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NUCLEOTIDE-BINDING SITES IN THE VOLTAGE-DEPENDENT ANION CHANNEL: CHARACTERIZATION AND LOCALIZATION*

Galit Yehezkel, Nurit Hadad, Hilal Zaid, Sara Sivan, and Varda Shoshan-Barmatz‡§

From the Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel

Running Title: VDAC nucleotide-binding sites. Address correspondence to: Varda Shoshan-Barmatz, Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel, Tel. 972-8-6461336; Fax: 972-8-6472992; E-mail: vardasb@bgu.ac.il.

In this study, we addressed the presence and location of nucleotide-binding sites in the voltage-dependent anion channel (VDAC). VDAC bound to reactive red-120 agarose, from which it was eluted by ATP, less effectively by ADP and AMP but not by NADH. The photoreactive ATP analog, benzoyl-benzoyl-ATP (BzATP), was used to identify and characterize the ATP-binding sites in VDAC. [αααα-32P]BzATP bound to pur ified VDAC at two or more binding sites with apparent high- and low-binding affinities. MALDI-TOF analysis of BzATP-labeled VDAC confirmed the binding of at least two BzATP molecules to VDAC. The VDAC-BzATP-binding sites showed higher specificity for purine than for pyrimidine nucleotides and higher affinity for negatively-charged nucleotide species. VDAC treatment with the lysyl residue-modifying reagent, fluorescein 5'-isothiocyanate, markedly inhibited VDAC labeling with BzATP. The VDAC nucleotide binding sites were localized using chemical and enzymatic cleavage. Digestion of [αααα-32P]BzATP-labeled VDAC with CNBr or with V 8 protease resulted in the appearance of ~17 and ~14 kDa labeled fragments. Further digestion, HPLC separation and sequencing of selected V8-resulted peptides suggested that the labeled fragments originated from two different regions of the VDAC molecule. MALDI-TOF analysis of BzATP-labeled, tryptic VDAC fragments indicated and localized three nucleotide binding sites, two of which were at the N- and C-termini of VDAC. Thus, the presence of two or more nucleotide binding sites in VDAC is suggested, and their possible function in the control of VDAC activity, and, thereby, of outer mitochondrial membrane permeability is discussed.

It has recently been recognized that there exists a metabolic coupling between the cytosol and the mitochondria, with the outer mitochondrial membrane (OMM)1, the boundary between these compartments, fulfilling an important function in this coupling (1-4). In the cross-talk between mitochondria and cytosol, mitochondrial ATP and Ca2+ play major roles. As the primary transporter of nucleotides, Ca2+ and other ions and metabolites across the OMM (reviewed in 5, 6), the voltage-dependent anion channel (VDAC) mediates a substantial portion of the OMM molecular traffic. It has been demonstrated that VDAC is permeable to Ca2+ and possesses Ca2+-binding sites that control its activity (reviewed in 5). VDAC permeability is also regulated by associated protein(s) (reviewed in (5-7) and by different ligands that bind to VDAC, such as glutamate, ruthenium red, La3+

and NADH (reviewed in 5, 6). Thus, by providing the pathway for anions, cations, ATP and other metabolites into and out of the mitochondria, as well as possessing ligand-binding sites, VDAC plays an important role in the regulation of mitochondrial functions. Indeed, VDAC has been suggested to control the OMM permeability, coupled respiration and cell survival (3, 4, 8).

It has been proposed that activation of the Ca2+-dependent mitochondrial permeability transition pore (PTP) is a key event committing the cell to an apoptotic fate (reviewed in 9-11). The PTP is a regulated, high-conductance, non-specific channel, which has been suggested to be composed of VDAC, located at the OMM, the adenine nucleotide translocase (ANT), located at the inner mitochondrial membrane (IMM), and cyclophilin D, located at the matrix (9, 12). ATP and ADP inhibition of PTP opening has prevalently been thought to be exerted via their interaction with the ANT (9). However, there is also evidence for the involvement of a second

http://www.jbc.org/cgi/doi/10.1074/jbc.M510104200The latest version is at JBC Papers in Press. Published on December 14, 2005 as Manuscript M510104200

Copyright 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

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adenine nucleotide-binding site, having different affinity, in PTP regulation (13-16, reviewed in 9, 10). Moreover, not only matrix ADP, but also external ADP affects PTP opening (see in 9, 10), suggesting the involvement of a cytoplasmically-facing nucleotide-binding site(s) (NBS(s)) in the modulation of PTP opening. As a mitochondrial protein facing the cytoplasm and as a PTP component, VDAC is a possible target for nucleotides regulating PTP opening. However, direct demonstration of the presence of nucleotide-binding properties in VDAC is lacking thus far.

The transport of adenine nucleotides via VDAC has been demonstrated using isolated mitochondria (3, 17) or VDAC reconstituted into liposomes (18) or into a planar lipid bilayer (PLB) (19-21). Moreover, not only does VDAC transport adenine nucleotides, but it is also regulated by them. NADH was found to regulate the gating of mammalian, fungal and plant VDACs (3, 22), and ATP was shown to modify Neurospora crassa VDAC channel conductance in a biphasic manner, leading to the suggestion that VDAC contains at least one binding site for purine nucleotides, located inside the channel's pore (19, 20). Additionally, mammalian VDAC binds to ATP-agarose resin and is eluted from it with ATP (23). Molecular and biochemical evidence also indicate that VDAC possesses one or more NBS(s) (22, 23). Based on homology with known nucleotide-binding sequences, Zizi et al. (22) proposed a C-terminal NBS in mammalian and fungal VDACs, and a possible N-terminal NBS in mammalian (but not fungal) VDAC. The latter suggestion is supported by 32P-ATP labeling of mammalian VDAC and of a human VDAC fragment containing the putative N-terminal NBS (GYGFG) (23).

Despite these findings, characterization and precise location of the VDAC NBS(s) have not yet been presented. Accordingly, in this study, several approaches, including photo-affinity labeling with the ATP-analog, benzoyl-benzoyl-ATP (BzATP) and mass-spectral analysis, were used to identify at least two NBSs in VDAC, characterize their binding properties and localize them within the VDAC molecule. The possible function of these sites in the regulation of OMM permeability is discussed.

EXPERIMENTAL PROCEDURES

Materials – cyanogen bromide (CNBr),

reactive red-120 agarose (RRA), EGTA,

fluorescein 5' isothiocyanate (FITC), Mops, nucleotides, Tris, trypsin and Staphylococcus aureus protease (V8) were obtained from Sigma (St. Louis, MO). Hydroxyapatite (Bio-Gel HTP) was purchased from Bio-Rad Labs (Hercules, CA) and celite was obtained from the British Drug Houses (UK). Radiolabeled 3'-O-(4-benzoyl)-benzoyl-ATP ([α-32P]BzATP) was synthesized and purified as described by Williams and Coleman (24), with some modifications to scale down the amount of BzATP synthesized.

