The Contribution of Uncoupling Protein and ATP Synthase to State 3 Respiration in Acanthamoeba Castellanii Mitochon - Dria*

Vol. 51 No. 2/2004

533–538

QUARTERLY

Communication

The contribution of uncoupling protein and ATP synthase to state 3 respiration in Acanthamoeba castellanii mitochon - dria*.

Wiesława Jarmuszkiewicz1½, Małgorzata Czarna1, Claudine Sluse-Goffart2 and Francis E. Sluse2

1Laboratory of Bioenergetics, Adam Mickiewicz University, Poznań, Poland, 2Laboratory of Bioenergetics, Department of Life Sciences, University of Liěge, Sart Tilman, Liěge, Belgium Received: 30 April, 2004; accepted: 07 May, 2004

Key words: mitochondria, uncoupling protein, energy-dissipating, Acanthamoeba castellanii

Mitochondria of the amoeba Acanthamoeba castellanii possess a free fatty acid-activated uncoupling protein (AcUCP) that mediates proton re-uptake driven by the mitochondrial proton electrochemical gradient. We show that AcUCP activity diverts energy from ATP synthesis during state 3 mitochondrial respiration in a fatty acid-dependent way. The efficiency of AcUCP in mitochondrial uncoupling increases when the state 3 respiratory rate decreases as the AcUCP contribution is constant at a given linoleic acid concentration while the ATP synthase contribution decreases with respiratory rate. Respiration sustained by this energy-dissipating process re-

*This work was presented in poster form at the 29th Congress of the Federation of European Biochemi- cal Societies, Warsaw, Poland, 26 June–1 July 2004. .This research was supported by the State Committee for Scientific Research (KBN, Poland) grant No. 0290/P04/2003/25, and the Fonds National de la Recherche Scientifique (FNRS, Belgium). ½ To whom correspondence should be addressed: Wies³awa Jarmuszkiewicz, Laboratory of Bioenergetics, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, A. Fredry 10, 61-701 Poznań, Poland; phone: (48 61) 829 4531; fax: (48 61) 829 4503; e-mail: [email protected] Abbreviations: AcUCP, uncoupling protein of A. castellani; BHAM, benzohydroxamate; BSA, bovine serum albumine; DmH+, proton electrochemical gradient; DY, mitochondrial transmembrane electrical potential; DY3, membrane potential during state 3; FFA, free fatty acids; LA, linoleic acid; RCR, respiratory control ratio; V3, state 3 respiration; VAcUCP, contribution of AcUCP activity-sustained respiration in V3; VATP synthase, contribution of ATP synthesis-sustained respiration in V3; UCP, uncoupling protein. 534 W. Jarmuszkiewicz and others 2004

mains constant at a given linoleic acid concentration until more than 60% inhibition of state 3 respiration by n-butyl malonate is achieved. The present study supports the validity of the ADP/O method to determine the actual contributions of AcUCP (activated with various linoleic acid concentrations) and ATP synthase in state 3 respiration of A. castellanii mitochondria fully depleted of free fatty acid-activated and describes how the two contributions vary when the rate of succinate dehydrogenase is decreased by succinate uptake limitation.

