Meredith F Ross

Tracy A Prime

Irina Abakumova

Andrew M James

Carolyn M Porteous §

Robin A J Smith

Dr Michael P Murphy

Rapid and extensive uptake and activation of hydrophobic triphenylphosphonium cations within cells

Keywords: plasma membrane potential, ESI-MS, electrospray ionization mass spectrometry, FCCP, carbonylcyanide p-(trifluoromethoxy)phenylhydrazone, FCS, fetal calf serum, IDTP, 10-iododecyltriphenylphosphonium iodide, MitoQ, MitoQuinone, MitoQH 2 , MitoQuinol, PBS, phosphate buffered saline, PMPI, Plasma Membrane Potential Indicator, ROS, reactive oxygen species, TFA, trifluoroacetic acid, TMA-OH, tetramethylammonium hydroxide, TPB, tetraphenylborate, TPMP, methyltriphenylphosphonium, TPP, triphenylphosphonium cation mitochondria, lipophilic cations, MitoQ, membrane potential, mitochondrial targeting

Mitochondria-targeted molecules comprising the lipophilic triphenylphosphonium (TPP) cation covalently linked to a hydrophobic bioactive moiety are used to modify and probe mitochondria in cells and in vivo. However it is unclear how hydrophobicity affects the rate and extent of their uptake into mitochondria within cells, making it difficult to interpret experiments because their intracellular concentrations in different compartments are uncertain. To address this issue, we compared the uptake into isolated mitochondria, and mitochondria within cells, of two hydrophobic TPP derivatives, [ 3 H]-MitoQ and [ 3 H]-DecylTPP, with the more hydrophilic TPP cation [ 3 H]-TPMP. Uptake of MitoQ by mitochondria and cells was described by the Nernst equation and was ~5-fold greater than that for TPMP, due to its greater binding within the mitochondrial matrix. DecylTPP was also taken up extensively by cells, indicating that increased hydrophobicity enhanced uptake. Both MitoQ and DecylTPP were taken up very rapidly into cells, coming to a steady state within 15 min, compared to ~8 h for TPMP. This far faster uptake was due to the increased rate of passage of hydrophobic TPP molecules through the plasma membrane. Within cells MitoQ was predominantly within mitochondria where it was rapidly reduced to the ubiquinol, consistent with its protective effects in cells and in vivo being due to the ubiquinol antioxidant. The strong influence of hydrophobicity on TPP cation uptake into mitochondria within cells facilitates the rational design of mitochondria-targeted compounds to report on and modify mitochondrial function in vivo.

INTRODUCTION

Mitochondria are central to many aspects of biomedical science; consequently there is considerable interest in the development of small molecules that can be targeted to mitochondria in cells and in vivo to report on and modify mitochondrial function [START_REF] Murphy | Targeting antioxidants to mitochondria by conjugation to lipophilic cations[END_REF]. Lipophilic cations such as triphenylphosphonium (TPP) have been linked covalently to small molecule thiol probes, spin traps, peptide nucleic acid oligomers, superoxide probes and antioxidants to deliver them to mitochondria, driven by the mitochondrial membrane potential (Δψ m ) [START_REF] Burns | Synthesis and characterization of thiobutyltriphenylphosphonium bromide, a novel thiol reagent targetted to the mitochondrial matrix[END_REF][START_REF] Smith | Targeting an antioxidant to mitochondria[END_REF][START_REF] Hardy | Mito-DEPMPO synthesized from a novel NH 2 -reactive DEPMPO spin trap: a new and improved trap for the detection of superoxide[END_REF][START_REF] Xu | Synthesis and ESR studies of a novel cyclic nitrone spin trap attached to a phosphonium group-a suitable trap for mitochondriagenerated ROS?[END_REF][START_REF] Robinson | Selective fluorescent imaging of superoxide in vivo using ethidium-based probes[END_REF][7][START_REF] Filipovska | Synthesis and characterization of a triphenylphosphonium-conjugated peroxidase mimetic: Insights into the interaction of ebselen with mitochondria[END_REF][START_REF] Dhanasekaran | Mitochondria superoxide dismutase mimetic inhibits peroxide-induced oxidative damage and apoptosis: role of mitochondrial superoxide[END_REF][START_REF] Muratovska | Targeting peptide nucleic acid (PNA) oligomers to mitochondria within cells by conjugation to lipophilic cations: implications for mitochondrial DNA replication, expression and disease[END_REF][START_REF] Lin | Specific modification of mitochondrial protein thiols in response to oxidative stress: a proteomics approach[END_REF][START_REF] Murphy | Superoxide activates uncoupling proteins by generating carbon-centered radicals and initiating lipid peroxidation: studies using a mitochondria-targeted spin trap derived from alpha-phenyl-N-tert-butylnitrone[END_REF][START_REF] Kelso | Selective targeting of a redox-active ubiquinone to mitochondria within cells[END_REF]. The delocalised positive charge on the lipophilic TPP cation promotes their Δψ m -dependent accumulation into mitochondria and their direct passage through phospholipid bilayers, consequently these molecules are useful in a range of mitochondrial studies [START_REF] Murphy | Targeting antioxidants to mitochondria by conjugation to lipophilic cations[END_REF][START_REF] Ross | Lipophilic triphenylphosphonium cations as tools in mitochondrial bioenergetics and free radical biology[END_REF].

Mitochondrial oxidative damage contributes to many pathologies because mitochondria are a source of reactive oxygen species (ROS) and are also susceptible to oxidative damage [START_REF] Balaban | Mitochondria, oxidants, and aging[END_REF][START_REF] Finkel | Opinion: Radical medicine: treating ageing to cure disease[END_REF]. To prevent oxidative damage, mitochondria-targeted antioxidants have been developed [START_REF] Jauslin | Mitochondriatargeted antioxidants protect Friedreich Ataxia fibroblasts from endogenous oxidative stress more effectively than untargeted antioxidants[END_REF][18][START_REF] Smith | Delivery of bioactive molecules to mitochondria in vivo[END_REF]. The prototypical mitochondria-targeted antioxidant is MitoQ (Figure 1), which comprises a ubiquinone moiety linked covalently by an aliphatic ten-carbon chain to the TPP cation [START_REF] Kelso | Selective targeting of a redox-active ubiquinone to mitochondria within cells[END_REF][START_REF] James | Interactions of mitochondria-targeted and untargeted ubiquinones with the mitochondrial respiratory chain and reactive oxygen species. Implications for the use of exogenous ubiquinones as therapies and experimental tools[END_REF][START_REF] James | Interaction of the mitochondria-targeted antioxidant MitoQ with phospholipid bilayers and ubiquinone oxidoreductases[END_REF]. MitoQ protects cells in culture against oxidative damage [START_REF] Kelso | Selective targeting of a redox-active ubiquinone to mitochondria within cells[END_REF][START_REF] Jauslin | Mitochondriatargeted antioxidants protect Friedreich Ataxia fibroblasts from endogenous oxidative stress more effectively than untargeted antioxidants[END_REF][START_REF] Kalivendi | Doxorubicin activates nuclear factor of activated T-lymphocytes and Fas ligand transcription: role of mitochondrial reactive oxygen species and calcium[END_REF], is taken up into organs following oral administration [START_REF] Smith | Delivery of bioactive molecules to mitochondria in vivo[END_REF], protects against tissue damage in animal models of disease [18,23,[START_REF] Neuzil | Mitochondria transmit apoptosis signalling in cardiomyocyte-like cells and isolated hearts exposed to experimental ischemia-reperfusion injury[END_REF] and is now undergoing Phase II clinical trials [25].

The interaction of MitoQ with the mitochondrial respiratory chain in simple systems is reasonably well understood. MitoQ is readily reduced to the ubiquinol form (MitoQH 2 ) by complex II, but is a poor substrate for isolated complex I, isolated electron transfer flavoprotein-ubiquinone oxidoreductase and complex III [START_REF] James | Interactions of mitochondria-targeted and untargeted ubiquinones with the mitochondrial respiratory chain and reactive oxygen species. Implications for the use of exogenous ubiquinones as therapies and experimental tools[END_REF][START_REF] James | Interaction of the mitochondria-targeted antioxidant MitoQ with phospholipid bilayers and ubiquinone oxidoreductases[END_REF]. MitoQ adsorbs extensively to phospholipid bilayers, with its TPP moiety localised to a potential energy well near the membrane surface, while the chain allows access of the ubiquinone moiety to the membrane core and the active site of complex II [START_REF] Ross | Lipophilic triphenylphosphonium cations as tools in mitochondrial bioenergetics and free radical biology[END_REF][START_REF] James | Interactions of mitochondria-targeted and untargeted ubiquinones with the mitochondrial respiratory chain and reactive oxygen species. Implications for the use of exogenous ubiquinones as therapies and experimental tools[END_REF][START_REF] James | Interaction of the mitochondria-targeted antioxidant MitoQ with phospholipid bilayers and ubiquinone oxidoreductases[END_REF]. In isolated mitochondria, MitoQ is rapidly taken up and converted to the ubiquinol form that prevents lipid peroxidation [START_REF] Kelso | Selective targeting of a redox-active ubiquinone to mitochondria within cells[END_REF][START_REF] Asin-Cayuela | Fine-tuning the hydrophobicity of a mitochondria-targeted antioxidant[END_REF]. However, little is known about the rate and extent of MitoQ uptake into mitochondria within cells and whether MitoQ is substantially reduced to its ubiquinol form.

Most mitochondria-targeted TPP probes are similar to MitoQ, therefore the rate and extent of their uptake into cells is also unknown. Basic questions remain, such as whether increasing hydrophobicity increases or decreases rate and extent of uptake relative to simple TPP cations such as methyltriphenylphosphonium (TPMP; Figure 1). The uptake of simple cations such as TPMP in response to a Δψ is described by the Nernst equation whereby cation uptake increases 10-fold for every 61.5 mV at 37˚C. ! "# (mV ) = 61.5log [START_REF] Muratovska | Targeting peptide nucleic acid (PNA) oligomers to mitochondria within cells by conjugation to lipophilic cations: implications for mitochondrial DNA replication, expression and disease[END_REF] [TPP cation] in [TPP cation] out [START_REF] Murphy | Targeting antioxidants to mitochondria by conjugation to lipophilic cations[END_REF] However, it is unclear if the significant potential-independent binding to membranes of hydrophobic TPP cations causes deviations from Nernstian behavior [START_REF] Kelso | Selective targeting of a redox-active ubiquinone to mitochondria within cells[END_REF]. These uncertainties hamper interpretation of data obtained using TPP molecules and restrict the rational development of further mitochondria-targeted compounds. Here we report on the uptake of the hydrophobic TPP cations MitoQ and DecylTPP (Figure 1) into mitochondria and cells and compare these with TPMP, a simple, hydrophilic TPP cation. We found that the uptake of hydrophobic TPP cations into mitochondria is described by the Nernst equation and that their uptake into cells is more extensive and rapid than TPMP. These findings facilitate interpretation of experiments using mitochondria-targeted molecules and indicate how these compounds can be modulated rationally to optimize their properties.