Mitochondria and VDAC purification – Mitochondria were isolated from rat liver as described previously (25), except that 0.1 mM PMSF and 0.5 µg/ml leupeptin were added to all solutions as protease inhibitors. VDAC was purified from rat liver mitochondria by two-step chromatography using hydroxyapatite:celite (HA:C) and RRA in the presence of Triton X-100, as previously described (5). Purified VDAC, eluted from the RRA with a solution containing 0.3% Triton X-100, 10 mM Tris-HCl, pH 7.2 and 0.4 M NaCl, was dialyzed against 10 mM Tris-HCl, pH 7.2 to avoid salt interference with subsequent BzATP labeling (see "Results"). Mitochondrial protein concentration was determined using the Buiret method, and VDAC concentration by densitometric estimation of Coomassie blue-stained gel-bands (using ovalbumin as a standard). It should be noted that the VDAC preparations mostly contain VDAC1 (see Ref. 26); as also supported by the mass spectral analyses described below).

Photoaffinity labeling by [α-32P]BzATP – Mitochondria (1 mg/ml) or purified VDAC (~0.1 mg/ml) were irradiated with a 15W-ultraviolet lamp for 3 min in the presence of 0.2 to 500 µM of [α-32P]BzATP (4 x 106 to 107 cpm/nmol) in 40 µl of 10 mM Tris-HCl, pH 7.2. The UV-irradiated samples were immediately diluted with SDS-PAGE sample buffer, incubated for 3 min at 90°C, and subjected to SDS-PAGE as described below. The dried gels were exposed to Kodak X-Omat film (Eastman Kodak Co) for autoradiography. Quantitative analysis of the labeled protein bands was performed by densitometric scanning of the autoradiogram, using a Molecular Dynamics Personal Scanning Densitometer.

Gel electrophoresis and immunoblotting – SDS-PAGE was performed according to Laemmli (27). Gels were stained with either Coomassie blue or silver, or electroblotted onto

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nitrocellulose membrane and subjected to Western blot analysis using a monoclonal anti-VDAC antibody (clone no. 173/045, Calbiochem-Novobiochem, Nottingham, UK) and alkaline phosphatase-conjugated anti-mouse secondary antibody. The colored reaction product was developed with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium.

V8 digestion and CNBr cleavage of VDAC – For Staphylococcus aureus (V8) digestion, purified [α-32P]BzATP-labeled VDAC was subjected to SDS-PAGE (10%) followed by Coomassie staining. VDAC bands were excised from the gel and rinsed in a solution of 0.125 M Tris-HCl, pH 6.8, 0.1% SDS and 1 mM EDTA for 30 min. Proteolysis was carried out as described previously (28). Briefly, squashed slices of the excised protein band were applied to the bottom of the well in the stacking portion of a second SDS-PAGE gel (7 to 20% acrylamide). V8, prepared in the same buffer containing 20% glycerol, was then introduced into each buffer-filled well. Electrophoresis was performed until the bromophenol dye front approached the bottom of the stacking gel. The current was then turned off for 30 min, following which it was resumed for several hours. The gel was subjected to Coomassie staining and autoradiography or was electroblotted and subjected to immunostaining. For CNBr cleavage, [α-32P]BzATP-labeled VDAC was dissolved in 0.4 ml of 80% (v/v) formic acid containing CNBr (8 or 32 mg/ml). The tube was sealed under nitrogen and incubated overnight in the dark at room temperature, after which 1 ml of water was added, and the sample was lyophilized twice, dissolved in sample buffer and subjected to SDS-PAGE (15% acrylamide), autoradiography, silver staining or immunoblotting.

Sequence analysis of VDAC fragments – Briefly, purified VDAC was subjected to V8 digestion as described above and the obtained fragment bands of about 17 and 14 kDa were excised from the gel and subjected to in-gel proteolysis with LysC. The resulting peptides were resolved by reverse-phase chromatography and electrosprayed into an ion-trap mass spectrometer and identified by liquid-chromatography/mass spectrometry (LC/MS-MS) (LCQ, Finnigan, San Jose, CA). Ten of the resultant peptides were identified in rat, mouse and human VDAC1 sequences. Selected peptides were then sequenced by an automated

sequencer (The Protein Center, Technion, Israel).

Matrix-assisted laser desorption ionization–time-of-flight (MALDI-TOF) analysis – Purified VDAC (25 µl, at 1mg/ml) was UV irradiated in the presence or absence of 80 µM BzATP (see above), dialyzed against 5 mM Tris-HCl, pH 7.2 for 6 hours at room temperature and dried using Speed-Vac. The samples were re-dissolved in an aqueous solution of 0.1% trifluoroacetic acid, diluted 1:1 with matrix solution (a saturated solution of α-cyano-4-hydroxycinnamic acid in isopropanol : formic acid : DDW (2:1:3, v/v)), and 1 µl of the mixture was spotted on the MALDI target plate and air dried. The obtained results were analyzed as described below.

To localize the VDAC-NBS(s), unlabeled and BzATP-labeled VDAC (~25 µg) were precipitated with acetone (at -70°C) so that the detergent would not disrupt the MALDI analysis. The pellets were re-solubilized in 50 mM NH4HCO3, pH 8, containing 1M urea, and incubated for 8 hours at 37°C. The samples were then diluted 1:3 with the same buffer without urea and subjected to digestion with trypsin (TPCK-treated, type XIII from bovine pancreas, Sigma), at a ratio of 1:20 enzyme:protein (w/w). Digestion was carried out for 24 hours at 37°C, and then aliquots (~0.6 µg protein) were dried by Speed-Vac and prepared for the MALDI analysis as described previously (29).

MALDI-TOF analysis was performed at the National Institute for Biotechnology in the Negev (BGU), Beer-Sheva, Israel. Mass spectra of VDAC and VDAC fragments, bound or not bound to BzATP, were acquired using a MALDI-TOF mass spectrometer Reflex IV (Bruker, Bremen, Germany) equipped with a 337 nm nitrogen laser, with delayed extraction. The acceleration voltage was 20.0 kV. All spectra were obtained in positive-ion mode. For each sample, 300–500 laser shots were accumulated in a linear mode and processed by XMASS 5.1.5 software (Bruker, Bremen, Germany).

RESULTS

Demonstration and characterization of

nucleotide-binding site(s) in VDAC – VDAC, purified from rat liver mitochondria by chromatography on HA:C, was found to bind to RRA, previously shown to bind nucleotide-binding proteins (30). Accordingly, RRA-bound

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VDAC was eluted with ATP (Fig. 1) and ADP (not shown), whereas AMP was less effective (Fig. 1) and NADH was ineffective in releasing VDAC from the RRA (not shown). These results thus suggest the presence of nucleotide-binding site(s) in VDAC.

To identify and characterize the NBS(s) in the VDAC molecule, the photoreactive ATP analog, [α-32P]BzATP, was used. A representative autoradiogram of purified VDAC labeled with increasing [α-32P]BzATP concentrations is shown in Figure 2C. Quantitative analysis of VDAC labeling with relatively high [α-32P]BzATP concentrations showed saturation with half-maximal binding (K0.5) at about 70 µM (Fig. 2B). Scatchard plot analysis of the data revealed a non-linear correlation representing high- and low-affinity binding sites, with apparent Kd values of 15 µΜ and 53 µM, respectively (Fig. 2A, inset), suggesting binding of [α-32P]BzATP to two or more classes of NBSs in the VDAC molecule. Further analysis of the high-affinity binding site revealed saturation and an apparent binding affinity of 4.98 µM ± 0.74 (n=10) (Fig. 2B), whereas the apparent binding affinity of the low-affinity site(s) (Fig. 2A) ranged between 55 and 110 µM (n=5).