Most mitochondria possess free energy-dis- using pair measurements of ADP/O ratios in sipating systems that decrease the yield of ox- the absence or presence of various concentra- idative phosphorylation. Ubiquinol alterna- tions of linoleic acid (LA) (Jarmuszkiewicz et tive oxidase dissipates redox energy in mito- al., 2000; Sluse et al., 2000). In the present chondria of plants and some unicellulars study, measurements in the presence of (Siedow & Umbach, 2000). Uncoupling pro- benzohydroxamate (BHAM) to exclude oxygen tein (UCP) dissipates proton electrochemical consumption by alternative oxidase and with gradient energy (DmH+) in animal, plant and an increasing concentration of n-butyl some fungal and protist mitochondria (Sluse malonate (an inhibitor of succinate uptake) in & Jarmuszkiewicz, 2002). Their activities order to decrease the rate of the quinone-re- have the same final effect; that is a decrease ducing pathway (succinate oxidation) have of ATP production per oxygen consumed. Un- been performed. They have allowed us to de- coupling proteins, forming a subfamily within scribe evolution of the UCP and ATP synthase the mitochondrial anion carrier protein fam- contributions when state 3 respiration is de- ily, dissipate DmH+ through a free fatty acid creased in A. castellanii mitochondria. (FFA)-activated purine nucleotide-inhibited H+ cycling process driven by membrane potential (DY) and DpH (both constituting MATERIAL AND METHODS DmH+). Indications that UCP is present in unicellulars are mainly based on functional Cell culture and mitochondrial isola- studies and the cross-reactivity of an around tion. The soil amoeba Acanthamoeba 32 kDa mitochondrial protein with antibodies castellanii, strain Neff, was cultured as de- developed against plant UCP. In mitochon- scribed by Jarmuszkiewicz et al. (1997). dria of the nonphotosynthetic amoeboid pro- Trophozoites of the amoeba were collected be- tozoan Acanthamoeba castellanii, the action of tween 44–48 h following inoculation at the UCP (AcUCP) has been shown to mediate middle exponential phase (at a density of FFA-activated, poorly purine nucleotide-in- about 5–6 x 106 cells/ml). Mitochondria were hibited H+ re-uptake driven by DmH+ that in isolated and purified on a self-generating state 3 respiration can divert energy from oxi- Percoll gradient (31%) as described earlier dative phosphorylation (Jarmuszkiewicz et (Jarmuszkiewicz et al., 1997). The presence of al., 1999). 0.4% bovine serum albumin (BSA) in isolation Both UCP and ATP synthase are able to con- media allowed FFA to be chelated from the sume DmH+ built up by the mitochondrial re- mitochondrial suspension and mitochondria spiratory chain. They may be considered as fully depleted of FFA to be obtained. For each two branching pathways: UCP as the DmH+ en- mitochondrial preparation, full depletion of ergy-dissipating path, and ATP synthase as the FFA was tested by measuring the effect of DmH+ energy-conserving path. The ADP/O BSA on the LA-induced respiration as de- method has been applied to calculate the con- scribed by Jarmuszkiewicz et al. (1998). Mito- tributions of UCP activity and ATP synthesis chondrial protein concentration was deter- in state 3 respiration of tomato mitochondria, mined by the biuret method. Vol. 51 AcUCP contribution to phosphorylating respiration 535

Oxygen uptake and membrane poten- consumed during state 3 respiration was used tial. Oxygen uptake was measured polaro- for calculation of the ratio. A prepulse of ADP graphically using a Rank Bros. (Cambridge, (80 mM) was always applied before the main U.K.) oxygen electrode or a Hansatech oxy- pulse to ensure that a true state 4 had been gen electrode in 1.4 ml or 2.7 ml (respec- achieved and to activate succinate dehydro- tively) of standard incubation medium (25°C) genase by the produced ATP. Increasing con- containing: 120 mM KCl, 20 mM Tris/HCl pH centrations of n-butyl malonate were used to 7.4, 3 mM KH2PO4, and 0.5 mM MgCl2, with decrease steady-state 3 respiration. Measure- 1–1.7 mg of mitochondrial protein. Mem- ments of DY allowed fine control of the dura- brane potential of mitochondria was mea- tion of state 3 respiration. sured simultaneously with oxygen uptake using a tetraphenylphosphonium-specific electrode according to Kamo et al. (1979). For cal- RESULTS AND DISCUSSION culation of the DY value the matrix volume of amoeba mitochondria was assumed as 2.0 State 3 respiratory rates and ADP/O ratios ml/mg protein. were measured during ADP pulses in the ab- All measurements were made in the pres- sence or presence of different concentrations ence of 7 mM succinate, 1.5 mM benzohydro- of LA. Figure 1 shows an example of the ef- xamate (BHAM), 170 mM ATP, 4 mM rote- fect of 9.1 mM LA on the coupling parameters none, 0–12.6 mM LA, and 0–30 mM n-butyl in isolated A. castellanii mitochondria. In malonate. The ADP/O ratio was determined resting state (state 4), LA increased the respi- by the ADP pulse method with 250–500 ratory rate and decreased DY. On the other nmoles of ADP. The total amount of oxygen hand, LA scarcely modified phosphorylating