EXPERIMENTAL Synthesis and purification of radiochemicals

To synthesize [ 3 H]-MitoQ a solution of idebenol mesylate (900 mg, 2.15 mmol) [27] was prepared by reacting idebenone with methane sulfonyl chloride and triethylamine followed by catalytic hydrogenation. This was added to CH 2 Cl 2 (5 mL) in a Nalgene tube which contained [ 3 H]-triphenylphosphine [START_REF] Smith | Targeting coenzyme Q derivatives to mitochondria[END_REF] (220 mg, 0.84 mmol, 19.3 mCi), stirred to give an homogeneous solution and then evaporated under a nitrogen flow at 40˚C. The tube was flushed with argon, sealed and stirred for 29 h at ~85˚C. The tube was then cooled and the contents dissolved in CH 2 Cl 2 (~2 mL) and stirred vigorously while CH 3 COOC 2 H 5 was added in three portions (2, 3 and 5 mL) to precipitate the product. The mixture was stirred for ~5 min and then the CH 2 Cl 2 was evaporated by bubbling with nitrogen for 15 min. Once the solution volume was less than 10 mL, the mixture was allowed to settle for 10 min. The CH 3 COOC 2 H 5 layer was decanted, the semi-solid residue dissolved in CH 2 Cl 2 (~2 mL) and precipitated with CH 3 COOC 2 H 5 (10 mL) as before. This precipitation process was repeated once more. After the CH 3 COOC 2 H 5 layer was decanted, the remaining solvent was removed by a nitrogen stream to give mitoquinol mesylate as a white powder (380 mg, 66%, 10.3 mCi). Oxygen gas was then bubbled through a solution of tritiated mitoquinol mesylate (200 mg, 0.29 mmol) in 1:2 H 2 O/CH 3 CN (5 mL) for 30 min. The solution was then stirred in air for ~3 days at room temperature. The solution was evaporated under nitrogen at ~30˚C to ~5 mL. H 2 O (~3 mL) was added and it was then freeze-dried to give a sticky orange solid (~200 mg). Column chromatography on silica gel (5 g) and elution with acetonitrile gave mitoquinone mesylate (118 mg, 0.17 mmol, 60% yield, 93% radiopurity by HPLC). All [ 3 H]-MitoQ preparations were further HPLC-purified to >97% radiopurity, as described below, before use in experiments.

To synthesize [ 3 H]-DecylTPP a solution of 1-bromodecane (5.49 mg, 24.8 µmol) in N,N-dimethylformamide (13 µL) was added to [ 3 H]-triphenylphosphine [START_REF] Smith | Targeting coenzyme Q derivatives to mitochondria[END_REF] (1.3 mg, 5 µmol, 11.9 µCi) in a 300 µL reaction vial. The solution was sparged with argon, stirred at 120˚C for 24 h, allowed to cool to room temperature and the residue dissolved in minimum CH 2 Cl 2 (~60 µL) and transferred to a pre-weighed 2.5 mL screw cap glass vial. Addition of C 2 H 5 OC 2 H 5 (2 mL) gave a precipitate and the tube was then centrifuged at 600 x g for 10 min and the supernatant decanted. The residue was re-dissolved in minimum CH 2 Cl 2 and precipitated with C 2 H 5 OC 2 H 5 again. This process was repeated a total of four times. The final solid (~1 mg, 0.58 µCi) was dried under a stream of nitrogen.

[ 3 H]-MitoQ, [ 3 H]-DecylTPP and [ 3 H]-TPMP iodide (60 Ci/mmol, from American Radiolabeled Chemicals) were further purified before use by RP-HPLC. [ 3 H]-MitoQ (<100 nmol; 10-20 Ci/mol) was loaded in 1 mL 45% acetonitrile/0.1% TFA onto a C18 column (Jupiter 5 µm 300 Å, 250 x 4.6 mm, Phenomenex) and was eluted using a gradient of acetonitrile/0.1% TFA (4.5-54% over 5 min, then 54-72% over 20 min) at 1 mL/min. The A 220 of the column eluant was detected using a Gilson UV/VIS 151 spectrophotometer, and 0.5 mL fractions were collected around the [ 3 H]-MitoQ peak, transferred to scintillation vials containing 3 mL Fluoransafe (BDH) and the radioactivity quantified using a Packard Tri-Carb 2800TR liquid scintillation analyser with appropriate quench corrections. Radioactive MitoQ fractions were pooled, lyophilized, resuspended in ethanol at 0.5-2 mM, and stored at -20˚C. Radiopurity was determined by RP-HPLC as above by comparing the radioactivity of product fractions with 0.5 mL fractions collected over the entire gradient. Greater than 97% Synthesis of 10-iododecyltriphenylphosphonium iodide (IDTP) 1,10-Diiododecane (2.4 g, 6.1 mmol) and triphenylphosphine (0.262 g, 1 mmol) were stirred in a Kimax tube at 75-80˚C overnight. The tube was cooled, the reaction dissolved in minimum CH 2 Cl 2 (4 mL) and added dropwise to C 2 H 5 OC 2 H 5 (100 mL). The crude product was isolated by filtration and purified by silica gel chromatography. Elution with 1:1 acetonitrile/CH 2 Cl 2 and acetonitrile gave a product (0.559 g, 0.85 mmol, 85%) that was 97.6% pure as judged by HPLC.

Uptake of [ 3 H]-MitoQ by isolated mitochondria.

Rat liver mitochondria were prepared by homogenization followed by differential centrifugation at 4˚C in 250 mM sucrose, 5 mM HEPES, and 1 mM EGTA, pH 7.2 [29]. For the 86 Rb + uptake experiments buffer pH was adjusted using tetramethylammonium hydroxide (TMA-OH) in place of KOH. The protein concentration was determined by the biuret assay using BSA as a standard [30].

The simultaneous accumulation of 86 Rb + and [ 3 H]-MitoQ or [ 3 H]-TPMP was measured as described [START_REF] Ross | Accumulation of lipophilic dications by mitochondria and cells[END_REF]. Briefly, mitochondria (2 mg protein.mL -1 ) were incubated for 5 min at 30˚C in 250 µl of 250 mM sucrose, 5 mM HEPES, 1 mM EGTA, pH 7.2 (TMA-OH) supplemented with 4 µg.mL -1 rotenone, 0.2 nmol valinomycin.mg protein -1 , 10 mM succinate (TMA salt), 86 RbCl (5 nCi.ml -1 ) and 1 µM [ 3 H]-MitoQ or [ 3 H]-TPMP (3-10 nCi.ml -1 ). The membrane potential was varied by addition of KCl (0-20 mM) ± carbonylcyanide p-(trifluoromethoxy)phenylhydrazone (FCCP; 0.5 µM). Experiments to determine mitochondrial binding in the absence of Δψ m were carried out in the presence of 0.5 µM FCCP/20 mM KCl, and 0.3-1 µM [ 3 H]-MitoQ. After incubation mitochondria were pelleted by centrifugation, and 400 µL supernatant and pellet 3 H and 86 Rb content assayed [START_REF] Ross | Accumulation of lipophilic dications by mitochondria and cells[END_REF]. In parallel experiments the non-mitochondrial sucrose space in the pellets was determined by incubation with [ 14 C]sucrose (50 nCi.mL -1 ) and was used to correct the apparent pellet spaces for non-mitochondrial 86 Rb + or 3 H. These values were then divided by the mitochondrial volume under these conditions (0.6 µL.mg protein -1 ) to give the accumulation ratio (ACR) [START_REF] Brand | Measurement of mitochondrial protonmotive force[END_REF].

! ACR = 400 " DPM pellet DPM supernatant # $ % % & ' ( ( ) sucrose space µL " mg protein )1 ( ) * + , , - . / / mitochondrial volume µL " mg protein )1 ( ) (2)

Cell culture and incubations

Cells were cultured at 37˚C under humidified 95% air/5% CO 2 , in medium supplemented with penicillin (100 U.mL -1 ) and streptomycin (100 µg.mL -1 ). The Jurkat human T lymphocyte cell line was grown in suspension in RPMI medium (Invitrogen) supplemented with 10% fetal calf serum (FCS), and cells were routinely maintained at densities of 0.2-2 x 10 6 cells.mL -1 . C2C12 cells (mouse myoblast; ECACC) were maintained at subconfluence in Dulbecco's modified Eagle medium (DMEM; Invitrogen) supplemented with 10% FCS.

To test the acute toxicity of MitoQ Jurkat cells were seeded at 1 x 10 6 .mL -1 (2 mL) in six-well plates, and incubated with vehicle (DMSO, 0.1% v/v) or mitochondria-targeted compound (0-50 µM) for 24 h. Cell integrity was measured by propidium iodide exclusion [33]. MitoQ and DecylTPP began to affect Jurkat cell viability at 1-2 µM, therefore concentrations of 0.5 µM were used for most experiments. For uptake studies cells were seeded at 3 x 10 6 .mL -1 and supplemented with 500 nM [ 3 H]-MitoQ, [ 3 H]-DecylTPP or [ 3 H]-TPMP, ± FCCP (5 µM), oligomycin/myxothiazol (2 µM of each; to abolish Δψ m selectively) or oligomycin (2 µM)/myxothiazol (2 µM)/gramicidin (10 µg.mL -1 ) (to abolish both plasma membrane potential (Δψ p ) and Δψ m ). All additions were from stocks in DMSO or ethanol, and the final DMSO or ethanol in all incubations was ≤ 0.2% (v/v). For oligomycin/myxothiazol ± gramicidin incubations, cells were incubated with the inhibitors for 30 min before addition of MitoQ. Cell density was measured in parallel incubations containing 500 nM unlabelled MitoQ, and showed negligible cell growth over the incubations shown, therefore cell density at t = 0 was applied at all time points to calculate cellular uptake. Samples (1 mL) were taken at various time points and cells were pelleted by centrifugation (16000 x g for 1 min). Cell uptake was quantified as described [START_REF] Ross | Accumulation of lipophilic dications by mitochondria and cells[END_REF]. The amount of [ 3 H]-MitoQ in the pellet was expressed in pmol per million cells, or as an accumulation ratio, calculated assuming a water volume of 0.2 pL for a Jurkat cell [START_REF] Scarlett | Changes in mitochondrial membrane potential during staurosporineinduced apoptosis in Jurkat cells[END_REF].

Measurement of plasma membrane potential

Jurkat cells (3 x 10 6 /mL) in Dulbecco's PBS supplemented with CaCl 2 and MgCl 2 , glucose (25 mM) and FCS (0.5%) were incubated for 1 h at 37˚C with the proprietary Plasma Membrane Potential Indicator (PMPI; a component of the FLIPR Membrane Potential Assay Kit, Explorer format -blue; Molecular Devices), added to 0.5 µL.mL -1 from a stock solution reconstituted according to the manufacturer's instructions. PMPI fluorescence in stirred cell suspensions in a 3 mL cuvette was detected over time in a Shimadzu RF 5301-PC fluorimeter (λ ex = 542 nm, slit width 3 nm; λ em = 553 nm, slit width 5 nm), and the effect of adding oligomycin, myxothiazol (both 5 µM) and gramicidin (10 µg.mL -1 ) was measured.

Extraction of MitoQ from cells and mitochondria

To determine the redox state of [ 3 H]-MitoQ in cells, Jurkat cells (3 x10 6 cells/mL) were incubated in 6 mL of medium supplemented with 500 nM [ 3 H]-MitoQ. Where the effect of respiratory inhibitors was tested, cells were incubated with inhibitors or vehicle (ethanol 0.2% v/v) for 30 min prior to [ 3 H]-MitoQ addition. Aliquots (4 mL) of the cell suspension were then transferred to two 2 mL Eppendorf tubes and pelleted by centrifugation (16000 x g for 1 min). The supernatant was aspirated, pellets were briefly centrifuged (10 s), and residual supernatant removed with a piece of rolled-up tissue paper, then 250 µL of 90% acetonitrile/0.1% TFA was added and the mix was immediately vortexed vigorously. Samples were centrifuged (16000 x g for 1 min) and 200 µL supernatant removed to fresh tubes, pooling within samples to give a total of 400 µL, which was then recentrifuged and samples (300 µL) were snap-frozen on dry ice. For HPLC samples were diluted with 0.1% TFA to give 45% acetonitrile/0.1% TFA, and analyzed immediately by RP-HPLC. Fractions (0.25 mL) were collected over a window where both redox forms of MitoQ eluted. MitoQuinol (MitoQH 2 ) eluted at ~14.5 min/64% acetonitrile and MitoQuinone eluted at ~16.5 min/67% acetonitrile. Fractions were transferred to a scintillation vial containing 3 mL Fluoransafe scintillant and the radioactivity determined. To determine the redox state of MitoQ in the extracellular medium, 1 mL of the supernatant obtained after pelleting cells was added to 1 mL acetonitrile/0.2% TFA, vortexed vigorously and centrifuged (16 000 x g for 5 min). Supernatant (1.96 mL) was removed to a fresh tube, centrifuged again and a sample (1.92 mL) analyzed immediately by RP-HPLC. Mock extractions of chemically reduced [ 3 H]-MitoQH 2 added to cell pellets immediately after addition of 250 µL 90% acetonitrile/0.1% TFA showed negligible oxidation of [ 3 H]-MitoQH 2 during extraction and freeze-thawing.