To demonstrate the presence of NBS(s) in membrane-embedded VDAC, representing VDAC in its native state, mitochondria were UV-irradiated in the presence of [α-32P]BzATP. Several proteins were labeled, amongst them VDAC, as identified by immunostaining using a specific antibody (Fig. 2D I). To further confirm membrane-embedded VDAC labeling, VDAC was purified from [α-32P]BzATP pre-labeled mitochondria. Indeed, as shown in Fig. 2D II , the purified VDAC was labeled with [α-32P]-BzATP.

The binding specificity of the VDAC-NBS(s) was analyzed by VDAC labeling with [α-32P]BzATP in the presence of various nucleotides. Quantitative analysis of autoradio-grams obtained from different experiments (representative results are shown in Figure 3A) revealed that the labeling was decreased by the nucleotides in the following order of effectiveness: GTP = GDP ≥ ATP = ADP > AMP-PNP > UDP > AMP > UTP, whereas CDP and CTP were much less effective (Fig. 3B). On the other hand, the effect of NADH on VDAC labeling varied among different VDAC preparations, where in three of six experiments, NADH showed an inhibitory effect, lower by

~15% than that of ATP, while in the other three experiments, NADH had no effect (data not shown). To conclude, the labeling results indicate that the VDAC-NBS(s) recognize(s) purine nucleotides (i.e., AMP, ADP, ATP, GDP, GTP and AMP-PNP) better than pyrimidine nucleotides (CDP, CTP, UDP and UTP) (p<0.0001, Student's t-test for the indicated purine group vs. the pyrimidine group).

The effect of various divalent cations (Me2+) and EDTA on [α-32P]BzATP labeling of VDAC was considered next. As presented in Table I, all divalent cations tested inhibited VDAC labeling. In contrast, EDTA increased [α-32P]BzATP binding. Characterization of the effects of Mn2+ and Mg2+ on [α-32P]BzATP labeling of VDAC showed that both cations inhibited [α-32P]BzATP labeling with IC50 values of 10 ± 2 and 92 ± 14 µM, respectively (Fig. 4A). Analysis of [α-32P]BzATP labeling of VDAC as a function of the relative amount of free nucleotide, or of the nucleotide•Mg complex (Fig. 4B), clearly indicated that binding increased with increasing concentrations of the free nucleotide, and decreased with increased Mg•[α-32P]BzATP concentration. Furthermore, when all of the [α-32P]BzATP was complexed with Mg2+, the extent of VDAC labeling was only 20% of that obtained when the same concentration of free [α-32P]BzATP was employed. Thus, VDAC seems to have about a 5-fold lower affinity for the Mg•[α-32P]BzATP complex than for [α-32P]BzATP. This suggests that the negatively-charged nucleotide, rather than the Me2+•[α-32P]BzATP complex, is the species bound by VDAC. High concentrations of NaCl, LiCl and KCl (10–100 mM) also inhibited [α-32P]BzATP labeling of VDAC (data not shown). Since, at solutions with similar ionic strength, NaCl displayed a 13-fold lower inhibition of [α-32P]BzATP labeling than MgCl2 (data not shown), it is most likely that the labeling inhibition is not due to non-specific effects of ionic strength. Furthermore, the effect of the monovalent cations on [α-32P]BzATP labeling was not due to the formation of Me1+•[α-32P]BzATP complexes (as calculated from the association constant), since only a small percentage of BzATP forms such complexes, but rather was the result of direct interaction with VDAC. To conclude, the inhibitory effect of divalent cations on [α-32P]-BzATP labeling appears to result from the reduction of the concentration of the VDAC bound species, i.e., free [α-32P]BzATP.

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FITC modification of the VDAC-NBS(s) – Having shown that VDAC possesses NBSs, we further tested whether lysine residues present in or proximal to Walker-type nucleotide-binding motifs (31) are involved in [α-32P]-BzATP binding to VDAC, using FITC, which covalently binds to the ε-amino group of lysine (Fig. 5). FITC strongly inhibited [α-32P]BzATP labeling of VDAC, with half-maximal inhibition occurring at about 18 µM and maximal inhibition (85%) at about 80 µM FITC (Fig. 5A). The linear relationship between FITC labeling of VDAC, as assayed by the fluorescence intensity of FITC-VDAC, and FITC inhibition of [α-32P]-BzATP labeling of VDAC (Fig. 5B) could reflect modification of a lysine residue(s) located in the ATP-binding site(s), or in a distinct site that indirectly affects nucleotide binding to VDAC. The relation between the site of FITC modification and the site of ATP binding was further demonstrated by the protection afforded by ATP and ADP against FITC inhibition of [α-32P]BzATP binding to VDAC (data not shown).

Localization of the VDAC-NBSs – In order to localize the NBSs in VDAC, [α-32P]-BzATP-labeled protein was subjected to chemical or enzymatic cleavage (Figs. 6 and 7), followed by SDS-PAGE, immunoblotting and autoradiography. As expected from the presence of a single methionine in rat VDAC, CNBr treatment of [α-32P]BzATP-labeled VDAC resulted in cleavage of the 31 kDa-protein into two fragments of about 16.4 and 14.7 kDa, each labeled with [α-32P]BzATP (Fig. 6, A and B). The decrease in the intensity of [α-32P]BzATP labeling of VDAC observed after 24 hr-incubation with formic acid (Fig. 6B, second lane) probably resulted from partial hydrolysis of the covalent bond between the protein and [α-32P]BzATP that occurred during the low-pH treatment. Finally, use of an antibody raised against the VDAC N-terminal led to immunostaining of the uncleaved VDAC and of the 16.4 kDa VDAC fragment, but not the 14.7 kDa fragment (Fig. 6C). Thus, the fragments seem to correspond to the N- and C-termini of VDAC, respectively. These results support the existence of at least two NBSs, at least one located in each of the N- and C- terminal halves of VDAC.

Similarly, V8 digestion of [α-32P]-BzATP-labeled VDAC yielded two major, labeled fragments of about 17 and 14 kDa (Fig. 7B). While these fragments did not cross-react

with an antibody raised against the N-terminal 19 residues of VDAC, a smaller unlabeled fragment, migrating near the gel front, was immunostained (Fig. 7C, arrow), suggesting that the NBS is not restricted within this region. Each of the two [α-32P]BzATP-labeled V8-generated fragments was subjected to LysC digestion, with the resulting peptides being separated by reverse phase-HPLC. Selected peptides were applied to a sequenator, yielding three peptide sequences from the 17 kDa fragment (WNTDNTLGTEIT-VEDQLAR; LTFDSSFSPNTGKK; EHINLGC-DVDFDIAGPSIR) and two from the 14 kDa fragment (YQIDPDACFSAK; LGLGLEFQA) (Fig. 9, Yellow and blue boxes, respectively). Comparison of these sequences revealed that they originated from two distinct, non-overlapping VDAC fragments, and thus supported the presence of two or more NBSs in VDAC.

