Hexokinase-I Protection against Apoptotic Cell Death Is Mediated via Interaction with the Voltage-dependent Anion Channel-1

In brain and tumor cells, the hexokinase isoforms HK-I and HK-II bind to the voltage-dependent anion channel (VDAC) in the outer mitochondrial membrane. We have previously shown that HK-I decreases murine VDAC1 (mVDAC1) channel conductance, inhibits cytochrome c release, and protects against apoptotic cell death. Now, we define mVDAC1 residues, found in two cytoplasmic domains, involved in the interaction with HK-I. Protection against cell death by HK-I, as induced by overexpression of native or mutated mVDAC1, served to identify the mVDAC1 amino acids required for interaction with HK-I. HK-I binding to mVDAC1 either in isolated mitochondria or reconstituted in a bilayer was inhibited upon mutation of specific VDAC1 residues. HK-I anti-apoptotic activity was also diminished upon mutation of these amino acids. HK-I-mediated inhibition of cytochrome c release induced by staurosporine was also diminished in cells expressing VDAC1 mutants. Our results thus offer new insights into the mechanism by which HK-I promotes tumor cell survival via inhibition of cytochrome c release through HK-I binding to VDAC1. These results, moreover, point to VDAC1 as a key player in mitochondrially mediated apoptosis and implicate an HK-I-VDAC1 interaction in the regulation of apoptosis. Finally, these findings suggest that interference with the binding of HK-I to mitochondria by VDAC1-derived peptides may offer a novel strategy by which to potentiate the efficacy of conventional chemotherapeutic agents. In brain and tumor cells, the hexokinase isoforms HK-I and HK-II bind to the voltage-dependent anion channel (VDAC) in the outer mitochondrial membrane. We have previously shown that HK-I decreases murine VDAC1 (mVDAC1) channel conductance, inhibits cytochrome c release, and protects against apoptotic cell death. Now, we define mVDAC1 residues, found in two cytoplasmic domains, involved in the interaction with HK-I. Protection against cell death by HK-I, as induced by overexpression of native or mutated mVDAC1, served to identify the mVDAC1 amino acids required for interaction with HK-I. HK-I binding to mVDAC1 either in isolated mitochondria or reconstituted in a bilayer was inhibited upon mutation of specific VDAC1 residues. HK-I anti-apoptotic activity was also diminished upon mutation of these amino acids. HK-I-mediated inhibition of cytochrome c release induced by staurosporine was also diminished in cells expressing VDAC1 mutants. Our results thus offer new insights into the mechanism by which HK-I promotes tumor cell survival via inhibition of cytochrome c release through HK-I binding to VDAC1. These results, moreover, point to VDAC1 as a key player in mitochondrially mediated apoptosis and implicate an HK-I-VDAC1 interaction in the regulation of apoptosis. Finally, these findings suggest that interference with the binding of HK-I to mitochondria by VDAC1-derived peptides may offer a novel strategy by which to potentiate the efficacy of conventional chemotherapeutic agents. Accumulating evidence indicates that the mitochondrially bound isoforms of hexokinase, HK-I and HK-II, play pivotal roles in promoting cell growth and survival in rapidly growing, highly glycolytic tumors (1Rempel A. Mathupala S.P. Griffin C.A. Hawkins A.L. Pedersen P.L. Cancer Res. 1996; 56: 2468-2471PubMed Google Scholar). As such, HK-I and HK-II were found to be overexpressed in many types of cancer, including colon, prostate, lymphoma, glioma, gastric adenomas, carcinomas, and breast cancers (2Bryson J.M. Coy P.E. Gottlob K. Hay N. Robey R.B. J. Biol. Chem. 2002; 277: 11392-11400Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 3Gottlob K. Majewski N. Kennedy S. Kandel E. Robey R.B. Hay N. Genes Dev. 2001; 15: 1406-1418Crossref PubMed Scopus (766) Google Scholar, 4Pastorino J.G. Shulga N. Hoek J.B. J. Biol. Chem. 2002; 277: 7610-7618Abstract Full Text Full Text