To determine the redox state of [ 3 H]-MitoQ after incubation with mitochondria, rat liver mitochondria (250 µg protein.mL -1 ) were incubated in 250 mM sucrose, 5 mM HEPES, and 1 mM EGTA, pH 7.4 supplemented with 500 nM [ 3 H]-MitoQ and with other substrates and inhibitors as indicated, for 5 min at 37˚C. The mitochondria were then pelleted by centrifugation (16000 x g for 5 min) and the pellets were extracted as described for cells, except that all centrifugation steps were 1 min; under these conditions there was negligible oxidation of added [ 3 H]-MitoQH 2 during extraction, but freeze-thawing caused some oxidation of [ 3 H]-MitoQH 2 , so mitochondrial extracts were analyzed by HPLC immediately after extraction.

To identify metabolites of [ 3 H]-MitoQ, cells (3 x 10 6 .mL -1 ) were incubated with 500 nM [ 3 H]-MitoQ for up to 72 h, before pelleting the cells and extracting MitoQ and its metabolites from the pellet. RP-HPLC was done as above, except that 60 x 0.5 mL fractions were collected, spanning the initial flowthrough and the entire gradient. The radioactivity in each fraction was quantified by scintillation counting of 50 µL of the eluant. Any fractions other than [ 3 H]-MitoQ and [ 3 H]-MitoQH 2 that contained >5% of the total eluted radioactivity after 48 h incubation were noted, and the same fractions collected from a parallel cell incubation with nonradioactive MitoQ. These fractions were analyzed by ESI-MS by direct infusion into a 4000 QTrap LC/MS/MS (Applied Biosystems) in positive ion mode and analysis at 150˚C, ion spray voltage 4500 V. Calibration was by polyethylene glycol. Data were analyzed using Analyst software.

Measurement of serum binding of MitoQ and TPMP

Binding of [ 3 H]-MitoQ or [ 3 H]-TPMP to serum, which was present at 10% (v/v) for all cell incubations, were determined by equilibrium dialysis [START_REF] Nobes | Non-ohmic proton conductance of the mitochondrial inner membrane in hepatocytes[END_REF]. Two dialysis cassettes (Pierce; molecular weight cut-off 3500 Da) containing 2 mL 10% FCS/PBS were placed in 500 mL PBS supplemented with [ 3 H]-MitoQ (specific activity 5.6 Ci/mol; 500 nM) or [ 3 H]-TPMP (specific activity 10.4 Ci/mol; 200 nM), and incubated with stirring at 37˚C for up to 48 h. Samples (500 µL) were taken from the PBS medium and from dialysis cassettes, mixed with 3 mL Fluoransafe scintillant and the radioactivity quantified and compared to determine the binding to serum. [ 3 H]-MitoQ and [ 3 H]-TPMP uptake into the dialysis cassettes stabilized within 48 h. At this time point, 78% of [ 3 H]-MitoQ was bound to serum while there was negligible binding of [ 3 H]-TPMP to serum.

Confocal microscopy

Fibroblasts were grown to semi-confluence on 22 mm diameter glass cover slips in 6-well culture plates. Cells were incubated with IDTP (1 µM) for 3.5 h ± FCCP (5 µM). MitoTracker Orange (Molecular Probes; 25 nM) was added at 3 h. Immunocytochemistry was carried out as described [START_REF] Ross | Cellpenetrating peptides do not cross mitochondrial membranes even when conjugated to a lipophilic cation: evidence against direct passage through phospholipid bilayers[END_REF], and cells were visualized on a Zeiss LSM 510 Meta inverted confocal microscope using a 63x objective, 488 and 543 nm laser lines and 515/30 and 560LP filter sets. The uptake of MitoQ by mitochondria is described by the Nernst equation Hydrophobic lipophilic cations such as MitoQ (Figure 1) are extensively internalized by mitochondria driven by the membrane potential (Δψ m ), but it is unclear if their significant potential-independent binding to membranes causes deviations from Nernstian behavior [START_REF] Kelso | Selective targeting of a redox-active ubiquinone to mitochondria within cells[END_REF].

RESULTS

Biochemical

To address this we measured the uptake and binding of [ 3 H]-MitoQ by isolated mitochondria over a range of Δψ m values while simultaneously measuring 86 Rb + uptake (Figure 2). In the presence of valinomycin 86 Rb + uptake is solely determined by Δψ m [START_REF] Brand | Measurement of mitochondrial protonmotive force[END_REF][START_REF] Brown | Thermodynamic control of electron flux though mitochondrial cytochrome bc 1 complex[END_REF]. Dual isotope counting enabled the apparent pellet spaces of 86 Rb + and [ 3 H]-MitoQ to be determined simultaneously over a Δψ m range set by various amounts of KCl [START_REF] Ross | Accumulation of lipophilic dications by mitochondria and cells[END_REF][START_REF] Brown | Thermodynamic control of electron flux though mitochondrial cytochrome bc 1 complex[END_REF]. From the known mitochondrial volume, and parallel measurement of the distribution of membraneimpermeant [ 14 C]sucrose to calculate the non-mitochondrial pellet space, the accumulation ratios (= [compound] mitochondria /[compound] supernatant ) for [ 3 H]-MitoQ and 86 Rb + were calculated [START_REF] Ross | Accumulation of lipophilic dications by mitochondria and cells[END_REF]. Applying the Nernst equation to the 86 Rb + accumulation ratio enabled the MitoQ accumulation ratio to be plotted over a range of known Δψ m values.

In Figure 2A the accumulation ratio of the simple lipophilic cation [ 3 H]-TPMP is plotted against that for 86 Rb + (lower x-axis) over a range of Δψ m values (upper x-axis). As expected, the relationship is linear up to the highest 86 Rb + accumulation ratio (~900, Δψ m ~174 mV) indicating that [ 3 H]-TPMP uptake is described by the Nernst equation. The slope is greater than unity because a fixed proportion of matrix TPMP is bound. A slope of ~2.1 means that ~53% of intramitochondrial TPMP is membrane-bound and the intercept with the y-axis close to the origin indicates that its binding to unenergized mitochondria is negligible [START_REF] Brand | Measurement of mitochondrial protonmotive force[END_REF][START_REF] Brown | Thermodynamic control of electron flux though mitochondrial cytochrome bc 1 complex[END_REF]. Plotting the [ 3 H]-MitoQ accumulation ratio against that of 86 Rb + also gave a straight line (Figure 2B), but this line passed through the y-axis well above the origin (accumulation ratio ~8600) due to substantial binding of [ 3 H]-MitoQ to unenergized mitochondria (Figure 2B). The slope was 10.3 indicating that >90% of intramitochondrial MitoQ is bound with <10% free in the matrix. This is consistent with the greater hydrophobicity of MitoQ (the octan-1-ol:PBS partition coefficients for MitoQ and TPMP are ~3000 and ~0.35, respectively [START_REF] Asin-Cayuela | Fine-tuning the hydrophobicity of a mitochondria-targeted antioxidant[END_REF]) and with its high affinity for phospholipid bilayers [START_REF] James | Interactions of mitochondria-targeted and untargeted ubiquinones with the mitochondrial respiratory chain and reactive oxygen species. Implications for the use of exogenous ubiquinones as therapies and experimental tools[END_REF]. Thus, the Δψ m -dependent uptake of MitoQ by mitochondria is fully described by the Nernst equation.

To separate Δψ m -independent and -dependent binding we incubated unenergized mitochondria with 0.3-1 µM [ 3 H]-MitoQ and measured free and bound [ 3 H]-MitoQ, which gave a binding constant of 5.1 (pmol.mg protein -1 ).nM -1 (Figure 2C). As the mitochondrial volume is 0.6 µl.mg protein -1 this corresponds to an accumulation ratio of 8500 for uncoupled mitochondria, consistent with the intercept with the y-axis in Figure 2B. The TPMP binding constant was <1% of that for MitoQ (~0.038 (pmol.mg protein -1 ).nM -1 ) (Figure 2C). From this binding constant and the known free concentration of MitoQ we used the data from Figure 2B to calculate the Δψ m -independent mitochondrial binding of MitoQ, which was subtracted from the total accumulated to generate the Δψ m -dependent mitochondrial accumulation (Figure 2D). This plot was also a straight line of slope 10.3, further confirming that MitoQ uptake was Nernstian. Note that the y-axis intercept in Figure 2B depends solely on the free concentration of MitoQ and the mitochondrial amount, as described by Figure 2C, whereas the accumulation ratio-dependence on Δψ m is independent of [MitoQ].

From these data it was possible to model the Δψ m -dependent uptake of MitoQ and TPMP as a function of Δψ m at an external free concentration of 10 nM (Figure 2E), and as a function of external free concentration at Δψ m = 150 mV (Figure 2F). These show that the Δψ m -dependent MitoQ uptake by mitochondria was always ~5-fold greater than that of TPMP, even though the Nernst equation predicts identical ratios of free MitoQ and TPMP inside and outside mitochondria at a given Δψ m . This occurs because the greater binding of MitoQ within the mitochondrial matrix increases its Δψ m -dependent uptake. Therefore the Δψ m -dependent uptake of hydrophobic TPP compounds such as MitoQ is fully described by the Nernst equation and is greater than more hydrophilic TPP compounds.

Rapid and extensive uptake of MitoQ and DecylTPP by cells

It was unknown if the greater hydrophobicity of TPP cations such as MitoQ increases or decreases the rate and extent of their uptake into cells compared to more hydrophilic cations such as TPMP. To address this we compared the uptake of MitoQ and DecylTPP with that of TPMP into Jurkat T lymphocytes. Use of a suspension cell line facilitates uptake studies because they can be rapidly isolated from the incubation medium by centrifugation [START_REF] Scarlett | Changes in mitochondrial membrane potential during staurosporineinduced apoptosis in Jurkat cells[END_REF]. We incubated cells with [ 3 H]-MitoQ and isolated cells at various times over 24 h and measured the [ 3 H]-MitoQ in the cell pellet and supernatant (Figure 3A). This experiment was repeated over a shorter time scale to show uptake over the first 40 min of incubation (Figure 3B). [ 3 H]-MitoQ uptake into cells was rapid, with a steady state reached within ~12 min and remaining stable for at least 24 h (Figs. 3A &B). In contrast, uptake of TPMP was slow, taking ~6-8 h to reach a steady state (Figure 3A,B). To see if these differences in uptake were due to hydrophobicity or were specific for the ubiquinone moiety of MitoQ, we measured the uptake of another hydrophobic TPP derivative DecylTPP, which lacks a ubiquinone (Figure 1). Uptake of DecylTPP was as rapid as that of MitoQ, quickly reaching a steady state (Figure 3B) and thereafter remaining stable (data not shown). Therefore increasing the hydrophobicity of TPP cations greatly increases their rate of uptake into cells.

To see if increasing the hydrophobicity of TPP cations also affected their efflux rate, we incubated cells with MitoQ or TPMP for 3 or 6 h, respectively, and then abolished Δψ m with FCCP and measured efflux over 90 min (Figure 3C). Efflux of MitoQ was rapid and was complete within 10 min, while efflux of TPMP took an hour (Figure 3C). Efflux showed first order kinetics, enabling rate constants and half lives (t 1/2 ) to be calculated: k = 0.16 min -1 and t 1/2 ~4 min for MitoQ; k = 0.03 min -1 and t 1/2 ~24 min for TPMP (Figure 3C, inset). This 6fold faster efflux of MitoQ compared with TPMP is consistent with the relative rates of cell uptake of the two compounds and suggests that the rate of movement of hydrophobic TPP compounds into and out of cells is significantly faster than for more hydrophilic compounds such as TPMP.

