a) or presence (b) of oxamate. In the former case, oxygen consump- tion was restored, but in the latter, l-lactate addition failed to restore oxygen uptake, thus showing that l-lactate itself was not responsible for AOX activation. It is likely that in the absence of oxamate, activation of AOX was due to the newly formed pyruvate. In a par- allel experiment, the ability of l-lactate to cause oxygen uptake by PTM was investigated. We found that addi- tion of 10 mml-lactate resulted in oxygen uptake at a rate of 20 nmol O 2 Æmin )1 Æmg )1 protein. As expected, this uptake was not stimulated by 0.2 mm ADP, and was completely prevented following addition of SHAM (not shown). Control experiments showed that SHAM did not affect O 2 uptake due to either NADH or succi- nate in the absence of CN – (not shown). L-Lactate transport in PTM The experiments reported above raise the question of how l-lactate produced in the cytosol can cross the mitochondrial membrane. To gain insight into this, swelling experiments were carried out as in de Bari et al. [25]; the results are shown in Fig. 5. PTM Fig. 3. Assay of LDH activity in PTM solubilized with Triton X-100. Pyruvate was added at the indicated concentrations to PTM treated with Triton X-100 (0.2%). The rates (v o ) of NADH oxidation, calcula- ted as difference of rate in traces (b) and (a) of Fig. 2C, are expressed as nmol pyruvate reducedÆmin )1 Æmg )1 protein. L-Lactate metabolism in PTM G. Paventi et al. 1462 FEBS Journal 274 (2007) 1459–1469 ª 2007 The Authors Journal compilation ª 2007 FEBS suspended in 0.18 m ammonium l-lactate showed spontaneous swelling, but with a rate and to an extent significantly lower than those found with ammonium d-lactate, as judged by statistical analysis of five swell- ing experiments using Student’s t-test (P<0.02). This shows that both d-lactate and l-lactate can enter PTM, but that the uptake is stereospecific. The results indicate that l-lactate enters mitochondria in a proton- compensated manner. The metabolite transport para- digm proposed in Passarella et al. [21] suggests that net carbon uptake by mitochondria is accompanied by efflux of newly synthesized compound ⁄ s. We wished to determine whether this applies in the case of l-lactate. In particular, in the light of the occurrence of an l-lac- tate ⁄ pyruvate shuttle in mammalian mitochondria, the possible efflux of pyruvate as a result of l-lactate addi- tion to PTM was investigated (Fig. 6A). The concen- tration of pyruvate outside PTM was negligible, as shown by the minimal change in absorbance at 334 nm found when commercial LDH was added along with the NADH to complete the pyruvate- detecting system (for details, see Experimental proce- dures). On the other hand, in the presence of l-lactate (10 mm), the absorbance at 334 nm decreased rapidly, which is indicative of the appearance of pyruvate in the extramitochondrial phase. This can be explained on the basis that the l-lactate imported into the mito- chondria forms pyruvate via the mitochondrial LDH, Fig. 4. Effect of L-lactate addition to PTM. Change in the redox state of pyridine nucle- otides (A), failure to cause membrane poten- tial generation (inset), and activation of AOX (B). (A) PTM (0.2 mg protein) were incuba- ted in 2 mL of the standard medium (see Experimental procedures), and the fluores- cence (k ex 334 nm, k em 456 nm) was con- tinuously monitored. At the times indicated by the arrows, L-lactate (10 mm), SHAM (1 m M), and oxamate (OXAM, 10 mM) were added. The numbers alongside the traces refer to the rate of reduction of NAD(P) + in nmolÆmin )1 Æmg )1 protein. Inset: PTM (0.2 mg of protein) were incubated in 2 mL of the standard medium in the presence of 2.5 l M safranin, and fluorescence (k ex 520 nm, k em 570 nm), measured as arbitrary units (a.u.), was continuously monitored. Where indicated by S, L-lactate ( L-LAC, 10 mM), D-lactate (D-LAC, 10 mM)or succinate (5 m M) were added separately; where indicated, FCCP (1 l M) was added. (B) PTM (0.2 mg protein) were suspended at 25 °C in 1 mL of respiratory medium, and the amount of residual oxygen was meas- ured as a function of time. Where indicated, the following additions were made: succi- nate (SUCC, 5 m M), oxamate (10 mM), ADP (0.2 m M), cyanide (CN – ,1mM), L-lactate ( L-LAC, 10 mM), pyruvate (PYR, 5 mM), and SHAM (1 m M). Numbers along the curves are rates of oxygen uptake expressed as nmol O 2 Æmin )1 Æmg )1 mitochondrial protein. G. Paventi et al. L-Lactate metabolism in PTM FEBS Journal 274 (2007) 1459–1469 ª 2007 The Authors Journal compilation ª 2007 FEBS 1463 and that the pyruvate exits in exchange for further l-lactate. As expected, externally added oxamate (5 mm) was found to prevent pyruvate efflux, further confirming that PTM produce pyruvate from l-lactate via LDH. It was found that oxamate under the same conditions did not impair pyruvate detection via the pyruvate-detecting system. In the same experiment, the addition of phenylsuccinate, a nonpenetrant compound that inhibits a variety of carriers ([21] and refs therein), resulted in strong inhibition of the rate of NADH oxidation. In contrast, the pyruvate carrier inhibitor a-cyanocinnamate did not affect pyruvate efflux, thus ruling out the involvement of such a transporter in the observed process. To find out whether the rate of NADH oxidation mirrors the transport across the mitochondrial membrane, we investigated the depend- ence of the inhibition of the rate of NADH oxidation on increasing phenylsuccinate concentration (Fig. 6A,a). Significantly, the y intercept of the line fitting the experimental points measured in the presence of the inhibitor coincided with the experimental values meas- ured in the absence of inhibitor. In accordance with the control strength analysis [21], this shows that phe- nylsuccinate controls the rate of the measured process; that is, the rate of decrease of the absorbance of NADH reflects the rate of pyruvate efflux. The data in the inset were also plotted as 1 ⁄ i against 1 ⁄ [inhibitor], where the fractional inhibition i is 1 ) v i ⁄ v o (inset b). The y inter- cept was 1, showing that phenylsuccinate could com- pletely prevent l-lactate ⁄ pyruvate exchange, and that no pyruvate efflux from mitochondria can occur either by diffusion or via a carrier insensitive to phenylsuccinate. Figure 6B shows the results of measurements of the rate of pyruvate efflux as a function of increasing l-lactate concentration. The dependence was sig- moidal, with a K 0.5 of about 27 mm. Discussion In this article, we show for the first time the occur- rence of mitochondrial l-lactate metabolism in plants arising from the presence of a mitochondrial LDH. In particular, we show that l-lactate can be transported into mitochondria from potato tubers, and metabo- lized therein. The sequence of events involved in mitochondrial metabolism of l-lactate (Scheme 1) is envisaged as: uptake into mitochondria of l-lactate, synthesized in the cytosol by anaerobic glycolysis; oxidation of the l-lactate to pyruvate by the mito- chondrial LDH located in an inner mitochondrial compartment; activation of AOX by the newly syn- thesized pyruvate and oxidation of the intramito- chondrial NAD(P)H via AOX; and efflux of pyruvate via a putative l-lactate ⁄ pyruvate antiporter and the oxi- dation of cytosolic NADH in a non-energy-competent l-lactate ⁄ pyruvate shuttle. The results that we have reported are entirely con- sistent with this scheme. Existence of a mitochondrial LDH has been shown both by western blotting and by enzymatic assay (Figs 1 and 2). Note that the occurrence of LDH in plant mitochondria cannot be predicted by informatics analysis in Arabidopsis thali- ana, in which the occurrence of LDH in chloroplasts is suggested. As LDH activity can be assayed only after addition of Triton X-100 to PTM, we conclude that this enzyme is localized on the inner side of the mitochondrial inner membrane or in the matrix space. The experiment with dichloroindophenol in Fig. 2 rules out the possibility that l-lactate is oxid- ized on the external face of the inner membrane, with electrons transferred to the inner surface. Mito- chondria can take up l-lactate with net carbon uptake in a proton-compensated manner, as shown by swelling experiments. Whether l-lactate uptake occurs in a carrier-mediated manner remains to be established. The mitochondrial LDH is an NAD(P)- dependent enzyme, as shown by reduction of the intramitochondrial pyridine nucleotide. In this regard, the LDH of PTM is similar to the enzymes found in mitochondria