Mass-spectral evidence lent further support to the existence of one or more VDAC-NBSs. MALDI-TOF spectrum of VDAC showed a sharp peak of 30857 m/z (Fig. 8, dashed line), in accordance with the VDAC1 calculated molecular mass of 282 amino acids (excluding the first methionine). MALDI-TOF spectrum of BzATP-photolabeled VDAC (Fig. 8, solid line) showed a broad peak with an average of 31653 m/z. This mass shift of 796 Da between the two peaks corresponds to the molecular mass of one BzATP molecule (i.e., ~715.4 Da) plus a phosphate group (HPO3, 80.1 Da). The VDAC-bound phosphate group could reflect the presence of phosphorylated VDAC (32, 33) in our purified protein preparations (see below), or could be a result of BzATP hydrolysis. Moreover, the broad peak obtained for BzATP-labeled VDAC could represent a mixed population of VDAC with different molar ratios of BzATP and VDAC. The different BzATP adduct species of VDAC could not be resolved due to poor resolution of the MALDI in this molecular mass range.

Next, VDAC and BzATP-labeled VDAC were subjected to trypsin digestion and MALDI-TOF analysis. For both samples, the masses of the obtained fragments were compared to three databases (NCBInr, Swiss-Prot, Mascot-MSDB) and peptides matching 39-65% of VDAC1 (from human or rat)/porin31HM sequence were identified. In addition, of about 40 peptides found only in BzATP-labeled VDAC, three were suspected to be BzATP-binding peptides since subtraction of

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the BzATP mass (715.4 Da) from theirs yielded masses comparable with peptides obtained in the unlabeled VDAC sample and with masses expected from virtual digestion of VDAC (see Table II). These three VDAC peptides corresponded to the following amino acid sequences (numbering including the first methionine): 13–28, 110–119 and 257–274. Interestingly, the VDAC sample also contained a peptide of 1385.8 m/z, corresponding to the 1386.8 m/z expected for phosphorylated trypsin-resulting peptide 110–120 (addition of ~80 Da to the peptide theoretical mass of 1306.8 Da). Consistently, only the sample of BzATP-labeled VDAC included a peptide of 2103.1 m/z, corresponding to the mass of 2102.2 m/z expected from both phosphate group and BzATP bound to the 110–120 peptide. These findings are in accordance with the MALDI result described above (Fig. 8), and together they further support the existence of a VDAC-NBS around the 110–119 region (Table II).

Based on the above findings and comparison to known nucleotide binding sequences (31, 34-36), three VDAC-NBSs were proposed, as shown in Figure 9 (see "Discussion").

DISCUSSION

The presence of nucleotide-binding

site(s) in VDAC has been previously suggested (19-23). In this study, using a more direct approach, i.e. affinity-labeling with photoactivated BzATP, we have demonstrated, characterized and localized the NBSs in VDAC.

Characterization of the VDAC NBS(s) – [α-32P]BzATP labeling of purified VDAC saturated at two concentration ranges, thus revealing high- and low-affinity NBSs, having apparent Kd values of 15 µΜ and 53 µM, respectively (Fig. 2). VDAC labeling decreased in the presence of various nucleotides, in a manner indicating that the binding site is specific for purine nucleotides and that nucleotide tri- and di-phosphates are better recognized than monophosphates (Fig. 3). The nucleotide-binding specificity of VDAC was also demonstrated by the efficient elution of RRA-bound VDAC with ATP, yet only weakly with AMP (Fig. 1) and not with NADH (not shown). These results are fairly consistent with the ability of various nucleotides to generate ion current fluctuations ("noise") through PLB-

reconstituted VDAC (resulting from their interaction with VDAC) (21).

Divalent cations inhibited [α-32P]BzATP labeling of VDAC with the following ranked order of potency: Mn2+ > Mg2+ > Ca2+ > Sr2+ (Table I), in correlation with the formation of Me2+•[α-32P]BzATP complexes. This, and the finding that when all [α-32P]BzATP is complexed with Mg2+, the extent of VDAC labeling is only 20% of that obtained with the same concentration of free [α-32P]BzATP (Fig. 4), suggest that the inhibitory effect of divalent cations is due to their binding to [α-32P]BzATP, thus pointing to the charged nucleotide as the VDAC-bound species. This suggestion is consistent with the correlation found between the ability of nucleotides to generate current noise through VDAC and the charge of the nucleotides (i.e., ATP4- > ADP3- > AMP2- and NADPH4- > NADH2- > NAD1-) (21). Additional support for the binding of negatively-charged nucleotides to VDAC comes from nucleotide transport studies. Nucleotide transport into and out of the mitochondria involves two linked transport systems: the ANT in the IMM (37) and VDAC in the OMM (4, 8, 17, 19, 20). Binding and transport of negatively-charged nucleotides by VDAC suggested here and in other studies (3, 17-19, 21-23) is in accordance with the binding and transport of such species by the ANT (37).

The lysyl-modifying reagent, FITC, was used to further characterize the VDAC-NBS(s). Lysine residues are a common feature of NBSs in many ATP-binding proteins, including ATPases, kinases and transporters (31, 34-36), and FITC was shown to bind to NBSs in various proteins, including ATPases (i.e., Na+,K+-ATPase), kinases and the mitochondrial ANT and phosphate carrier, and to inhibit their activities (see 38 and refs. therein). FITC decreased the ability of VDAC to bind [α-32P]-BzATP (Fig. 5), while the concurrent presence of ATP or ADP protected against FITC-inhibition of [α-32P]BzATP binding to VDAC. Similarly, acetylation of lysine residues prevents nucleotide-labeling of the Neisseria porin, PorB (39). These results suggest the involvement of lysine residue(s) in binding of adenine nucleotides to VDAC, and indeed the proposed VDAC-NBSs contain lysine residues (see Fig. 9 and Table II).

The highly conserved ATP-binding motif A, G(X/G)XGXGKT, described by Walker et al. (31) contains a lysine residue that probably interacts with the phosphate groups of

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ATP (34). A proposed NBS in the VDAC1 N-terminal region, similar to inverted Walker-type A motif (TKGYGFG, positions 19–25), and another possible site, containing some residues characterizing the Walker motifs A and B, in the VDAC1 C-terminal region (GGHKLGLGLEFQA, positions 271–283) (see Fig. 9), each contain a lysine residue. An additional NBS (KNAKIKTGYKR, positions 110–120), containing residues characterizing the Walker motifs and including several lysines, is proposed. It is noteworthy that K20 is located at a specific, highly-conserved position in the N-terminal of VDACs from various species (6, 40), supporting the hypothesis that there is a significant function for this residue. Indeed, K20 mutation in yeast VDAC alters its channel properties (41). It has been previously shown that substitution of the lysine residue in Walker motif A generally results in complete or partial inhibition of ATP binding and hydrolysis (34-36). Accordingly, FITC-inhibition of [α-32P]-BzATP binding to VDAC supports the suggestion that the VDAC-NBS(s) contain(s) lysine residue(s). Thus, to establish the function of K20 in the regulation of VDAC activity by nucleotide binding, the study of the effects of site-directed mutation of K20 in VDAC is in progress (manuscript in preparation).