Substantially more MitoQ and DecylTPP were taken up by cells than TPMP (Figs. 3A &B). As the volume of Jurkat cells is ~0.2 pL per cell [START_REF] Scarlett | Changes in mitochondrial membrane potential during staurosporineinduced apoptosis in Jurkat cells[END_REF] the uptake of compounds can be expressed as an accumulation ratio (ACR = [TPP cation] cell /[TPP cation] supernatant ) and for MitoQ the accumulation ratio was 921 ± 104 at 3 h, 4.75-fold greater than that for TPMP (192 ± 56) (Figure 3D). When the uncoupler FCCP was added the accumulation ratios of MitoQ, DecylTPP and TPMP decreased by 81%, 87% and 79%, respectively (Figure 3D). These data are consistent with Δψ m -dependent uptake of all three TPP compounds into mitochondria after initial uptake into cells.

Therefore hydrophobic TPP compounds such as MitoQ and DecylTPP are more rapidly taken up into cells than the hydrophilic cation TPMP. These hydrophobic molecules are also 4-10 fold more extensively accumulated by cells than TPMP, consistent with their greater uptake into isolated mitochondria (Figure 2).

Within cells hydrophobic TPP cations predominantly localize to mitochondria

FCCP led to the release of the TPP compounds from the cell, indicating that uptake of intracellular MitoQ, DecylTPP and TPMP is driven by Δψ m (Figure 3D). However, directly demonstrating the mitochondrial localization of TPP compounds within cells is challenging, as they rapidly redistribute upon subcellular fractionation. To circumvent this, we investigated the uptake of a MitoQ analogue, IDTP (Figure 4). Like MitoQ and DecylTPP, IDTP contains the TPP targeting moiety and a ten-carbon aliphatic alkyl chain, but also has an iodo moiety that reacts slowly with protein thiols to form a stable thioether bond [START_REF] Lin | Specific modification of mitochondrial protein thiols in response to oxidative stress: a proteomics approach[END_REF] (Figure 1). Thus the extent of covalent binding of IDTP to intracellular proteins is a reflection of its steady-state concentration within a cytosolic compartment. The TPP moiety of IDTP can be detected using anti-TPP antiserum enabling the localization of IDTP binding by immunocytochemistry [START_REF] Lin | Specific modification of mitochondrial protein thiols in response to oxidative stress: a proteomics approach[END_REF]. IDTP was taken up rapidly into isolated mitochondria in a Δψ mdependent manner, similar to MitoQ and DecylTPP (data not shown). In fibroblasts IDTP localized exclusively to mitochondria and this uptake was prevented by FCCP (Figure 4). In the C2C12 myoblast cell line IDTP localization was also mitochondrial and was prevented by FCCP and by oligomycin/myxothiazol (data not shown). The reactivity of protein thiols (pK a typically 8-9) with IDTP within the mitochondrial matrix (pH ~8) is likely to be higher than in the cytosol (pH 7.2), because it is the thiolate form that reacts with IDTP [START_REF] Lin | Specific modification of mitochondrial protein thiols in response to oxidative stress: a proteomics approach[END_REF]. However, there was negligible cytoplasmic IDTP binding in fibroblasts after 6 h incubation, suggesting that the steady state concentration of IDTP in the cytosol is far lower than in mitochondria. Together these data strongly suggest that within cells hydrophobic TPP compounds are predominantly concentrated within mitochondria driven by the Δψ m .

Cell uptake of MitoQ is consistent with the Nernst equation

The uptake of hydrophilic TPP cations such as TPMP into cells occurs in response to both the plasma membrane potential (Δψ p ) and the mitochondrial membrane potential (Δψ m ) as described by the Nernst equation [START_REF] Scott | Energy transduction in intact synaptosomes[END_REF][START_REF] Brand | Membrane potential of mitochondria in intact lymphocytes during early mitogenic stimulation[END_REF]. Uptake is determined experimentally as an accumulation ratio (ACR = [TPP cation] cell /[TPP cation] supernatant ) and this is related to Δψ p and Δψ m by equation 3 [START_REF] Scott | Energy transduction in intact synaptosomes[END_REF], where V c and V m are the volumes of the cytosol and mitochondria, and a s , a c and a m are the activity coefficients of the compounds in the extracellular environment, cytosol and the mitochondrial matrix, respectively (the activity coefficient is the proportion of compound in that compartment that is free in solution and not bound).

! ACR =

[TPPcation]Cell [TPPcation]Supernant = as • 10

"#p 61.5 • V c a c + Vm am •10 "#m 61.5 $ % & ' & ( ) & * & V c + V m (3)
In Jurkat cells the plasma membrane potential is 40 mV (negative inside) [START_REF] Scarlett | Changes in mitochondrial membrane potential during staurosporineinduced apoptosis in Jurkat cells[END_REF] and the volume is 200 nL/10 6 cells with ~5% occupied by mitochondria [START_REF] Scarlett | Changes in mitochondrial membrane potential during staurosporineinduced apoptosis in Jurkat cells[END_REF], giving values for V m and V c of 10 nL/10 6 cells and 190 nL/10 6 cells, respectively. We determined that the serum binding of TPMP in these experiments was negligible, so a s = 1. The cytoplasmic binding of TPMP within hepatocytes, a c = 0.21 [START_REF] Brand | Measurement of mitochondrial protonmotive force[END_REF], was taken as the value for a c in Jurkat cells. The binding of TPMP within mitochondria, determined from the reciprocal of the slope of Figure 2A, gives a m = 0.47. These parameters, together with the TPMP ACR of 192 from Figure 3D, give Δψ m = 157 mV from equation 3, which is in the expected range [START_REF] Brand | Measurement of mitochondrial protonmotive force[END_REF]. Thus equation 3 describes the Nernstian uptake of TPP cations by cells in our experiments.

If the uptake of MitoQ into Jurkat cells is Nernstian then its ACR should also be described by equation 3. To assess this we require the activity coefficients of MitoQ in the three compartments. MitoQ was ~78% bound to serum in the incubation medium, consequently a s = 0.22. The intramitochondrial binding of MitoQ, from the reciprocal of the slope in Figure 2B, indicates that a m = 0.097. The extensive binding of MitoQ to deenergized cells makes it technically difficult to determine a reliable value for a c [START_REF] Nobes | Non-ohmic proton conductance of the mitochondrial inner membrane in hepatocytes[END_REF]. This would [START_REF] Nobes | Non-ohmic proton conductance of the mitochondrial inner membrane in hepatocytes[END_REF], as was done for mitochondria in Figure 2B. However for cells it is only possible to vary Δψ p over about 60 mV [START_REF] Nobes | Non-ohmic proton conductance of the mitochondrial inner membrane in hepatocytes[END_REF]. As can be seen in Figure 2B the extensive non-specific binding of MitoQ would render any Δψ p -sensitive uptake negligible relative to random errors in binding below Δψ p of ~120 mV.

To determine a c by an alternative approach we assume that equation 3 does describe MitoQ uptake, then we can use it to calculate a c , from Δψ m = 157 mV, the known values for V m , V c and Δψ p , and the values of a s and a m that we have derived for MitoQ. Entering these values, along with the ACR for MitoQ of 921 from Figure 3D, gives a c = 0.002. This indicates that our data on the uptake of MitoQ into cells are consistent with Nernstian uptake, provided ~99.8% of the MitoQ in the cytosol is bound.

To check if this estimate for extensive (>99%) cytosolic binding of MitoQ was valid, we calculated a c by an alternative method based on different starting assumptions. For the Jurkat cell incubation in Figure 3 the total [MitoQ] added was 500 nM which after equilibration reached a steady state total [MitoQ] in the extracellular medium of 260 ± 5 nM (Figure 3D and data not shown). As ~78 % of extracellular MitoQ is bound to serum the free concentration is 57 nM. Assuming Nernstian uptake into the cytosol and mitochondria driven by a Δψ p of 40 mV and a Δψ m of 157 mV, the cytosolic and mitochondrial free MitoQ concentrations will be 255 nM and 91 µM, respectively. From the cytosolic and mitochondrial volumes (190 nL and 10 nL/10 6 cells) this gives 0.048 and 0.91 pmol MitoQ/10 6 cells free in the cytosol and mitochondria, respectively. The total amount of cytosolic MitoQ can be estimated as that lost when cells incubated with myxothiazol and oligomycin to abolish Δψ m are further treated with gramicidin to abolish Δψ p (Figure 5C). The total amount of mitochondrial MitoQ can be estimated as that which is sensitive to abolishing Δψ m with myxothiazol and oligomycin (Figure 5A). From these experiments the total amounts of MitoQ present in the cytosolic and mitochondrial compartments are 16 pmol and 29.3 pmol MitoQ/10 6 cells, respectively. Comparing these values with the free concentration of MitoQ estimated from the Nernst equation indicates that in the cytosolic free MitoQ is ~0.3% of the total, with ~99.7% bound. In mitochondria ~3% of the total MitoQ is free with ~97% bound. This estimate of ~99.7% of cytosolic MitoQ bound is comparable to our earlier estimate of 99.8% bound, suggesting that a c = 0.002 for MitoQ is a reasonable estimate.

To summarize, uptake of MitoQ into cells is consistent with the Nernst equation, provided that binding of MitoQ in the cytosol is >99%. It was not possible to determine directly MitoQ binding in the cytosol, but two different calculations suggest that cytosolic MitoQ is >99% bound. MitoQ is far more hydrophobic than TPMP: octano-1-ol partition coefficients for MitoQ and TPMP are 2760 and 0.35, respectively [START_REF] Asin-Cayuela | Fine-tuning the hydrophobicity of a mitochondria-targeted antioxidant[END_REF]; affinity of MitoQ for unenergized mitochondrial membranes is 134-fold greater than that of TPMP (Figure 2C); and 78% of MitoQ was bound to serum in the incubation medium while binding of TPMP was negligible. In hepatocytes ~80% of cytoplasmic TPMP is bound [START_REF] Brand | Measurement of mitochondrial protonmotive force[END_REF], making >99% binding of the far more hydrophobic MitoQ plausible. Therefore our findings are consistent with uptake of MitoQ into cells being described by the Nernst equation with most intracellular compound being bound.

Different rates of uptake of TPP cations across the plasma membrane

The relatively hydrophobic TPP cations MitoQ and DecylTPP are far more rapidly taken up and released by cells than the hydrophilic TPMP cation (Figure 3). This discrepancy is not due to differences in rates of uptake into mitochondria as TPMP and MitoQ are both taken up rapidly into isolated mitochondria with >90 % of uptake complete within 1 min [START_REF] Asin-Cayuela | Fine-tuning the hydrophobicity of a mitochondria-targeted antioxidant[END_REF][START_REF] Smith | Targeting coenzyme Q derivatives to mitochondria[END_REF]. This suggests that the different uptake rates into cells are due to plasma membrane differences.

To measure the uptake of TPP compounds across the plasma membrane without the confounding effects of subsequent mitochondrial uptake, we abolished Δψ m with oligomycin and myxothiazol. While this loss of Δψ m decreased the extent of uptake of MitoQ (Figure 5A) and DecylTPP (data not shown), a steady state distribution was reached within 5 min (Figure 5A). Oligomycin and myxothiazol did not disrupt Δψ p over the time scale of these experiments, as measured by the fluorescent Δψ p indicator PMPI [40] (Figure 5B). Subsequent addition of the ionophore gramicidin collapsed Δψ p (Figure 5B) and further decreased uptake of MitoQ (Figure 5C) and DecylTPP (data not shown). Therefore the gramicidin-sensitive uptake of MitoQ and DecylTPP in the presence of myxothiazol and oligomycin is rapid and is primarily driven by Δψ p . In contrast, uptake of TPMP under these conditions was far slower than that of MitoQ or DecylTPP and was mostly prevented when Δψ p was abolished with gramicidin (Figure 5D). It is concluded that hydrophobic TPP compounds are taken up more rapidly into cells than TPMP because they cross the plasma membrane far more quickly.

Redox state of MitoQ within mitochondria

The reduction of MitoQ to its ubiquinol form MitoQH 2 by the respiratory chain is thought to be essential for it to prevent lipid peroxidation [START_REF] Kelso | Selective targeting of a redox-active ubiquinone to mitochondria within cells[END_REF][START_REF] Koopman | Inhibition of complex I of the electron transport chain causes O 2 -. -mediated mitochondrial outgrowth[END_REF]. In simple in vitro systems, MitoQ is rapidly reduced by complex II, but in cells and isolated mitochondria little is known about the redox state of MitoQ or its rate of equilibration with endogenous electron donors. We therefore set out to investigate reduction of [ 3 H]-MitoQ by mitochondria (Figure 6).