Figure 1. The effect of LA on coupling parameters of A. castellanii mitochondria. Mitochondria (mito) were incubated in the presence of 7 mM succinate, 170 mM ATP, 4 mM rotenone, 1.5 mM BHAM, in the absence (soild line) or presence of 9.1 mM LA (dashed line). After the ADP pulse, respiration was un- coupled and membrane potential was collapsed by 1 mM carbonylcyanide p-trifluoromethoxyphenylhydrazone –1 (FCCP). RCR, respiratory control ratio. Numbers on the traces refer to O2 consumption rates in nmol O2 x min per mg protein. 536 W. Jarmuszkiewicz and others 2004

Figure 2. Constancy of membrane potential in state 3 respiration. Assay conditions as described in Material and Methods. Oxidation rate of succinate in the presence or absence of 9.1 mM LA was gradually decreased by increasing concentrations of n-butyl malonate (2–30 mM). Data from 3 experiments are shown. The mean value of DY3 in the absence of LA is 163.2 ± 2.8 (S.D., n = 20), in the presence of 9.1 mM LA is 164.8 ± 2.2 (S.D., n = 11). The mean value of DY3(± LA) is 163.8 ± 2.7 (S.D., n = 31).

state 3 respiration (V3) and membrane poten- concentrations of the inhibitor both when tial (DY3). As a consequence, the ADP/O ra- AcUCP was activated (plus LA) and not (no tio and respiratory control ratio (RCR) were LA) (Fig. 2). DY3 was almost unchanged by clearly lowered in the presence of LA suggest- LA addition. However, like in tomato mito- ing activation of AcUCP (i.e. an increased chondria (Jarmuszkiewicz et al., 2000), a slight LA-induced AcUCP-mediated H+ re-uptake increase was systematically observed in the sustaining part of state 3 respiration). presence of LA. The mean value of DY3(± 9.1 In order to describe how the contributions of mM LA) obtained with different mitochondrial AcUCP and ATP synthase change with varia- preparations and n-butyl malonate concentrations in state 3 respiration, the rate of the tions was 163.8 ± 2.7 (S.D., n = 31). Figure 3 ubiquinone-reducing pathway (succinate shows the effect of three concentrations of LA dehydrogenase) was decreased by n-butyl on the ADP/O ratio plotted against the recip- malonate, an inhibitor of succinate uptake. rocal of state 3 respiration which was varied Pair measurements of ADP/O ratios and respi- with n-butyl malonate. In the absence of LA, ratory rates in the absence or presence of a the ADP/O ratio remained constant when V3 given LA concentration were performed with was decreased (up to about 65%) and the mean increasing concentrations of n-butyl malonate value was 1.403 ± 0.021 (S.D., n = 31) (Fig. 3). (up to 30 mM). V3 decreased with increasing This constancy is one of the requirements for