from rat heart [7] and rat liver [8], rather than to that from Euglena mitochondria [26], with the major difference that in PTM, NAD(P)H is not reoxidized in the cytochrome pathway, but by the AOX. Uptake of l-lactate by PTM was investigated using spectroscopic techniques under conditions in which the mitochondria were metabolically active; consequently, mitochondrial reactions and traffic of newly synthes- ized substrates across the mitochondrial membrane could be monitored. Fig. 5. Mitochondrial swelling in ammonium D-lactate and L-lactate solutions. PTM (0.2 mg protein) were rapidly added at 25 °Cto 2 mL of sucrose (SUCR, 0.36 M), ammonium L-lactate (NH 4 -L-LAC, 0.18 M), ammonium D-Lactate (NH 4 -D-LAC, 0.18 M), and ammonium phosphate (NH 4 -Pi, 0.13 M), and mitochondrial swelling was monit- ored as described in Experimental procedures. L-Lactate metabolism in PTM G. Paventi et al. 1464 FEBS Journal 274 (2007) 1459–1469 ª 2007 The Authors Journal compilation ª 2007 FEBS As in Valenti et al. [7], the l-lactate ⁄ pyruvate shuttle was reconstructed in vitro. At present, we would sug- gest that the l-lactate ⁄ pyruvate shuttle makes use of both cytosolic and mitochondrial LDHs and of a puta- tive l-lactate ⁄ pyruvate carrier. Application of control strength criteria showed that oxidation of the NADH outside PTM was limited by the rate of pyruvate efflux, at least at a lower l-lactate concentration (10 mm). However, the dissection of the steps involved in pyruvate efflux requires further work. Whatever the detailed mechanism, our findings that plant mitochondria can metabolize l-lactate requires a detailed revision of all the metabolic path- ways dealing with l-lactate metabolism in plants. There are also obvious important implications for understanding how plants respond to hypoxic stress. In this regard, the reconstructed l-lactate ⁄ pyruvate shuttle appears to have the unique characteristic of providing a non-energy-competent mechanism for the oxidation of cytosolic NADH, perhaps active under hypoxic conditions. Under conditions that limit oxy- gen availability, complete substrate oxidation is restricted by the lack of an electron acceptor. Conse- quently, oxygen deficiency causes a decrease of Fig. 6. Appearance of pyruvate in the extra- mitochondrial phase induced by the addition of L-lactate (L-LAC) to PTM. (A) PTM (0.1 mg protein) were suspended at 25 °C in 2 mL of standard medium in the pres- ence of the pyruvate-detecting system (0.2 m M NADH plus LDH 2 eu), and the absorbance (A 340 ) was continuously monit- ored. L-Lactate (10 mM) was added both in the absence and in the presence of phenyl- succinate (PheSUCC, 10 m M), or oxamate (5 m M), or a-cyanocinnamate (a-CCN – , 0.1 m M). Inset (a) is a Dixon plot of the inhi- bition by phenylsuccinate of the rate of pyruvate efflux due to externally added L-lac- tate; the L-lactate concentration was 10 mM, and the rate of pyruvate appearance, meas- ured as described above, was determined as a function of increasing phenylsuccinate concentrations and expressed as nmolÆ min )1 Æmg )1 protein. Inset (b) shows the plot of 1 ⁄ i against 1 ⁄ [phenylsuccinate], where i ¼ 1 ) v i ⁄ v o , v i and v o being the rate of L-lactate uptake in the presence and in the absence of phenylsuccinate, respectively. (B) Dependence of the rate of pyruvate efflux on increasing concentrations of L-lac- tate. The experiments and measurements were carried out as in (A). G. Paventi et al. L-Lactate metabolism in PTM FEBS Journal 274 (2007) 1459–1469 ª 2007 The Authors Journal compilation ª 2007 FEBS 1465 mitochondrial respiration, which is partly compensa- ted by increased glycolytic flux. As a result, ATP levels decrease and NADH levels increase. It is tempting to propose that in addition to other proces- ses, involving nitrate, nitric oxide and hemoglobin, which contribute to plant adaptation to hypoxia, a similar role is played by mitochondrial metabolism of l-lactate [27]. Scheme 1. L-Lactate metabolism in PTM. For an explanation see the text. ALA, alanine; AOX, alternative oxidase; GLU, glutamate; GPT, glu- tamate pyruvate transaminase; aKG, a-ketoglutarate; cLDH, cytosolic lactate dehydrogenase; mLDH, mitochondrial lactate dehydrogenase; L-LAC, L-lactate; MAL, malate; ME, malic