After incubation of mitochondria with [ 3 H]-MitoQ, the ubiquinone ([ 3 H]-MitoQ) and ubiquinol ([ 3 H]-MitoQH 2 ) forms were extracted into acidified acetonitrile, separated by HPLC, and quantitated by scintillation counting (Figure 6A). The ratio of the area under the [ 3 H]-MitoQH 2 peak to the total amount recovered gave the percent reduction of MitoQ. Control experiments with exogenous [ 3 H]-MitoQH 2 showed that this extraction and analysis procedure preserved the MitoQ redox state. In mitochondria energized with succinate, 75 ± 8% of [ 3 H]-MitoQ was reduced (Figure 6B). Succinate-mediated reduction was largely abolished by the succinate dehydrogenase inhibitor malonate (17 ± 5% reduced), similar to the 24 ± 4% reduced found in the absence of exogenous substrate. In glutamate/malateenergized mitochondria, 61 ± 16% of MitoQ was reduced, and this was significantly decreased by rotenone (30 ± 3% reduced). FCCP abolished the Δψ m , thereby decreasing the amount of MitoQ associated with mitochondria (data not shown), but led to only a slight decrease in the extent of MitoQ reduction (Figure 6B).

As respiratory inhibitors abolish Δψ m they affect MitoQ uptake as well as inhibiting potential electron donors, complicating interpretation of their effects on MitoQ redox state. To overcome this we incubated mitochondria with MitoQ in the presence of FCCP, along with various inhibitors (Figure 6C). Under these conditions the complex III inhibitor myxothiazol further increased MitoQ reduction in succinate-energised mitochondria, and counteracted the inhibition of MitoQ reduction by malonate (Figure 6C). Similarly in glutamate/malate-energised mitochondria, myxothiazol also increased MitoQ reduction and counteracted inhibition of this reduction by rotenone (Figure 6C). As myxothiazol increases the level of reduction of the Coenzyme Q (CoQ) pool, these findings suggest that in mitochondria MitoQ becomes reduced in response to the redox state of the endogenous CoQ pool. Direct exchange between MitoQ and the endogenous CoQ pool is thermodynamically restricted in the low dielectric environment of the membrane [START_REF] Murphy | Slip and leak in mitochondrial oxidative phosphorylation[END_REF], so the interaction between MitoQ is known to be a good substrate for complex II making it a good candidate for this reaction [START_REF] James | Interactions of mitochondria-targeted and untargeted ubiquinones with the mitochondrial respiratory chain and reactive oxygen species. Implications for the use of exogenous ubiquinones as therapies and experimental tools[END_REF][START_REF] James | Interaction of the mitochondria-targeted antioxidant MitoQ with phospholipid bilayers and ubiquinone oxidoreductases[END_REF]. However a contribution from complex I in the presence of the endogenous CoQ pool is also possible, even though the isolated complex is unreactive [START_REF] James | Interaction of the mitochondria-targeted antioxidant MitoQ with phospholipid bilayers and ubiquinone oxidoreductases[END_REF].

Redox state and metabolism of MitoQ within cells

When [ 3 H]-MitoQ was added to cells it was rapidly taken up and converted to the ubiquinol form within 10 min and then remained stable at 50-60% reduced (Figure 7A), similar to the redox state in isolated mitochondria. The redox state of [ 3 H]-MitoQ in the extracellular medium also became reduced over time, equilibrating with the redox state of intracellular MitoQ within 2 h (Figure 7A), presumably by molecular exchange.

To see whether MitoQ reduction within cells was set by cell metabolism, we added the reduced form, [ 3 H]-MitoQH 2 . [ 3 H]-MitoQH 2 was rapidly oxidized and within 20 min reached a stable steady state level of 40-60% reduction, the same as that following addition of oxidized MitoQ (data not shown). The redox state of [ 3 H]-MitoQ was also unaffected when the amount of added MitoQ was varied from 125 nM to 1 µM, or when the [O 2 ] was decreased from 21% to 3 % (data not shown). The reduction of exogenous MitoQ was not cell type-specific, as [ 3 H]-MitoQ added to C2C12 cells rapidly reached a steady state level of reduction similar to that of Jurkat cells (data not shown). When cells were incubated with [ 3 H]-MitoQ in the presence of rotenone, the [ 3 H]-MitoQ became less reduced (30 ± 4%) while addition of the complex III inhibitor antimycin caused the [ 3 H]-MitoQ to become more reduced (87 ± 7%) (Figure 7B). Together these data indicate that the redox state of MitoQ within cells responds to that of the mitochondrial CoQ pool, and that reduction of MitoQ occurs rapidly, consistent with its fast uptake into mitochondria within cells.

To see how stable MitoQ was within cells, we incubated Jurkat cells with [ 3 H]-MitoQ for 1, 24 and 48 h, and assayed for metabolites of [ 3 H]-MitoQ by separating cell extracts by RP-HPLC and measuring the radioactivity in all fractions (Figure 7C). Over 48 h [ 3 H]-MitoQ remained predominantly (~60-80%) in the reduced form (data not shown). After 1 h 99% of the radioactivity co-eluted with [ 3 H]-MitoQH 2 or [ 3 H]-MitoQ (Figure 7C), with no detectable MitoQ metabolites. At 24 and 48 h one new peak that accounted for 1-2% of the 3 H radioactivity at 24 h and 12 ± 6 % at 48 h appeared on the HPLC trace. To identify the metabolite, equivalent fractions from parallel 48 h incubations with unlabelled MitoQ were isolated by HPLC and analyzed by ESI-MS. The metabolite peak contained a species of m/z = 665.3 that by tandem mass spectrometry readily fragmented to MitoQH 2 (m/z = 585.4) with a loss of 79.9 Da. The MitoQ mass was further fragmented to give a daughter ion of 262 Da, diagnostic of the TPP moiety confirming that the metabolite was a derivative of MitoQ. These data are consistent with the metabolite being sulfated MitoQ (m/z = 665.2 Da) which on loss of a sulfate (80.05 Da) regenerates MitoQuinol (Figure 1). This is in agreement with animal and cell data where monosulfated MitoQ was a major metabolite [START_REF] Li | Quantitation and metabolism of mitoquinone, a mitochondria-targeted antioxidant, in rat by liquid chromatography/tandem mass spectrometry[END_REF][START_REF] Li | Transport and metabolism of MitoQ 10 , a mitochondria-targeted antioxidant, in Caco-2 cell monolayers[END_REF]. Therefore, within cells MitoQ is rapidly reduced and attains a stable steady state level of the ubiquinol over long term incubations. Over 48 h some MitoQ is metabolised to a sulfated derivative, but the majority remains in the active ubiquinol form.

DISCUSSION

The targeting of probes and bioactive molecules to mitochondria by conjugation to lipophilic cations such as TPP plays an increasing role in reporting on and modifying mitochondrial function. These molecules generally comprise relatively bulky and hydrophobic groups attached to a TPP moiety that drives their selective uptake into mitochondria. However, it was unknown how these hydrophobic additions to the TPP molecule affected the rate and extent of their uptake into mitochondria within cells. Here, by comparing the uptake of MitoQ and DecylTPP into mitochondria and cells with the hydrophilic TPP cation TPMP, we have shown how the hydrophobicity of TPP cations affects their uptake.

The uptake of MitoQ into mitochondria and cells was described by the Nernst equation. Previous preliminary studies suggested that saturation of binding might lead to deviations from Nernstian behavior [START_REF] Kelso | Selective targeting of a redox-active ubiquinone to mitochondria within cells[END_REF]. However, comparing the uptake of MitoQ with that of 86 Rb + showed that MitoQ uptake into mitochondria is fully described by the Nernst equation and that MitoQ bound extensively (>90%) within the mitochondrial matrix. Due to this binding, the total amount in mitochondria is about 10-fold greater than simple equilibration of the free compound with the Δψ m , and was ~5-fold greater than that of TPMP. This binding is most likely to be to the matrix facing surface of the inner membrane, with the TPP compounds adsorbed as a monolayer on the membrane with the TPP cation in a potential energy well close to the surface with the hydrophobic side chain inserted into the membrane [START_REF] Ross | Lipophilic triphenylphosphonium cations as tools in mitochondrial bioenergetics and free radical biology[END_REF][START_REF] James | Interaction of the mitochondria-targeted antioxidant MitoQ with phospholipid bilayers and ubiquinone oxidoreductases[END_REF]. Such a location for hydrophobic TPP derivatives is fortuitous as many mitochondrial activities are associated with the inner membrane.

The extent of uptake of MitoQ into mitochondria within cells was also 4-5-fold greater than that of TPMP and was also consistent with the Nernst equation. In the cytosol most MitoQ was bound. The uptake was selective for mitochondria with the majority of MitoQ in the cell being concentrated within mitochondria due to the Δψ m .

Increasing the hydrophobicity of TPP molecules increased their rate of uptake into cells several-fold. This increase was due to the far more rapid permeation of the plasma membrane by the hydrophobic TPP compounds. The reason for this is likely to be the greater hydrophobicity of MitoQ and DecylTPP lowering their activation energies for plasma membrane passage (octan-1-ol:PBS partition coefficients: MitoQ ~3000, DecylTPP ~5000, TPMP = 0.35) [START_REF] Asin-Cayuela | Fine-tuning the hydrophobicity of a mitochondria-targeted antioxidant[END_REF]. The activation energy for transport of lipophilic cations through a phospholipid bilayer is made up of an unfavorable Born energy due to the charge of the TPP moiety that is the same for all TPP cations and a favorable hydrophobic force that is the main difference between molecules such as MitoQ and TPMP [START_REF] Ross | Lipophilic triphenylphosphonium cations as tools in mitochondrial bioenergetics and free radical biology[END_REF]. That the plasma membrane acts as a significant kinetic barrier to movement of hydrophilic TPP cations is strongly supported by the fact that the lipophilic tetraphenylborate anion (TPB) is routinely added to speed up equilibration of TPMP into cells and does so by lowering the activation energy for membrane transport [START_REF] Ross | Lipophilic triphenylphosphonium cations as tools in mitochondrial bioenergetics and free radical biology[END_REF][START_REF] Brand | Membrane potential of mitochondria in intact lymphocytes during early mitogenic stimulation[END_REF][START_REF] James | Altered mitochondrial function in fibroblasts containing MELAS or MERRF mitochondrial DNA mutations[END_REF]. TPB is not required to assist the uptake of TPMP through the mitochondrial inner membrane suggesting that the activation energy for transport across this membrane is lower. Lipophilic dications are also more readily taken up through the mitochondrial inner membrane than the plasma membrane and increasing their hydrophobicity increases their uptake through the plasma membrane [START_REF] Ross | Accumulation of lipophilic dications by mitochondria and cells[END_REF]. Several factors may contribute to this difference in activation energies between the mitochondrial and plasma membranes, including their different phospholipid compositions, lack of cholesterol and presence of cardiolipin in the inner membrane, the difference in Δψ values and the very high protein content of the inner membrane which may increase its dielectric constant [START_REF] Murphy | Slip and leak in mitochondrial oxidative phosphorylation[END_REF].

Once past the plasma membrane MitoQ and DecylTPP were taken up rapidly into mitochondria, as evidenced by the predominantly mitochondrial localisation of the MitoQ analogue IDTP and by the fact that the uptake of MitoQ and DecylTPP into cells was greatly decreased by FCCP. MitoQ was rapidly reduced to its ubiquinol form by the mitochondrial respiratory chain, further supporting mitochondrial localization within cells. As this reduction had the same kinetic profiles as uptake into cells, it is concluded that these compounds are rapidly accumulated by mitochondria once within cells .