Figure 3. The effect of different LA concentrations on the ADP/O ratio plotted versus 1/V3. Assay conditions as in Material and Methods. Oxi- dation rate of succinate was gradually decreased by increasing concentrations of n-butyl malonate (2–30 mM). ADP/O ratios were determined in the presence of 0, 4.5, 9.1, and 12.6 mM LA. Data from 3 experiments are shown. Mean value of ADP/O ratio in the absence of LA is 1.403 ± 0.021 (S.D., n = 31). In the presence of LA, values of intercepts with ordinate axis (A) of the least square regression lines (Y = A + BX, where Y = ADP/O and X = 1/V3 x 103) are: 1.391 ± 0.014 (S.D., n = 13), 1.429 ± 0.014 (S.D., n = 13), and 1.422 ± 0.053 (S.D., n = 8) for 4.5, 9.1, and 12.6 mM LA, respectively. Vol. 51 AcUCP contribution to phosphorylating respiration 537 the validity of the ADP/O method and has The contributions of ATP synthesis and been already verified with tomato mitochon- AcUCP activity to state 3 respiration were cal- dria (Jarmuszkiewicz et al., 2000). In the pres- culated from the pair measurements of ence of LA, the ADP/O ratio was lowered and ADP/O and V3 in the absence or presence of decreased with decreasing V3 (increasing LA (as described by Jarmuszkiewicz et al., n-butyl malonate concentration). Thus, the 2000, see also legend of Fig. 4) and plotted lowering of the electron supply to the against V3 in order to emphasize their behav- cytochrome pathway amplified the decrease in iour during titration of respiration by n-butyl ADP/O induced by LA, suggesting an increas- malonate. As a result, at a fixed LA concen- ing relative contribution of the AcUCP-sus- tration, the AcUCP activity remained con- tained respiration to the overall state 3 respi- stant and ATP synthesis decreased linearly ration. At a given V3, the higher the LA con- with decreasing state 3 respiration (Fig. 4). centration was the lower ADP/O ratio was ob- Using higher concentrations of LA, UCP ac- served, thereby indicating a higher AcUCP tivity increased at the expense of ADP contribution (Fig. 3). A linear relationship was phosphorylation without an increase in respi- obtained between ADP/O and the reciprocal ration. Namely, at a given V3, increasing LA of V3, with identical ordinates at origin (inde- decreased the ATP synthase contribution and pendently of the LA concentration) and slopes increased the AcUCP activity contribution. which increased negatively when LA concen- These results show how efficiently the UCP tration was increased. In the presence of LA, activity can divert energy from oxidative the ordinates at the origin (obtained using lin- phosphorylation, especially when state 3 ear regression, see legend of Fig. 3) were not respiration is progressively inhibited. significantly different for the mean ADP/O The protonophoric action of FFA can be me- value in the absence of LA. Such behaviour, diated not only by UCP but also, at least in observed also for tomato mitochondria part, by several members of the mitochon- (Jarmuszkiewicz et al., 2000; Sluse et al., drial carrier family such as the ADP/ATP, 2000), indicates that (i) at a fixed LA concen- dicarboxylate, aspartate/glutamate, and tration, VUCP remains constant during titra- phosphate carriers (Andreyev et al., 1989; tion by n-butyl malonate, and (ii) LA does not Wieckowski & Wojtczak, 1997; Samartsev et affect the intrinsic stoichiometry of oxidative al., 1997; ða ková et al., 2000). However, this phosphorylation (ratio of H+/O and H+/ FFA-dependent mitochondrial uncoupling, ADP). being a side function of these carriers, is ex-