enzyme; mim, mitochondrial inner membrane; NAD(P)H DH int, internal NAD(P)H dehydrogenase; PDH, pyruvate dehydrogenase; PYR, pyruvate; SHAM, salicyl hydroxamic acid; UQ, ubiquinone; +, activation; –, inhibition. L-Lactate metabolism in PTM G. Paventi et al. 1466 FEBS Journal 274 (2007) 1459–1469 ª 2007 The Authors Journal compilation ª 2007 FEBS Experimental procedures Materials ADP, antimycin A, BSA, CN – , FCCP, bovine heart LDH (EC 1.1.1.27), dichloroindophenol, dithiothreitol, EDTA, EGTA, mannitol, NADH, NAD + , phenazine methosul- fate, phenylmethanesulfonyl fluoride, Tris, Triton X-100, Tween-20, ascorbic acid, glycerol 3-phosphate, d-lactic acid, l-lactic acid, pyruvic acid, SHAM and succinic acid were obtained from Sigma-Aldrich Chemie (Steinheim, Germany); phenylsuccinate and skimmed milk powder were obtained from Fluka (Mallinckrodt, Buchs, Switzer- land). Sucrose was obtained from Baker (Deventer, the Netherlands). All chemicals were of the purest grade available, and were used as Tris salts at pH 7.0–7.4, adjusted with Tris or HCl. SHAM, antimycin A and FCCP were dissolved in ethanol. Both primary (goat polyclonal anti-LDH, goat polyclonal anti-b-tubulin and rabbit polyclonal anti-COX IV) and secondary (anti-goat and anti-rabbit horseradish per- oxidase-conjugated) sera were obtained from Abcam plc (Cambridge, UK). Potato tubers were initially obtained either from local farmers (who use no chemical additives during plant growth and harvest) or from local markets. It was found, however, that concurrent experiments carried out with isolated mitochondria from potato tubers obtained from the markets gave conflicting, often quantitatively differ- ent, results. This could be attributed to either different ages of the tubers or to the use of chemical agents in their growth or harvest, or to both of these factors. For this reason, we have preferred, in the work described here, to use only tubers obtained from farmers who do not use chemical agents in their production process. It was also necessary to carry out experiments over a short time interval (2–3 weeks), as the tubers showed significant changes in l-lactate metabolism with the time postharvest [28]. Isolation of PTM and preparation of the cytosolic fraction PTM were isolated as in Pastore et al. [29], free of sub- cellular contamination as determined in Neuburger & Douce [30], and checked for their intactness as in Douce et al. [16]. Mitochondrial protein content was determined by the method of Lowry as in Harris [31], using BSA as a standard. The cytosolic fraction was obtained by centrifugation (105 000 g for 60 min at 4 °C, Kontron Ultracentrifuge Centrikon T2170, fixed-angle rotor TFT 65.13) of the supernatant obtained during isolation of PTM. Glucose-6- phosphate dehydrogenase (EC 1.1.1.49) was assayed as in Loh & Waller [32]. Immunoblot analysis Immunoblot analysis was performed on total mitochondrial or total cytosolic protein by using antibodies raised against LDH, COX IV and b-tubulin. Polyclonal antibodies recog- nizing COX IV and b-tubulin were used as markers of mitochondria and cytosol, respectively. Both purified PTM and cytosolic protein were solubi- lized in 1% Triton X-100, 500 mm NaCl, 50 mm Tris ⁄ HCl (pH 7.5), 1 mm EGTA, 1 mm EDTA, 0.5 mm dithiothreitol and 0.1 m m phenylmethanesulfonyl fluoride for 30 min on ice. Protein content was determined using the Bradford reagent (Bio-Rad Laboratories, Hercules, CA, USA), with BSA as a standard. Solubilized proteins (30 and 40 lg) were subjected to electrophoresis on 12% SDS ⁄ polyacrylamide gel [33]. Following electrophoresis, protein blots were transferred to a poly (vinylidene difluoride) membrane. The membrane was blocked with 5% nonfat milk in Tris buffer solution, and incubated overnight with the corresponding primary antibodies in the blocking solution at 4 °C. After being washed three times with Tris buffer solution plus Tween-20 (0.3%), the membrane was incubated at room temperature for 1 h with horseradish peroxidase-conjugated secondary anti- body. The detected protein signals were visualized with enhanced chemiluminescence western blotting reagents (Amersham, ECL, Little Chalfont, UK). Relative absor- bances and areas of bands were quantified using a GS- 700 Imaging Densitometer implemented with molecular