In cells MitoQ rapidly becomes 50-60% reduced to the ubiquinol form. MitoQ is stably retained in the reduced form within cells over at least 48 h. Over this time there was some metabolism to a sulfated derivative, but most of the added MitoQ persisted and was predominantly reduced. These findings are consistent with the protective effects seen with MitoQ in cell and animal models being due to MitoQH 2 preventing lipid peroxidation within mitochondria [START_REF] Koopman | Inhibition of complex I of the electron transport chain causes O 2 -. -mediated mitochondrial outgrowth[END_REF].

To summarise, uptake of lipophilic TPP cations into mitochondria is described by the Nernst equation, is rapid compared to its metabolism and it equilibrates rapidly with the CoQ pool. A model for the uptake of MitoQ by cells that is consistent with our findings is illustrated in Figure 8. The Nernst equation relates free, unbound pools of compound that equilibrate with the Δψ p and Δψ m . Extracellular MitoQ is ~ 78% bound to serum, and the free MitoQ then equilibrates with the free concentration within the cytosol in response to the Δψ p . This free concentration is then in equilibrium with the >99% of cytosolic MitoQ that is bound. The free MitoQ in the cytosol is in turn in equilibrium with the free concentration within the mitochondrial matrix, and this relationship is determined by the Δψ m . The free pool of MitoQ in the matrix is in equilibrium with a 10-fold excess that is bound to the matrix face of the mitochondrial inner membrane. These findings suggest that mitochondria-targeted compounds can be optimised by adjusting their hydrophobicity to modulate their rates of uptake into cells, and the extent of their uptake and concentration in the matrix by altering their binding to the mitochondrial inner membrane. Through such modifications it should be possible to design rationally further compounds to manipulate and report on particular mitochondrial functions. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 25 Feb 2008 as manuscript BJ20080063 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080063 S t a g e 2 ( a ) P O S T -P R I N T of eluted radioactivity co-eluted with MitoQ, with less than 0.2% in any other fraction. The specific activity of [ 3 H]-MitoQ was determined within each sample by counting of 2 x 200 µL of suspension immediately after addition of [ 3 H]-MitoQ. [ 3 H]-DecylTPP and [ 3 H]-TPMP were purified in the same way.

  Journal Immediate Publication. Published on 25 Feb 2008 as manuscript BJ20080063 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080063 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

  Journal Immediate Publication. Published on 25 Feb 2008 as manuscript BJ20080063 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080063 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

  Journal Immediate Publication. Published on 25 Feb 2008 as manuscript BJ20080063 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080063 S t a g e 2 ( a ) P O S T -P R I N T require abolishing Δψ p and Δψ m and comparing the [ 3 H]-MitoQ ACR relative to that for 86 Rb + over a range of Δψ p values

Biochemical

  Journal Immediate Publication. Published on 25 Feb 2008 as manuscript BJ20080063 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080063 S t a g e 2 ( a ) P O S T -P R I N T MitoQ and the endogenous CoQ pool is most likely mediated through an enzyme active site.

Biochemical

  Journal Immediate Publication. Published on 25 Feb 2008 as manuscript BJ20080063 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080063 S t a g e 2 ( a ) P O S T -P R I N T

Figure 1

 1 Figure 1Structures of MitoQ, MitoQH 2 , DecylTPP, TPMP, IDTP and a sulfated metabolite of MitoQ While MitoQ is sulfated on only one phenolic oxygen it is not known which.

Figure 2

 2 Figure 2Uptake of [ 3 H]-MitoQ and [ 3 H]-TPMP by mitochondria over a range of Δψ m values (A) The TPMP accumulation ratio (ACR) within isolated mitochondria is a linear function of the 86 Rb + accumulation ratio: TPMP ACR = 2.1 x Rb + ACR. (B) The MitoQ accumulation ratio within isolated mitochondria is also a linear function of the Rb + accumulation ratio: MitoQ ACR = 10.3 x Rb ACR + 8600. (C) Quantitation of MitoQ binding to mitochondria in the absence of Δψ m . MitoQ bound to mitochondria the absence of Δψ m is proportional to the concentration of MitoQ free in solution, and is described by the relationship: MitoQ(bound; pmol.mg protein -1 ) = 5.1 x MitoQ (free; nM). The bound/free ratio for 1 µM TPMP (0.038 (pmol.mg protein -1 ).nM -1 ) is also shown. (D) Relationship between MitoQ ACR and Rb + ACR when Δψ m -independent binding is accounted for. The concentration of free MitoQ (i.e. MitoQ in the supernatant after isolation of mitochondria) in the samples shown in (B) was used to quantitate Δψ m -independent binding of MitoQ using the equation from Figure2C. This was subtracted from the total amount of mitochondria-associated MitoQ in the samples shown in (B), and a new ACR calculated. The data for the TPMP accumulation ratio from Figure2Aare included for comparison. (A-D) show representative data from single experiments that were repeated at least three times. Lines of best fit were determined with Excel software. (E) Variation of mitochondrial uptake of TPMP and MitoQ with Δψ m at a fixed extramitochondrial concentration of 10 nM. (F) Variation of mitochondrial uptake of TPMP and MitoQ with extramitochondrial concentration at a fixed Δψ m of 150 mV. For (E) and (F) mitochondrial uptake was modelled using the equations generated in (D).

Figure 3 [ 3 H

 33 Figure 3 [ 3 H]-MitoQ and [ 3 H]-DecylTPP uptake into cells is rapid, extensive and FCCP-sensitive (A) MitoQ and TPMP uptake by cells over 23 h. Jurkat cells (3 x 10 6 .mL -1 ) were incubated with 500 nM [ 3 H]-MitoQ or [ 3 H]-TPMP for up to 23 h. Data are means of duplicate samples from a representative experiment that was repeated three times. (B) Uptake of [ 3 H]-MitoQ, [ 3 H]-DecylTPP and [ 3 H]-TPMP into cells over 40 min. The conditions were as for (A). (C) Rates of MitoQ and TPMP efflux from Jurkat cells. Cells were incubated with [ 3 H]-MitoQ or [ 3 H]-TPMP as above for 3 and 6 h, respectively, then 5 µM FCCP was added and the amounts of [ 3 H]-MitoQ or [ 3 H]-TPMP left in the cells were measured at the indicated times.The inset shows a plot of the natural log of the efflux data for the two compounds. The slopes of these lines gave the first order rate constants and half-lives for the FCCP-induced efflux of MitoQ and TPMP from Jurkat cells. (D) MitoQ, DecylTPP and TPMP uptake by cells ± FCCP. Jurkat cells were incubated for 3 h with 500 µM [ 3 H]-MitoQ, [ 3 H]-TPMP, or for 9 h with [ 3 H]-TPMP ± 5 µM FCCP. Data are means ± SD from at least three independent experiments, each determined in duplicate.

Figure 4 IDTP

 4 Figure 4IDTP localises predominantly to mitochondria within cultured human fibroblasts Cells were incubated with 1 µM IDTP for 3.5 h ± 5 µM FCCP. MitoTracker Orange was added for the last 30 min. The cells were then fixed and treated with antiserum against the TPP moiety of IDTP and a secondary antibody linked to Oregon Green 488. Scale bar = 20 µm.

Figure 5 [ 3 H

 53 Figure 5 [ 3 H]-MitoQ and [ 3 H]-TPMP uptake into cells driven by the p (A) MitoQ uptake into cells driven by Δψ p over 40 min. Jurkat cells (3 x 10 6 .mL -1 ) were incubated with 500 nM [ 3 H]-MitoQ for up to 40 min in the presence or absence of the mitochondrial inhibitors oligomycin and myxothiazol. Data are means of duplicate samples from a representative experiment that was repeated once. (B) Stability of Δψ p in the presence of oligomycin and myxothiazol. PMPI was used to follow changes in Jurkat cell Δψ p . Additions of oligomycin and myxothiazol (both 5 µM) were made which showed no effect on Δψ p . Gramicidin (5 µg.mL -1 ) was added after 45 min to abolish the Δψ p . The approximate values of the resting Δψ p and of Δψ p in the presence of gramicidin are shown. Traces of the PMPI fluorescence of cells without addition of oligomycin and myxothiazol were similar to the trace shown. (C) Uptake of MitoQ into cells lacking a Δψ m (oligomycin/myxothiazoltreated). Quantification of MitoQ uptake after 40 min, in the presence of oligomycin/myxothiazol ± gramicidin. Data are means ± SD of three experiments each determined in duplicate; *p < 0.05 Student's two-tailed t test. (D) Uptake of TPMP into cells lacking a Δψ m . Jurkat cells (3 x 10 6 .mL -1 ) were incubated with 500 nM [ 3 H]-TPMP for up to 150 min in the presence or absence of the mitochondrial inhibitors oligomycin and myxothiazol ± gramicidin. Data are means of duplicate samples from a representative experiment.

Figure 6 Figure 7

 67 Figure 6 Redox state of MitoQ on incubation with mitochondria (A) Detection and quantitation of [ 3 H]-MitoQuinone ([ 3 H]-MitoQ) and [ 3 H]-MitoQuinol ([ 3 H]-MitoQH 2 ) following extraction from a mitochondrial incubation, separation by RP-HPLC and scintillation counting of collected fractions. (B) Redox poise of [ 3 H]-MitoQ in energized mitochondria. Rat liver mitochondria respiring on succinate (10 mM) in the presence of rotenone (4 µg.ml -1 ) or glutamate and malate (5 mM of each) were incubated in the presence of 500 nM [ 3 H]-MitoQ for 5 min. [ 3 H]-MitoQuinone and [ 3 H]-MitoQuinol were extracted and their ratio determined as described in (A). The effects of the uncoupler FCCP (0.5 µM) or of the inhibitors malonate (20 mM) and rotenone (4 µg.mL -1 ) are shown. (C) MitoQ redox poise in intact mitochondria, in the presence of the uncoupler FCCP. [ 3 H]-MitoQ was incubated with mitochondria as in (B) for 5 min in the presence of FCCP along with rotenone and succinate, or glutamate/malate, for 5 min and then the [ 3 H]-MitoQuinone and [ 3 H]-MitoQuinol were extracted and their ratio determined as described in (A). The effect of preincubation with the inhibitors myxothiazol (2 µM), malonate (20 mM), and rotenone (4 µg.mL -1 ) on the redox poise of MitoQ were then determined. Data in (B & C) are means ± SD n = 3-8 *p < 0.05 **p < 0.005 ***p < 0.0005; n.s. p > 0.05 by Student's two-tailed t test.

Figure 8 21 Biochemical 22 Biochemical

 82122 Figure 8Uptake and distribution of MitoQ and related compounds in cells The equilibrium distribution of free MitoQ within a cell with a Δψ p of 40 mV and a Δψ m of 150 mV was calculated from the Nernst equation. The indicated proportions of MitoQ bound were determined from the binding constants derived in the text. The values in square brackets are the approximate relative concentration free or bound in the different compartments. The total amounts of MitoQ present in each compartment will vary depending on the cell and mitochondrial volumes.

Figure 4 Scale 24 Biochemical

 424 Figure 3

  Figure 6

[ 3 28 Biochemical

 328 Figure 7

  1 H NMR δ 7.67-7.87 (m, 15 H, PPh 3 ) 3.74 (m, 2 H, -CH 2 PPh 3 ), 3.171 (t, 2 H, 7 Hz, -CH 2 I), 1.52-1.83 (m, 6 H), 1.18-1.39 (m, 10 H) ppm. 31 P NMR δ 25.57 ppm. (+)ESI-MS found 529.153 (C 28 H 35 PI + requires 529.152).

  3 H]-MitoQH 2 [ 3 H]-MitoQ

	MitoQ redox state (% reduced)
	+MALON	+FCCP	no subs.		+ROT	+FCCP
				+ROT
	Succinate/rotenone		Glutamate/malate
	MitoQ redox state (% reduced)
	+MYXO	+MALON +MALON	+MYXO	+ROT	+ROT
			+MYXO		+MYXO
	Succinate/rotenone/FCCP	Glutamate/malate/FCCP

S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

This work was supported by the Medical Research Council (UK), Antipodean Pharmaceuticals Inc. and by the European Community's 6 th Framework Programme for Research, Priority 1 "Life sciences, genomics and biotechnology for health" Contract LSHM-CT-2004-503116. We thank Dr. Abdul-Rahman bin Manas and Pauline Bandeen for technical assistance with the synthetic chemistry, Stephanie Brown for technical assistance and Martin Brand for helpful discussions on the manuscript.