VDAC possesses two or more NBSs – In this report, [α-32P]BzATP labeling, controlled proteolysis or chemical cleavage, immunolabe-ling and peptide sequencing, as well as MALDI-TOF analysis (Figs. 2, 6–8, Table II), all suggest the existence of two or more NBSs in VDAC1. Scatchard plot analysis of [α-32P]-BzATP binding to VDAC points to the existence of two or more classes of binding sites that possess different affinities. The existence of two or more VDAC-NBSs is also reflected in the shift of the MALDI spectrum of BzATP-labeled VDAC, which seems to correspond to the binding of two or more BzATP molecules (Fig. 8), and in the generation of two [α-32P]BzATP-labeled, distinct, non-overlapping VDAC fragments by V8 or CNBr cleavage (Figs. 6–7). In terms of their locations, it seems that no NBS is within the first 19 amino acids of the N-terminal of VDAC1, the region specifically recognized by the anti-VDAC antibody used here, since neither of the [α-32P]BzATP-labeled, V8-generated 17 and 14 kDa fragments was immunostained (Fig. 7C), and a peptide observed in the gel's front that was recognized by this antibody was not labeled with [α-32P]BzATP.

The proposed locations of the NBSs are illustrated in the membrane topology model of rat VDAC1 based on the human VDAC1 model suggested by Song and Colombini (6, 40) (Fig. 9). The first NBS suggested by MALDI analysis of trypsin-digested, BzATP-labeled VDAC (Table II) pointed to positions 13–28 as a BzATP-binding region and thus supports the localization of the NBS in the protein's N-terminal. Indeed, this site corresponds to a sequence in the N-terminal region of mammalian VDAC1 reminiscent of the Walker A motif (31), but in inverted sequence (TKGYGFG, positions 19–25). The second NBS (KNAKIKTGYKR, positions 110–120) proposed by MALDI includes several lysine residues, glycine and threonine; all are residues typical of the Walker A motif (31). MALDI analysis not only indicated that this peptide binds BzATP (Table II), but also suggested it could be phosphorylated (see "Results" and Fig. 8). Indeed, the peptide includes a threonine and a tyrosine residues, and VDAC phosphorylation has been previously reported (32), specifically tyrosine phosphorylation (33). In addition, the second proposed NBS partly overlaps a peptide which is a part of the 14-kDa V8-generated radiolabeled fragment (positions 98–111, Fig. 9 second yellow box). MALDI data (Table II) also pointed to a third NBS (positions 257–274) at the C-terminal region of VDAC1. This region partially overlaps a sequence (GGHKLGLGL-EFQA, positions 271–283) containing a lysine residue and several glycine residues, similar to several Walker A-type NBSs in other proteins, and also includes residues (e.g., E, L, Q) present in or proximal to the Walker B motif (31, 34, 35). Moreover, the sequence in positions 271–283 includes a peptide which is a part of the 17-kDa V8-generated radiolabeled fragment (positions 275–283, Fig. 9 second blue box).

The locations of the predicted VDAC-NBSs with respect to the OMM vary according to each of the three main membrane topology models proposed for VDAC1. It has been proposed that the VDAC1 N-terminal α-helix crosses the membrane (6, 40), thus its NBS might face the mitochondrial inter-membrane space (IMS) and/or the pore lumen. Figure 9 is compatible with such a model. Alternatively, the N-terminal might protrude from the OMM to the cytosol (42), or associate with the cytosolic face of the OMM (43), and thus the NBS could be freely exposed to the cytosol. Previous studies were inconclusive, reporting reversible binding

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of ATP to the VDAC N-terminal (comprising a putative NBS – GYGFG, positions 20–24) located at the membrane surface (23), and attraction of ATP molecules to the channel's aqueous pore (20, 21). On the other hand, according to the three proposed VDAC topology models, the VDAC C-terminal is embedded in the membrane so that its NBS probably faces the IMS and/or the pore lumen (see Fig. 9). The putative location of the NBSs on both the cytosolic and IMS-oriented surfaces of VDAC or within the channel's lumen could reflect an important regulatory function for nucleotide binding.

Possible functions of the VDAC nucleotide-binding sites – The binding of several cytosolic kinases, including hexokinase (44, 45, reviewed in 7), creatine kinase (44) and glycerol kinase (46), to VDAC could reflect its ATP transport activity. The role of VDAC as a pathway for adenine nucleotides and as a nucleotide-binding protein provides a possible mechanism for the regulation of nucleotide fluxes into and out of the mitochondria. In this regulation, the state of the VDAC channel, i.e. "open" or "closed", serves as a key determinant: when VDAC is open, regulation of nucleotide fluxes through the channel by bound nucleotides controlling transport activity would be of great importance. Indeed, it has been shown that induction of VDAC closure (e.g., by addition of NADH or a specific inhibitor that alters VDAC’s gating) in reconstituted systems also decreases ADP-dependent type-3 respiration in mitochondria (3, 8, 17, 22) and reduces the activity of mitochondrial adenylate kinase (8). VDAC closure also prevents exchange of nucleotides and other metabolites between the cytosol and mitochondria, and this has been linked to the initiation of apoptosis (4). Moreover, the findings that NADH alters VDAC's gating (3, 22) and decreases the OMM permeability to ADP (3) could imply VDAC sensitivity to cytosolic NADH levels. This means that VDAC permeability may depend on the rate of glycolysis (producing NADH), and thus it can be involved in a molecular mechanism underlying the glucose-induced suppression of respiration seen in normal and transformed cells ("The Crabtree effect") (see 3, 22 and Refs. therein). Thus, limited transport of NADH and ATP/ADP (and other respiration substrates) can affect mitochondrial respiration and, thereby, the cell’s energy metabolism, growth, and survival.

Other implications of nucleotide binding to VDAC could be changes in its association with key metabolic enzymes, such as kinases that possess preferential accessibility to ATP (44, 45), or with pro- and anti-apoptotic proteins such as members of the Bcl-2 protein family. The ability of VDAC to bind ATP or ADP can also affect PTP opening, directly or indirectly, by affecting VDAC’s association with other PTP components or regulators, and thus could be implicated in the regulation of apoptosis.

In the cell, ATP concentration is in the millimolar range (3.1 mM), while ADP is present in the micromolar (30 µM) range (47), except under certain conditions that lead to ATP depletion. Channeling of ATP through VDAC, so as to expel the nucleotide to the cytosol (2), provides a possible regulation of energy production via ATP binding to VDAC and modulation of its nucleotide transport activity. It is expected that at high cytosolic ATP levels, the VDAC-NBS(s) would be occupied and VDAC conductance to nucleotides would be decreased (21). This would subsequently lead to decreased transport of ATP and ADP to the cytosol and mitochondria, respectively, and, in turn, reduced ATP production. However, when considering the effect of divalent cations on nucleotide binding to VDAC (Fig. 4), the relationship between ATP and ADP concentrations and VDAC transport activity becomes more complex. Changes in the concentration of ATP4-, the proposed nucleotide species interacting with VDAC (Fig. 4) (19-21, 23), could result from changes in intracellular free Mg2+. The concentration of free Mg2+ in the cell is about 1.1 mM, while complexed Mg2+ is found at 8 mM (47). In the mitochondrial matrix, Mg2+ is found in the 0.8-1.5 mM range (48). Thus, changes in free and complexed ATP levels and, concomitantly, in Mg2+ levels, would affect the modulation of VDAC activity by nucleotides. It has been postulated that free Mg2+ serves as the sensor of matrix volume (49) and plays an important role in the control of mitochondrial metabolic activities such as oxidative phosphorylation (50). These functions of Mg2+ might be mediated by its capacity to form complexes with ADP and ATP, and thereby, to affect VDAC conductance. Modulation of VDAC activity by ATP and ADP interaction with the NBSs, possibly facing the cytosol and the IMS, provides a regulatory mechanism for metabolite transport sensitive to the cell's energy demands.