analyst software (Bio-Rad Laboratories). LDH activity and other photometric assays The LDH assay was performed photometrically at 340 nm in the pyruvate-to-lactate direction as in Hoffman et al. [18], by means of a Jasco (Tokyo, Japan) V-560 spectro- photometer. Briefly, Triton X-100-solubilized PTM were incubated at 25 °C in 2 mL of the standard medium con- sisting of 0.125 m mannitol, 65 mm NaCl, 2.5 mm sodium phosphate, 0.33 mm Na-EGTA, and 10 mm Tris ⁄ HCl (pH 7.20), in the presence of 0.2 mm NADH. LDH activity was assayed by measuring the difference between the rate of decrease in absorbance at 340 nm due to the oxidation of NADH before and after pyruvate addition. The activity was expressed as nmol NADH oxidizedÆmin )1 Æmg )1 protein (e NADH ¼ 6.2 mm )1 Æcm )1 ). Glycerol-3-phosphate dehydrogenase and succinate dehy- drogenase activities were checked photometrically at 600 nm as in Atlante et al. [19]. Briefly, PTM were incuba- ted at 25 °C in 2 mL of the standard medium in the pres- ence of 30 lm phenazine methosulfate and 50 lm dichloroindophenol. Enzymatic activities were assayed by measuring the decrease in absorbance at 600 nm due to the reduction of dichloroindophenol that occurred when sub- strates were added to the sample. The activities were G. Paventi et al. L-Lactate metabolism in PTM FEBS Journal 274 (2007) 1459–1469 ª 2007 The Authors Journal compilation ª 2007 FEBS 1467 expressed as nmol dichloroindophenol reducedÆmin )1 Æmg )1 protein (e dichloroindophenol ¼ 21 mm )1 Æcm )1 ). Mitochondrial swelling was monitored photometrically at 546 nm. PTM (0.2 mg protein) were rapidly added to iso- tonic solutions of ammonium salts, the pH values of which were adjusted to 7.2, and the decrease in the absorbance was continuously recorded. Appearance of pyruvate outside the mitochondria was monitored as in Valenti et al. [7] in 2 mL of standard medium, using the pyruvate-detecting system consisting of 200 lm NADH plus 1 enzymatic unit (eu) of LDH. Oxida- tion of NADH consequent on addition of l-lactate externally was followed photometrically at 340 nm. l-Lactate itself had no effect on the enzymatic reactions or on the absorbance measured at 340 nm. Controls were carried out to ensure that none of the compounds used affected the enzymes used to reveal metabolite appearance outside mitochondria. The rates of pyruvate efflux were obtained by the difference in the oxidation rate of NADH before and after addition of l-lactate, and are expressed as NADH oxidizedÆ min )1 Æmg )1 mitochondrial protein (e NADH ¼ 6.2 mm )1 Æcm )1 ). Oxygen uptake studies Oxygen uptake measurements were carried out at 25 °C using a Rank Brothers Oxygraph (Cambridge, UK) equipped with a Clark electrode in 1 mL of the respiratory medium consisting of 0.3 m mannitol, 5 mm MgCl 2 ,10mm NaCl, 0.1% (w ⁄ v) defatted BSA, and 10 mm sodium phos- phate buffer (pH 7.20). Fluorimetric assays Changes in the redox state of mitochondrial nicotinamide nucleotide were monitored fluorimetrically at k ex 334 nm and k em 456 nm, as in Valenti et al. [7]. PTM (0.2 mg of protein) were incubated in 2 mL of standard medium, either in the presence or in the absence of 1 lm FCCP, and then treated with 1 mm CN – . NAD(P) + reduction due to l-lactate addition was observed as fluorescence increase, and the rate of reaction was calculated as the tangent to the initial part of the progress curve and expressed as nmol NAD(P) + reducedÆmin )1 Æmg )1 protein. Changes in mitochondrial membrane potential (DY) were followed by monitoring safranin O fluorescence changes (k ex 520 nm and k em 570 nm) at 25 °C, as in Moore & Bon- ner [34], by means of a Perkin-Elmer (Beaconsfield, UK) LS50B spectrofluorimeter in 2 mL of standard medium containing 2.5 lm safranin O and PTM (0.2 mg of protein). Acknowledgements The authors thank Professor Shawn Doonan for his critical reading. 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Hence, potato tuber mitochondria possess a non-energy-competent l-lactate ⁄ pyruvate shuttle. We maintain, therefore, that mitochondrial metabolism of l-lactate. exit from the mitochondria in a novel l-lactate ⁄ pyruvate shuttle operating in a non- energy-competent manner. Results The existence of LDH in mitochondria isolated from potato tubers In order to