Keywords: Lnk, c-Kit, adaptor protein, signal transduction, receptor tyrosine kinase, proteinprotein interaction

The adaptor protein Lnk is expressed in hematopoietic cells and plays a critical role in hematopoiesis. Animal model studies demonstrated that Lnk acts as a broad inhibitor of signalling pathways in hematopoietic lineages. Lnk belongs to a family of proteins sharing several structural motifs, including a Src homology 2 (SH2) domain which binds phosphotyrosines in various signal-transducing proteins. The SH2 domain is essential for Lnk-mediated negative regulation of several cytokine receptors (i.e. Mpl, EpoR, c-Kit). Therefore, inhibition of the binding of Lnk to cytokine receptors might lead to enhanced downstream signalling of the receptor and thereby to improved hematopoiesis in response to exposure to cytokines (i.e. erythropoietin in anemic patients). This hypothesis led us to define the exact binding site of Lnk to the stem cell factor receptor c-Kit. Pull-down experiments using GST-fusion proteins of the different domains of c-Kit showed that Lnk almost exclusively binds to the phosphorylated juxtamembrane domain. Binding of Lnk to the juxtamembrane domain was abolished by point mutation of the Y568 and was competed by peptides with a phospho-tyrosine in position 568. Co-immunoprecipitation with full-length wild-type or Y568F mutant c-Kit and Lnk confirmed these results, thus showing the importance of this phosphorylated tyrosine. Lnk bound directly to c-Kit without requiring other interacting partners. The identification of the binding site of Lnk to c-Kit will be useful to discover inhibitory molecules that prevent the binding of these two proteins, thus making hematopoietic cells more sensitive to growth factors.

Adaptor protein Lnk associates with Y568 in c-Kit

Saskia GUELLER * †1 , Sigal GERY * , Verena NOWAK * , Liqin LIU ‡ , Hubert SERVE † and H. Phillip KOEFFLER *

INTRODUCTION

The receptor for stem-cell factor (SCF), c-Kit, belongs to the class III family of receptor tyrosine kinases that also includes the platelet-derived growth factor receptors (PDGFR) and the macrophage colony stimulating factor receptor (CSF1R) [1;2]. These receptors are structurally characterized by an extracellular region consisting of five immunoglobulin-like domains, a transmembrane, juxtamembrane and an intracellular tyrosine kinase domain. The latter is split into a proximal and a distal region separated by an insert. In general, c-Kit is expressed on hematopoietic stem cells and their progenitor cells; and expression is lost during further differentiation. It plays an important role in self-renewal, survival and growth of hematopoietic stem cells. Therefore, homozygous c-Kit knock-out mice are nonviable due to anemia; and heterozygous deletional mice have reduced c-Kit tyrosine kinase activity causing anemia and reduced fertility [START_REF] Lennartsson | Normal and oncogenic forms of the receptor tyrosine kinase kit[END_REF]. Binding of SCF to c-Kit results in receptor dimerization and activation of the tyrosine kinase, which subsequently causes autophosphorylation of specific tyrosine residues. These phosphotyrosines can be recognized and bound by signal transduction molecules containing Src homolgy 2 (SH2) domains. Thereby, Grb2, Grb7, APS, Lnk, PI3K, PLCγ and other proteins can associate with activated c-Kit [START_REF] Roskoski | Signaling by Kit protein-tyrosine kinase--the stem cell factor receptor[END_REF]. Lnk is an adaptor protein expressed in hematopoietic tissues and has critical functions in hematopoiesis [START_REF] Ema | Quantification of self-renewal capacity in single hematopoietic stem cells from normal and Lnk-deficient mice[END_REF][START_REF] Nobuhisa | Regulation of hematopoietic development in the aorta-gonad-mesonephros region mediated by Lnk adaptor protein[END_REF][START_REF] Takaki | Control of B cell production by the adaptor protein lnk. Definition Of a conserved family of signal-modulating proteins[END_REF][START_REF] Takaki | Enhanced hematopoiesis by hematopoietic progenitor cells lacking intracellular adaptor protein[END_REF][START_REF] Takaki | Impaired lymphopoiesis and altered B cell subpopulations in mice overexpressing Lnk adaptor protein[END_REF][START_REF] Velazquez | Cytokine signaling and hematopoietic homeostasis are disrupted in Lnk-deficient mice[END_REF]. Together with APS and SH2-B, Lnk shares several structural motifs including a SH2 domain, a pleckstrin homology (PH) domain and a dimerization domain (DD). In response to ligand mediated autophosphorylation of tyrosine residues within either the erythropoietin receptor (EpoR) [START_REF] Tong | Lnk inhibits erythropoiesis and Epo-dependent JAK2 activation and downstream signaling pathways[END_REF], thrombopoietin receptor (Mpl) [START_REF] Tong | Lnk inhibits Tpo-mpl signaling and Tpo-mediated megakaryocytopoiesis[END_REF] or stem cell factor receptor (c-Kit) [6;8], Lnk associates with the receptors through its SH2 domain and subsequently inhibits their downstream signalling [START_REF] Huang | Cloning and characterization of Lnk, a signal transduction protein that links T-cell receptor activation signal to phospholipase C gamma 1, Grb2, and phosphatidylinositol 3-kinase[END_REF][START_REF] Li | Cloning and characterization of human Lnk, an adaptor protein with pleckstrin homology and Src homology 2 domains that can inhibit T cell activation[END_REF][START_REF] Takaki | Characterization of Lnk. An adaptor protein expressed in lymphocytes[END_REF]. The SH2 domain is essential for the inhibitory activity of Lnk [6;11;12]. Proliferation and differentiation of hematopoietic precursors is enhanced by decreased expression of Lnk and is inhibited by increased levels of Lnk [6;8]. Therefore, inhibition of Lnk binding to cytokine receptors makes hematopoietic progenitor cells more sensitive to the growth enhancing factors. The binding site of Lnk in c-Kit has not yet been determined. We wished to determine this site of interaction because we want to develop an assay to screen for agents that prevent binding of Lnk to cytokine receptors and thereby enhance the effect of endogenous or exogenous cytokines.

EXPERIMENTAL

Cell culture and transfections 293T cells were grown in Dulbecco's modified Eagle's medium with 10 % FBS and transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. 48 hours after transfection, cells were lysed in lysis buffer containing 0.5 % NP-40, 50 mM Tris-HCl (pH 7.5), 150 mM NaCl and Complete Protease Inhibitor Cocktail (Roche Applied Science, Indianapoils, IN).

Expression vectors

V5-tagged human Lnk cDNA was cloned into the pcDNA3.1 vector. The point mutation Lnk R392E was generated by PCR and confirmed by sequencing. DNA corresponding to the juxtamembrane (JXM, amino acids 544-577), proximal kinase (Kin1, amino acids 578-685), kinase insert (KI, amino acids 686-762), distal kinase (Kin2, amino acids 775-845), and carboxyl tail (CT, amino acids 926-976) domains of c-Kit cloned into pGEX-4T-1 were kindly provided by Johan Lennartsson (Ludwig Institute for Cancer Research, Uppsala University, Uppsala, Sweden). The point mutations Y568F and Y570F were generated by site-directed mutagenesis of the JXM domain, and the sequences confirmed by nucleotide sequencing. The expression vector (pDSRα22) for wild-type full-length c-Kit was kindly provided by Amgen. The point mutation Y568F was generated using the Quikchange Mutagenesis Kit (Strategene, La Jolla, CA) followed by nucleotide sequencing.

GST-fusion proteins

The pGEX-4T-1 constructs were transformed into TKX1 competent cells (Stratagene, La Jolla, CA) for production of GST-fusion proteins with phosphorylated tyrosines and into E. coli DH5α for generation of unphosphorylated GST-fusion proteins. Transformation, accumulation of the fusion proteins and induction of the tyrosine kinase were performed according to the manufacturer's manual. Briefly, after growing either TKX1 or DH5α bacteria overnight, production of the GST-fusion proteins was induced by 0.1 mM isopropylthio-β-galactoside (ITPG; Invitrogen) for 2.5 hours. Thereafter, only TKX1 bacteria were resuspended in tryptophan-free medium containing 10 µg/mL indoleacrylic acid (IAA; Sigma-Aldrich, St. Louis, MO) to activate the tyrosine kinase and incubated for another 2 hours. Harvesting and purification of phosphorylated and unphosphorylated GST-fusion proteins were carried out as described in the "GST Gene Fusion System" manual (Pharmacia Biotech, Uppsala, Sweden).

Cells were sedimented and disrupted by sonication, proteins were bound to Gluthatione Sepharose 4B (Amersham Biosciences, Piscataway, NJ) and were stored in PBS containing 15 % glycerol at -80 °C until their use for pull-down experiments.

In vitro translation

In vitro translated (IVT) Lnk was sythesized from the pcDNA3.1-Lnk expression vector using the TNT ® T7 Coupled Reticulocyte Lysate System (Promega, Madison, WI) according to the manufacturer's recommendations. Generation of proteins was carried out in reticulocyte lysate using the one-step transcription/translation system. Expression levels of IVT Lnk were determined by Western blot.

GST pull-down

GST pull-down assays were carried out with equivalent amounts of GST and GST-fusion proteins immobilized on glutathione-sepharose beads in lysis buffer. After 16-hour incubation at 4 °C with whole 293T cell lysates or IVT Lnk, respectively, precipitated proteins were washed with PBS, eluted with SDS sample buffer and resolved by SDS-PAGE. After transfer to PVDF membranes, the Lnk protein was detected with V5-tag antibodies (Applied Biological Materials Inc., Vancouver, Canada), phosphorylation of the GST-fusion proteins with phospho-tyrosine antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) and equal loading was confirmed either with GST-antibodies (Santa Cruz Biotechnology), Ponceau staining or Coomassie staining of the gel. Phosphorylation of TKX1-derived GST-fusion proteins was verified by Western blot using phospho-tyrosine antibodies (Santa Cruz Biotechnology).

Co-immunoprecipitation

For co-immunoprecipitation of full-length c-Kit and Lnk, 293T cells were co-transfected with c-Kit wild-type or Y568F mutant and Lnk wild-type cDNA. Before lysis, cells were serumstarved overnight and treated with 50 ng/mL human SCF (Peprotech, Rocky Hill, NJ) for 30 minutes. 500 µg of protein was precipitated using 1 µg of V5-tag (Invitrogen) or c-Kit (Santa Cruz Biotechnology) antibodies and 20 µL Protein A/G PLUS-Agarose (Santa Cruz Biotechnology) at 4 °C for 16 hours, then washed with PBS, eluted with SDS sample buffer and resolved by SDS-PAGE. Precipitated proteins were detected with c-Kit or V5-tag antibodies.

Competition assay

Synthetic peptides corresponding to the tyrosine-containing part of the JXM domain of c-Kit were purchased from Quality Controlled Biochemicals (Hopkinton, MA) including different phospho-tyrosines: GKGKGKEEINGNNYVYIDPTQL-OH (Y568/570), GKGKGKEEINGNN[pY]VYIDPTQL-OH (pY568), GKGKGKEEINGNNYV[pY]IDPTQL-OH (pY570) and GKGKGKEEINGNN[pY]V[pY]IDPTQL-OH (pY568/570). Competition pulldown assays were carried out in lysis buffer using a 10-or 100-fold molar excess of the peptides compared to the amount of GST-JXM fusion protein and 5 µg of protein from 293T cells overexpressing Lnk. The samples were incubated at 4 °C for 2.5 hours and then washed and processed as described for the pull-down assays.