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REFERENCES

1. Brown, G. C. (1992) Biochem. J. 284, 1-13 2. Brdiczka, D. (1994) Biochim. Biophys. Acta.

1187, 264-269 3. Lee, A. C., Xu, X., and Colombini, M.

(1996) J. Biol. Chem. 271, 26724-26731 4. Vander-Heiden, M. G., Li, X. X., Gottleib,

E., Hill, R. B., Thompson, C. B., and Colombini, M. (2001) J. Biol. Chem. 276, 19414-19419

5. Shoshan-Barmatz, V., and Gincel, D. (2003) Cell Biochem. Biophys. 39, 279-292

6. Colombini, M. (2004) Mol. Cell. Biochem. 256-257, 107-115

7. Shoshan-Barmatz, V., and Israelson, A. (2005) J. Membr. Biol. 204, 57-66

8. Liu, M. Y., and Colombini, M. (1992) Biochim. Biophys. Acta 1098, 255-260

9. Zoratti, M., and Szabo, I. (1995) Biochim. Biophys. Acta. 1241, 139-176

10. Crompton, M. (1999) Biochem. J. 341, 233-249

11. Kroemer, G., and Reed, J. C. (2000) Nature Med. 6, 513-519

12. Crompton, M., Virji, S., and Ward, J. M. (1998) Eur. J. Biochem. 258, 729-735

13. Lapidus, R. G., and Sokolove, P. M. (1994) J. Biol. Chem. 269, 18931-18936

14. Novgorodov, S. A., Gudz, T. I., Brierley, G. P., and Pfeiffer, D. R. (1994) Arch. Biochem. Biophys. 311, 219-228

15. Halestrap, A. P., Kerr, P. M., Javadov, S., and Woodfield, K. Y. (1998) Biochim. Biophys. Acta. 1366, 79-94

16. Haworth, R. A., and Hunter, D. R. (2000) J. Bioenerg. Biomembr. 32, 91-96

17. Benz, R., Wojtczak, L., Bosch, W., and Brdiczka, D. (1988) FEBS Lett. 231, 75-80

18. Bathori, G., Sahin-Toth, M., Fonyo, A., and Ligeti, E. (1993) Biochim. Biophys. Acta. 1145, 168-176

19. Rostovtseva, T., and Colombini, M. (1997) Biophys. J. 72, 1954-1962

20. Rostovtseva, T. K., and Bezrukov, S. M. (1998) Biophys. J. 74, 2365-2373

21. Rostovtseva, T. K., Komarov, A., Bezrukov, S. M., and Colombini, M. (2002) Biophys. J. 82, 193-205

22. Zizi, M., Forte, M., Blachly-Dyson, E., and Colombini, M. (1994) J. Biol. Chem. 269, 1614-1616

23. Florke, H., Thinnes, F. P., Winkelbach, H., Stadtmuller, U., Paetzold, G., Morys-Wortmann, C., Hesse, D., Sternbach, H.,

Zimmermann, B., and Kaufmann-Kolle, P. (1994) Biol. Chem. Hoppe Seyler. 375, 513-520

24. Williams, N., and Coleman, P. S. (1982) J. Biol. Chem. 257, 2834-2841

25. Johnson, D., and Lardy, H. (1967) Methods Enzymol. 10, 94-96

26. Huizing, M., Ruitenbeek, W., van den Heuvel, L. P., Dolce, V., Iacobazzi, V., Smeitink, J. A., Palmieri, F., and Trijbels, J. M. (1998) J. Bioenerg. Biomembr. 30, 277-284

27. Laemmli, U. K. (1970) Nature 227, 680-685 28. Cleveland, D. W., Fischer, S. G., Kirschner,

M. W., and Laemmli, U. K. (1977) J. Biol. Chem. 252, 1102-1106

29. Landry, F., Lombardo, C. R., and Smith, J. W. (2000) Anal. Biochem. 279, 1-8

30. Coll, R. J., and Murphy, A. J. (1984) J. Biol. Chem. 259, 14249-14254

31. Walker, J. E., Saraste, M., Runswick, M. J., and Gay, N. J. (1982) EMBO J. 1, 945-951

32. Bera, A. K., Subhendu, G., and Sudipto, D. (1995) Biochem. Biophys. Res. Comm. 209, 213-217

33. Liberatori, S., Canas, B., Tani, C., Bini, L., Buonocore, G., Godovac-Zimmermann, J., Mishra, O. P., Delivoria-Papadopoulos, M., Bracci, R., and Pallini, V. (2004) Proteomics 4, 1335-1340

34. Hishida, T., Iwasaki, H., Yagi, T., and Shinagawa, H. (1999) J. Biol. Chem. 274, 25335-25342

35. Hou, Y.-X., Cui, L., Riordan, J. R., and Chang, X.-B. (2000) J. Biol. Chem. 275, 20280-20287

36. Imagawa, T., Teramachi, S., and Taniguchi, K. (2003) Ann. NY Acad. Sci. 986, 240-241

37. Klingenberg, M. (1980) J. Membr. Biol. 56, 97-105

38. Majima, E., Ishida, M., Miki, S., Shinohara, Y., and Terada, H. (2001) J. Biol. Chem. 276, 9792-9799

39. Rudel, T., Schmid, A., Benz, R., Kolb, H. A., Lang, F., and Meyer, T. F. (1996) Cell 85, 391-402

40. Song, J., and Colombini, M. (1996) J. Bioenerg. Biomembr. 28, 153-161

41. Blachly-Dyson, E., Peng, S., Colombini, M., and Forte, M. (1990) Science 247, 1233-1236

42. Casadio, R., Jacoboni, I., Messina, A., and De Pinto, V. (2002) FEBS Lett. 520, 1-7

43. Reymann, S., Florke, H., Heiden, M., Jakob, C., Stadtmuller, U., Steinacker, P., Lalk, V.

by guest on April 6, 2018

http://ww

w.jbc.org/

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nloaded from

10

E., Pardowitz, I., and Thinnes, F. P. (1995) Biochem. Mol. Med. 54, 75-87

44. Beutner, G., Ruck, A., Riede, B., and Brdiczka, D. (1998) Biochim. Biophys. Acta. 1368, 7-18

45. Azoulay-Zohar, H., Israelson, A., Abu-Hamad, S., and Shoshan-Barmatz, V. (2004) Biochem. J. 377, 347-355

46. Adams, V., Griffin, L., Towbin, J., Gelb, B., Worley, K., and McCabe, E. R. (1991) Biochem. Med. Metab. Biol. 45, 271-291