Figure 4. The partitioning of state 3 respiratory rate (V3) between uncoupling protein activity (VUCP) and ATP synthesis (VATP synthase) at different LA concentrations. Assay conditions as in Fig. 3. As described by Jarmuszkiewicz et al. (2000), VATP synthase is equal to V3 x (ADP/O)+LA/(ADP/O)–LA and VUCP is equal to V3 – VATP synthase. Mean values of VUCP for different concentrations of LA are: 13.4 ± 1.1 (S.D., n = 12), 25.8 ± 1.3 (S.D., n = 12), 37.5 ± 4.5 (S.D., n = 8) for 4.5, 9.1, and 12.6 mM LA, respectively. 538 W. Jarmuszkiewicz and others 2004 clusively observed in the high-energy state of aries of the high energy status of mitochon- mitochondria (at high DY like in state 4) and dria to be crossed so as to focus on regulation can be inhibited by their substrates or by of UCP activity in state 3 respiration, thus their specific inhibitors. It is highly unlikely during ADP phosphorylation. that this unspecific uncoupling takes place during phosphorylating respiration where the particular carriers are employed in the im- REFERENCES port of ADP, succinate or phosphate. Thus, the first candidate to catalyze the LA-induced Andreyev AY, Bondareva TO, Dedukhova VI, H+ re-cycling observed in amoeba mitochon- Mokhova EN, Skulachev VP, Tsofina LM, dria in state 3 measurements is AcUCP. Volkov NL, Vygodina TV. (1989) Eur J. In isolated A. castellanii mitochondria de- Biochem.; 182: 585–92. pleted of FFA, the yield of oxidative phos- Jarmuszkiewicz W. (2001) Acta Biochim Polon.; phorylation (ADP/O ratio) remained con- 48: 145–55. stant when the respiratory rate was de- Jarmuszkiewicz W, Wagner AM, Wagner MJ, creased by a factor of 3 by the addition of Hryniewiecka L. (1997) FEBS Lett.; 411: n-butyl malonate. This constancy allows the 110–14. contribution of the LA-induced energy-dissipating pathway to be determined by the Jarmuszkiewicz W, Almeida AM, Sluse-Goffart CM, Sluse FE, Vercesi AE. (1998) J Biol ADP/O method. Linoleic acid decreases the Chem.; 273: 34882–6. yield of oxidative phosphorylation in a concentration dependent manner by a pure Jarmuszkiewicz W, Sluse-Goffart CM, protonophoric process (i.e., LA is not a slip in- Hryniewiecka L, Sluse FE. (1999) J Biol ducer, see Jarmuszkiewicz et al., 2000). Chem.; 274: 23198–202. Therefore, it can be concluded that in A. Jarmuszkiewicz W, Almeida AM, Vercesi AE, castellanii ADP/O measurements allow calcu- Sluse FE, Sluse-Goffart CM. (2000) J Biol lation of the part of respiration leading to Chem.; 275: 13315–20. ATP synthesis and the part of respiration sus- Kamo N, Muratsugu N, Hongoh R, Kobatake Y. + tained by the dissipative H re-uptake in- (1979) J Membr Biol.; 49: 105–21. duced by LA that can be attributed to AcUCP Sluse FE, Jarmuszkiewicz W. (2002) FEBS activity. Thus, the ADP/O method first veri- Lett.; 510: 117–20. fied in tomato mitochondria (Jarmuszkiewicz et al., 2000) can be applied widely in different Sluse FE, Jarmuszkiewicz W, Almeida AM, mitochondria including those of unicellulars. Vercesi AE, Sluse-Goffart CM. (2000) In An- Most of the investigations on the activity and imating the Cellular Map. Hofmeyr J-H, regulation of uncoupling proteins in isolated Rohwer JM, Snoep JL, eds, pp 135–40. Stellenbosch University Press, Stellenbosh, mitochondria are performed only in state 4 South Africa. (nonphosphorylating) respiration which im- plicates a high phosphate potential (no ADP) Samartsev VN, Mokhova EN, Skulachev VP. and a high reducing power (slow respiratory (1997) FEBS Lett.; 412: 251–7. substrate oxidation). Under these experimen- Siedow JN, Umbach AL. (2000) Biochim Biophys tal conditions, ubiquinone is highly reduced, Acta.; 1459: 432–9. production of superoxide by mitochondria is Dm + Wieckowski M, Wojtczak L. (1997) Biochem high and H is maximal (high membrane Biophys Res Commun.; 232: 414–7. potential). The present work describes an ex- perimental approach that allows the bound- ða ková M, Krämer R, Jeñek P. (2000) Int J Biochem Cell Biol.; 32: 499–508.