RESULTS

Lnk binds to the phosphorylated juxtamembrane domain of c-Kit

Phosphorylated GST-fusion proteins consisting of the five different domains of c-Kit were isolated from TKX1 bacteria, and expression and phosphorylation were confirmed by Western blot (Figure 1A). Some of the GST-proteins showed low expression, so that the amounts of GST input were adapted according to their expression to get equal loading. In pull-down experiments using the different c-Kit domains that were incubated with whole cell lysates of 293T cells overexpressing wild-type Lnk, we found that Lnk mainly bound to the juxtamembrane (JXM) domain of c-Kit (Figure 1B). As expected, Lnk did not bind to either the unphosphorylated JXM fusion protein or the control GST without fusion product.

Mutation of Y568 abolishes binding of Lnk

The SH2 domain of various adaptor proteins recognize and bind to phosphorylated tyrosine residues of their binding partners. Therefore, we tested if Lnk binds to a tyrosine residue in c-Kit. The JXM domain of the receptor carries two tyrosine residues, that are potential binding sites for Lnk: Y568 and Y570. By site-directed mutagenesis, we created vectors containing the sequences for the JXM domain of c-Kit containing either Y568F or Y570F point mutations, respectively. Pull-down experiments were carried out with wild-type or point mutated GST-fused JXM domains and whole lysates derived from 293T cells overexpressing Lnk. We found that the Y570F mutation did not influence the binding of Lnk, but the Y568F mutation abolished binding of Lnk to the JXM domain (Figure 2A). We conclude that Lnk selectively binds to the phosphorylated Y568 tyrosine residue in the JXM domain of c-Kit. To confirm the specifity of binding of Lnk to Y568 of c-Kit, competition assays using peptides with different phosphorylation patterns as competitors for GST-JXM were performed. The peptides were utilized in a 10-or 100-fold molar excess as compared to GST-JXM. As shown in Figure 2B, binding of Lnk to GST-JXM was competed in a dose-dependent manner by peptides harbouring a phospho-tyrosine in position 568 (pY568/570 and pY568), whereas the other peptides did not affect binding. Furthermore, co-immunoprecipitation assays were performed to prove the specific binding site in full-length c-Kit. With V5-tag or c-Kit antibodies, respectively, proteins from 293T lysates overexpressing either wild-type or Y568F mutant c-Kit and V5-tagged wildtype Lnk were precipitated and analyzed by SDS-Page. Figure 2C shows that SCF-induced phosphorylation of tyrosine residues in wild-type c-Kit expressing cells led to binding of Lnk to the receptor. The mutation Y568F completely abolished the interaction, thus showing the importance of this tyrosine as a docking site for Lnk.

Binding of human Lnk to human c-Kit depends on its SH2 domain

Binding of murine Lnk to the murine c-Kit receptor is abolished by point mutation of the arginine R364 of Lnk [START_REF] Nobuhisa | Regulation of hematopoietic development in the aorta-gonad-mesonephros region mediated by Lnk adaptor protein[END_REF]. We tested if the same arginine residue in the conserved SH2 domain of human Lnk is responsible for its binding to human c-Kit. R392 is the arginine residue in human Lnk which corresponds to R364 in murine Lnk. A point mutation R392E was inserted into the Lnk vector and it was expressed in 293T cells. Pull-down experiments were performed using GST-JXM of c-Kit and either wild-type or mutant Lnk lysates. As shown in Figure 3, R392E completely abolished binding of Lnk to c-Kit.

Binding of Lnk to c-Kit JXM is direct

Studies have shown that Lnk binds to c-Kit [START_REF] Nobuhisa | Regulation of hematopoietic development in the aorta-gonad-mesonephros region mediated by Lnk adaptor protein[END_REF], but no one elucidated if the binding is direct or if Lnk needs co-partners to interact with c-Kit. Therefore, pull-down assays were carried out using in-vitro translated protein as a source of Lnk instead of whole cell lysates. Similarly to Lnk from 293T lysates, in vitro translated Lnk was able to bind to GST-JXM. Y570F point mutated GST-JXM still bound Lnk, whereas the Y568F point mutation abolished binding (Figure 4).These data demonstrate that Lnk binds to c-Kit directly and independently of other binding partners.

DISCUSSION

In this study, we demonstrated for the first time that the adaptor protein Lnk binds to the juxtamembrane domain of c-Kit and specifically to the tyrosine Y568. These results are in agreement with data that showed that APS, a family member of Lnk, binds to the same tyrosine residue in c-Kit [START_REF] Wollberg | The adapter protein APS associates with the multifunctional docking sites Tyr-568 and Tyr-936 in c-Kit[END_REF]. In contrast to APS, Lnk binds to only one tyrosine and does not bind to the carboxy-tail of c-Kit; whereas APS binds to both the JXM domain, as well as the carboxy-tail. Both adaptor proteins lead to inhibition of downstream signalling of c-Kit upon stimulation with SCF. The juxtamembrane domain of c-Kit is known to have an autoinhibitory function. Transphosphorylation of Y568 and Y570 are the first steps that occur after ligand mediated dimerization of the receptor. As a result, the juxtamembrane segment no longer immobilizes the receptor in a static configuration, but allows conversion of the activation loop of c-Kit from a compact inactive form to an extended activated receptor. Subsequently, transphosphorylation of Y823 in the activation loop stabilizes the active form of the tyrosine kinase [START_REF] Roskoski | Structure and regulation of Kit protein-tyrosine kinase--the stem cell factor receptor[END_REF]. The SH2domain containing phosphatases 1 and 2 (SHP-1 and SHP-2) are known to bind to phosphorylated Y568 and Y570 and terminate the SCF signal by dephosphorylating the two tyrosine residues and thereby restoring the inactive conformation of the receptor [START_REF] Kozlowski | SHP-1 binds and negatively modulates the c-Kit receptor by interaction with tyrosine 569 in the c-Kit juxtamembrane domain[END_REF]. Thus, the juxtamembrane domain plays a major role both in activating as well as inhibiting c-Kit, which makes its tyrosine residues very important regulating elements. This is also reflected by the fact that several signal transduction molecules influencing the activity of the receptor (i.e. Src kinases, Chk and Shc) are known to bind to the same domain. We showed that binding of Lnk to this site is independent of binding of other proteins, but Lnk might still be an important factor that mediates binding of other regulating proteins to the receptor. APS has been shown to bind c-Cbl, a ubiquitin ligase which targets growth factor receptors for degradation in lysosomes [START_REF] Levkowitz | Ubiquitin ligase activity and tyrosine phosphorylation underlie suppression of growth factor signaling by c-Cbl/Sli-1[END_REF]. Thereby, loss of binding of APS to PDGFRβ leads to decreased recruitment of c-Cbl and to reduced degradation of the receptor, followed by enhanced c-fos activation. In contrast, low levels of c-Cbl had no effect alone on levels of the receptor, but in the presence of APS, enhanced recruitment of c-Cbl occurred resulting in degradation of the receptor [START_REF] Yokouchi | APS, an adaptor protein containing PH and SH2 domains, is associated with the PDGF receptor and c-Cbl and inhibits PDGF-induced mitogenesis[END_REF]. The Cterminal part of Lnk contains a c-Cbl binding site similar to APS, so the same mechanism could lead to its inhibitory function, whereas c-Cbl can also bind directly to c-Kit [START_REF] Masson | Direct binding of Cbl to Tyr568 and Tyr936 of the stem cell factor receptor/c-Kit is required for ligand-induced ubiquitination, internalization and degradation[END_REF]. Nevertheless, further exploration is necessary to elucidate the interaction between Lnk and c-Kit. Our data identifying the binding site of Lnk to c-Kit can help to develop an assay to screen for "small inhibitor molecules" to prevent the binding of these two proteins; and thus, potentially make hematopoietic cells more sensitive to either endogenous or exogenous hematopoietic growth factors offering a novel approach to stimulate blood cell production. Lnk binds to c-Kit directly without requirement of co-partners Pull-down assays were performed using in-vitro translated (IVT) protein as a source of Lnk. IVT Lnk bound to phosphorylated (TKX1) wild-type (WT) and Y570F mutated (570) GST-JXM, but not to either unphosphorylated (DH5α) or Y568F mutated (568) GST-JXM. Input shows expression of IVT Lnk, Coomassie staining confirms equal amounts of GST-fusion proteins.

Biochemical

  Journal Immediate Publication. Published on 30 Jun 2008 as manuscript BJ20080102 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080102 S t a g e 2 ( a ) P O S T -P R I N T

Figure 1

 1 Figure 1Lnk binds to the juxtamembrane domain of c-Kit (A) Expression (IB: GST, upper panel) and phosphorylation (IB: PY, lower panel) of GSTfusions of various domains of c-Kit: JXM (juxtamembrane), Kin1 (proximal kinase), KI (kinase insert), Kin2 (distal kinase), CT (carboxy-tail). GST indicates the GST control protein without fusion. The proteins obtained from the E. coli strain TKX1 are phosphorylated which is not the case for these proteins produced by regular DH5α E. coli. IB: immunoblot. Bands below the stars (*) highlight target protein fragments. (B) Lnk pull-down from a lysate of 293T cells overexpressing Lnk. The input lane shows Lnk expression in the lysate. The anti-V5 immunoblot (IB: Lnk, upper panel) reveals strong interaction of Lnk with the phosphorylated JXM domain of c-Kit. Coomassie staining of the SDS gel (lower panel) shows the relative amount of GST fusion proteins in each lane.

Figure 2 Phosphorylated

 2 Figure 2Phosphorylated Y568 is the binding site of Lnk in c-Kit (A) GST pull-down of Lnk with mutated c-Kit JXM proteins. Input lane shows Lnk expression in the lysate. GST indicates the GST control protein without fusion. The anti-V5 immunoblot (IB: Lnk, upper panel) reveals strong interaction of Lnk with the phosphorylated wild-type (WT) and Y570F point mutated (570) JXM domain of c-Kit. Interaction is abolished by the Y568F (568) point mutation. Phosphorylation and equal loading of the GST-fusion proteins is confirmed by reprobing the blot with phospho-tyrosine antibodies (IB: PY) and Coomassie staining (lower panels). (B) Binding of Lnk to phosphorylated GST-JXM (TKX1) can be competed by peptides harbouring a phospho-tyrosine in position 568. For competition pull-down experiments, peptides containing the two tyrosine residues of c-Kit JXM with different phosphorylation patterns were used: phospho-tyrosines in position 568 and 570 (pY568/570), only in position 568 (pY568) or 570 (pY570) or without any phospho-tyrosines (no pY). Pull-downs were carried out without competition (no comp.) or with 10-or 100-fold excess (10x or 100x) of the indicated peptides. Competition of peptides with a phospho-tyrosine in position 568 is dose-dependent, whereas peptides without pY568 does not compete. Ponceau staining shows equal loading of GST-JXM. (C) Co-immunoprecipitation (IP) of Lnk with wild-type and Y568F mutant full-length c-Kit. Lnk interacts with wild-type c-Kit (WT), but not with Y568F mutant c-Kit (568). "Input" shows expression of Lnk (IB: Lnk) or c-Kit (IB: c-Kit) in lysates from 293T cells that were used for IP with either V5-tag, c-Kit or isotype IgG antibodies. Precipitated proteins were detected with either c-Kit or V5-tag antibodies.

Figure 3

 3 Figure 3Binding of Lnk depends on R392 in its SH2 domain Pull-down experiments were performed with wild-type c-Kit JXM domain and either wild-type or R392E mutated Lnk lysates from 293T cells, respectively. Human Lnk with a point mutation of the SH2 domain lost the ability to bind to c-Kit. Input shows expression of Lnk proteins in the lysates, Ponceau staining indicates equal amounts of GST-JXM.

Figure 4

 4 Figure 4Lnk binds to c-Kit directly without requirement of co-partners Pull-down assays were performed using in-vitro translated (IVT) protein as a source of Lnk. IVT Lnk bound to phosphorylated (TKX1) wild-type (WT) and Y570F mutated (570) GST-JXM, but not to either unphosphorylated (DH5α) or Y568F mutated (568) GST-JXM. Input shows expression of IVT Lnk, Coomassie staining confirms equal amounts of GST-fusion proteins.

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S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

ACKNOWLEDGEMENTS

This work was supported in part by the Deutsche Krebshilfe (SG) and the Tower Cancer Research Foundation Fellowship (SG).