47. Traut, T. W. (1994) Mol. Cell Biochem. 140, 1-22

48. Rutter, G. A., Osbaldeston, N. J., McCormack, J. G., and Denton, R. M. (1990) Biochem. J. 271, 627-634

49. Garlid, K. D. (1980) J. Biol. Chem. 255, 11273-11279

50. Rodriguez-Zavala, J. S., and Moreno-Sanchez, R. (1998) J. Biol. Chem. 273, 7850-7855

51. Dawson, R. M. C., Elliott, D. C., Elliott, W. H., and Jones, K. M. (1986) Data for biochemical research., 3rd Ed., Clarendon Press, Oxford

FOOTNOTES

* This research was supported by a grant from the Israel Science Foundation, administrated by The Israel Academy of Science and Humanities. ‡ The Hyman Kreitman Chair in Bioenergetics. 1 The abbreviations used are: OMM, outer mitochondrial membrane; VDAC, voltage-dependent anion channel; PTP, permeability transition pore; ANT, adenine nucleotide translocase; IMM, inner mitochondrial membrane; NBS(s), nucleotide-binding site(s); PLB, planar lipid bilayer; CNBr, cyanogen bromide; RRA, reactive red-120 agarose; EDTA, ethylene-diaminetetraacetate; FITC, fluorescein 5' isothiocyanate; V8, Staphylococcus aureus protease; BzATP, 3'-O-(4-benzoyl)-benzoyl adenosine 5'-triphosphate; HA:C, hydroxyapatite:celite; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; LC/MS, liquid-chromatography/mass spectrometry; MALDI-TOF, matrix-assisted laser desorption ionization–time-of-flight; IMS, intermembrane space.

FIGURE LEGENDS Fig. 1. VDAC binds to reactive red-agarose column and is eluted with ATP. VDAC was purified from rat liver mitochondria using HA:C and RRA chromatography. HA:C-purified VDAC was applied to an RRA column, which was washed with a solution of 0.3% Triton X-100, 10 mM Tris-HCl, pH 7.2 (fractions 1, 2), followed by a wash with identical solution containing 10 mM of AMP (fractions 4–13) or ATP (fractions 14–25), as indicated. A Triton X-100 (3%) mitochondrial extract (Triton ext.) and fractions obtained from the HA:C and RRA columns were subjected to SDS-PAGE and immunoblotting using anti-VDAC antibodies. A Coomassie Brilliant Blue stained gel is shown in A, and the corresponding immunoblot in B. Fig. 2. Purified and membrane-embedded VDAC are labeled with [α-32P]BzATP. Purified VDAC was labeled with the indicated concentrations of [α-32P]BzATP and subjected to SDS-PAGE and autoradiography followed by densitometric quantification of the autoradiograms. A, representative results (2 of 5 experiments) of VDAC labeling as a function of [α-32P]BzATP concentration. Inset shows the Scatchard plot of the data. B, quantitative analysis of [α-32P]BzATP binding to the high-affinity site (n=10). Inset shows the Scatchard plot of the data. The results represent mean ± S.E.M. C, a representative autoradiogram of VDAC labeling by [α-32P]BzATP corresponding to one set of results presented in B, where CBB indicates Coomassie Brilliant Blue staining. D, mitochondria (20 µg) were labeled with [α-32P]BzATP (1µM) and subjected to SDS-PAGE (CBB), autoradiography (Auto), and Western blot using anti-VDAC antibody (WB) (I). [α-32P]BzATP-labeled VDAC was purified from [α-32P]BzATP-labeled mitochondria using HA:C column, as analyzed by SDS-PAGE (CBB) and autoradiography (Auto) (II). . Fig. 3. Nucleotide specificity of the VDAC binding site(s). VDAC was labeled with [α-32P]BzATP (1 µM) in the absence or in the presence of the indicated concentration of various nucleotides. Samples were subjected to SDS-PAGE and autoradiography followed by quantitative analysis of the

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autoradiograms. Representative autoradiograms (A) and quantitative analysis (B) of VDAC labeling in the presence of 0.4 mM of the indicated nucleotides are presented. The results represent the mean ± S.E.M of 6 to 10 different experiments. CBB indicates Coomassie Brilliant Blue staining. Fig. 4. Divalent cations inhibit [α-32P]BzATP labeling of VDAC. VDAC was labeled with [α-32P]BzATP (1 µM) in the absence or in the presence of MgCl2 (50–500 µM) or MnCl2 (5–500 µM). A, quantitative analysis of VDAC labeling in the absence or the presence of Mg2+ or Mn2+. Inset shows a representative autoradiogram. B, VDAC labeling as a function of the calculated concentrations of free BzATP (●) and Mg•BzATP complex (○). The results represent the mean ± S.E.M of 3 to 4 different experiments. The concentrations of free BzATP and Mg•BzATP complex were calculated with the computer program WinMAXC 2.0.5, using a log K stability constant employed for the Mg•ATP complex (i.e., 4.63 at 25oC, Ref. 51), since a constant for the Mg•BzATP complex is not available. Fig. 5. FITC modification inhibits [α-32P]BzATP labeling of VDAC. Purified VDAC was incubated with the indicated concentrations of FITC in 10 mM Tris-HCl, pH 7.2. After 10 min at 30°C, [α-32P]BzATP was added and the samples were UV-irradiated for 3 min. Samples were subjected to SDS-PAGE and then, to visualize the FITC-labeled protein bands, the gels were illuminated with 365 nm UV, photographed (see inset in A) and subjected to Coomassie staining and autoradiography. Quantitative analyses of the autoradiograms and the FITC fluorescence were carried out by densitometry. The results represent the mean ± S.E.M of 10 to 14 different experiments. A, [α-32P]BzATP and FITC labeling of VDAC as a function of FITC concentration. B, for each FITC concentration used, the relative level of [α-32P]BzATP labeling of VDAC is plotted as a function of the fluorescence intensity of the FITC-labeled VDAC. Fig. 6. CNBr-cleavage of [α-32P]BzATP-labeled VDAC yields two radiolabeled fragments. VDAC was labeled with [α-32P]BzATP (1 µM) and treated with the indicated concentrations of CNBr, in the presence of 83% formic acid, as described under "Experimental Procedures". Protein samples were subjected to SDS-PAGE (16% acrylamide) and silver staining (A) or electroblotted onto nitrocellulose membrane and subjected to autoradiography (B) followed by immunoblot (C) using an anti-VDAC antibody. The positions of the 16.4- and 14.7-kDa CNBr-resulted VDAC fragments are indicated by arrows. Note, as expected, only one CNBr generated fragment (16.4 kDa) is recognized by the anti-VDAC antibodies. Fig. 7. V8 proteolysis of [α-32P]BzATP-labeled VDAC yields two radiolabeled fragments. VDAC was labeled with 2 µM [α-32P]BzATP and subjected to SDS-PAGE (10% acrylamide), followed by Coomassie blue staining. VDAC bands were excised from the gel and subjected to V8 digestion in a second gel (see "Experimental Procedures"), followed by SDS-PAGE and Coomassie Brilliant Blue staining (CBB) (A) and autoradiography (B), or electroblotting and immunoblot using an anti-VDAC antibody (C). The arrow in C points to a low molecular weight fragment interacting with the anti-VDAC antibody raised against the first 19 amino acids of the N-terminal of VDAC. The 17- and 14-kDa V8-generated fragments are also shown. Fig. 8. MALDI-TOF spectra of unlabeled and BzATP-labeled VDAC. Purified VDAC (dashed line) and VDAC labeled with BzATP (solid line) were subjected to MALDI-TOF mass analysis. Numbers above the peaks represent the molecular mass corresponding to the average masses [M+H]+ of the proteins. Fig. 9. Proposed membrane topology model of VDAC1 showing the localization of sequenced VDAC peptides from BzATP-labeled fragments and the predicted ATP-binding sites. This model of rat VDAC1 is based on the model of human VDAC1 proposed by Colombini (6). Rat VDAC1 amino acids that differ from human VDAC1 are indicated by purple squares. Yellow and blue boxes represent the peptide sequences obtained from the 17- and 14-kDa V8-proteolysis products, respectively (see "Experimental Procedures"). Putative N- and C- termini NBSs are colored in red. The proposed V8 (E) and CNBr (M) cleavage sites are indicated by green squares and arrows.

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TABLE I Divalent cations inhibit VDAC labeling by [α-32P]BzATP

Purified VDAC was UV-irradiated in the presence of 1 µM [α-32P]BzATP and in the absence or in the presence of the indicated compounds. Samples were subjected to SDS-PAGE and autoradiography. Quantitative analysis of the autoradiograms was carried out as described under "Experimental Procedures". The results represent the mean ± S.E.M of 3–5 different experiments carried out with different VDAC preparations.

Addition Concentration

mM [αααα-32P]BzATP labeling

% of control

None – 100

EDTA 1.0 188.7 ± 31.5

MnCl2 0.01 48.7 ± 9.6

MnCl2 0.1 18.1 ± 1.7

MgCl2 0.5 28.9 ± 8.1

CaCl2 0.5 42.0 ± 4.3

SrCl2 0.5 59.0 ± 9.9

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TABLE II Suspected BzATP-binding peptides based on MALDI-TOF MS comparison between tryptic-digested

BzATP-labeled and unlabeled VDAC. VDAC and BzATP-labeled VDAC were trypsin-digested and subjected to MALDI-TOF analysis (see "Experimental Procedures"). Based on mass (m/z) comparison, peptides present only in the BzATP-labeled VDAC were suspected to be BzATP-binding fragments. A, the obtained masses of the suspected peptides (observed) and their masses after subtracting the 715.4-Da BzATP mass (calculated). B, the obtained (observed) and the theoretical masses (acquired by virtual digestion) of VDAC peptides with masses comparable to those calculated in A. C, the sequences and the locations of the suspected BzATP-binding peptides. Asterisk denotes that peaks with similar masses were found in both BzATP-labeled and non-labeled VDAC samples, thus reflecting incomplete BzATP labeling of VDAC.

A. Masses (m/z) of BzATP-labeled VDAC peptides

B. Masses (m/z) of VDAC peptides

Observed Calculated Observed Theoretical

C. Peptide locations and sequences

1867.0 1151.6 1151.5 1150.7 110–119

KNAKIKTGYK

2472.4 1757.0 1756.7 1759.0 13–28

SARDVFTKGYGGFLIK

2524.2 1808.8 1807.9* 1808.0 257–274

LTLSALLDGKNVNAGGHK

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A. CBB B. Immunoblot

VDAC

Trit

on e

xt.

HA

AT

P

AM

P

1 4 13 17 23

RRA

1 4 13 17 23

Trit

on e

xt.

HA

AT

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Fig. 1

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Fig. 2

0 50 100 150 200 250 300 3500

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[α

-32P

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nd ,

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A. B. Nucleotide, mM 0 0.2 0.4 0.6 1.2

Aut

orad

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am

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ADP

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AMP-PNP

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GDPGTP

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[ αα αα-32

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zAT

P la

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BzATP (. ) or Mg-BzATP (ΟΟΟΟ) , µµµµM

0.0 .2 .4 .6 .8 1.00

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P]B

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P]B

zAT

P la

belin

g

% o

f con

trol

MnCl 2 , 0 2 5 10 20 50 100 250 µµµµM

100

80

60

40

20

0 0 100 200 300 400 500

A. B.

Fig. 4

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18

0 20 40 60 80 1000

20

40

60

80

100

[α-32

P]B

zAT

P la

belin

g %

of c

ontr

ol

FIT C fluorescence, relative units

A. B.

0 20 40 60 800

20

40

60

80

100

0

20

40

60

80

100

FITC , µM

FITC, µM 5 10 20 40 60 80

FIT

C fluorescence , relative u

nits

[α-32

P]B

zAT

P la

belin

g %

of c

ontr

ol

Fig. 5

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19

Fig. 6

A. Silver Stain B. [αααα-32P]BzATP C. Immunoblot

Formic acid , 83% - + + + - + + + - + + +

CNBr , mg/ml - - 8 32 - - 8 32 - - 8 32 kDa

16.4 14.7

32 VDAC

16.4

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Fig. 7

A. CBB B. [αααα-32P]BzATP C. Immunoblot

V8 , µµµµg 0 0.25 0.5 1 0 0.25 0.5 1 0 0.25 0.5 1

VDAC

14 17

kDa

31

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21

Mass , m/z30000 35000 40000 45000

0.2

0.4

0.6

0.8

1.0

1.2

Arb

itrar

y in

tens

ity

3085

6.7

3165

2.8

0

Mass , m/z30000 35000 40000 45000

0.2

0.4

0.6

0.8

1.0

1.2

Arb

itrar

y in

tens

ity

3085

6.7

3165

2.8

0

Fig. 8

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Cytosol

Inter membrane space

A

D

VPPT

G

K

YA

S

N

D

K

L

RA

D

V

E

FT

K

GY

GG

FL I

T

L

LKTKS

GLEF

SSGSAN

N

TT

TE K

V

GSLETKYRWT

EVTIETGLTND

E TNW

YG

KLT E

TF

DQ

LARG

KL

L

TFDSSFSPNTGKKN

AKIKTGYKREHINLGCD

D

DF

I AGPSIRG A

LVLGY E

GWLAGYQ

ATP bindingsite?

NF

T

KSRVTQ S N F A V

GYKTDEFQLHTNVNDGTEFGGS

I

YQK V NK K L ETAA

V N L

WAAK G FRT

I

N NS GATYQ

DPDACFS

KVNN

SSLIG

ALGYT

QTL

KPGIKLTLSALLDGK AN NV

GGHKLGLGLEFQA

AT

P bi

ndin

g si

te?

AT

P bi

ndin

g si

te?

Fig. 9

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Galit Yehezkel, Nurit Hadad, Hilal Zaid, Sara Sivan and Varda Shoshan-Barmatzlocalization

Nucleotide-binding sites in the voltage-dependent anion channel: Characterization and

published online December 14, 2005J. Biol. Chem. 

  10.1074/jbc.M510104200Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

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