PDF PubMed Scopus (559) Google Scholar, 5Wilson J.E. J. Exp. Biol. 2003; 206: 2049-2057Crossref PubMed Scopus (741) Google Scholar). The elevated levels of HK-I and HK-II allow tumor cells to evade apoptosis, thereby allowing proliferation to continue (6Azoulay-Zohar H. Israelson A. Abu-Hamad S. Shoshan-Barmatz V. Biochem. J. 2004; 377: 347-355Crossref PubMed Scopus (334) Google Scholar, 7Pedersen P.L. Mathupala S. Rempel A. Geschwind J.F. Ko Y.H. Biochim. Biophys. Acta. 2002; 1555: 14-20Crossref PubMed Scopus (298) Google Scholar). HK-I and HK-II dock onto the cytosolic surface of the outer mitochondrial membrane mainly through binding to the voltage-dependent anion channel (VDAC) 4The abbreviations used are: VDAC, voltage-dependent anion channel; HK, hexokinase; GFP, green fluorescent protein; FCS, fetal calf serum; PBS, phosphate-buffered saline; FACS, fluorescent-activated cell sorter; PLB, planar lipid bilayer; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PI, propidium iodide; STS, staurosporine. 4The abbreviations used are: VDAC, voltage-dependent anion channel; HK, hexokinase; GFP, green fluorescent protein; FCS, fetal calf serum; PBS, phosphate-buffered saline; FACS, fluorescent-activated cell sorter; PLB, planar lipid bilayer; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PI, propidium iodide; STS, staurosporine. (8Nakashima R.A. Mangan P.S. Colombini M. Pedersen P.L. Biochemistry. 1986; 25: 1015-1021Crossref PubMed Scopus (187) Google Scholar). It has been proposed that binding of HK to mitochondria allows a continuous ATP flux, providing energy for the phosphorylation of glucose, and thus an increased glycolytic rate (7Pedersen P.L. Mathupala S. Rempel A. Geschwind J.F. Ko Y.H. Biochim. Biophys. Acta. 2002; 1555: 14-20Crossref PubMed Scopus (298) Google Scholar). VDAC, also known as mitochondrial porin, functions as the major channel allowing passage of nucleotides, ions, Ca2+, and other metabolites between the intermembrane space and cytoplasm (9Colombini M. Mol. Cell Biochem. 2004; 256-257: 107-115Crossref PubMed Google Scholar, 10Shoshan-Barmatz V. Israelson A. Brdiczka D. Sheu S.S. Curr. Pharm. Des. 2006; 12: 2249-2270Crossref PubMed Scopus (273) Google Scholar, 11Shoshan-Barmatz V. Gincel D. Cell Biochem. Biophys. 2003; 39: 279-292Crossref PubMed Scopus (168) Google Scholar). In vitro and in vivo studies have shown that HK-I and HK-II play a clear role in protecting against mitochondrially regulated apoptosis through direct interaction with mitochondria (3Gottlob K. Majewski N. Kennedy S. Kandel E. Robey R.B. Hay N. Genes Dev. 2001; 15: 1406-1418Crossref PubMed Scopus (766) Google Scholar) and, more specifically, with VDAC (6Azoulay-Zohar H. Israelson A. Abu-Hamad S. Shoshan-Barmatz V. Biochem. J. 2004; 377: 347-355Crossref PubMed Scopus (334) Google Scholar). Several recent studies demonstrated that in tumor cells, HK-I (12Mathupala S.P. Rempel A. Pedersen P.L. J. Biol. Chem. 1995; 270: 16918-16925Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 13Nakashima R.A. Paggi M.G. Scott L.J. Pedersen P.L. Cancer Res. 1988; 48: 913-919PubMed Google Scholar, 14Rempel A. Bannasch P. Mayer D. Biochim. Biophys. Acta. 1994; 1219: 660-668Crossref PubMed Scopus (75) Google Scholar) and HK-II (15Sade H. Khandre N.S. Mathew M.K. Sarin A. Eur. J. Immunol. 2004; 34: 119-125Crossref PubMed Scopus (21) Google Scholar, 16Preston T.J. Abadi A. Wilson L. Singh G. Adv. Drug Deliv. Rev. 2001; 49: 45-61Crossref PubMed Scopus (91) Google Scholar) not only augment cellular energy supply and levels of glucose 6-phosphate, an intermediate metabolic in many biosynthetic pathways, but also protect against cell death. The molecular mechanisms by which mitochondrially bound HK promotes cell survival are not, however, fully understood. Studies relying on purified VDAC, isolated mitochondria, or cells in culture suggest that the anti-apoptotic activity of HK-I occurs via its interaction with VDAC1 and modulation of the mitochondrial phase of apoptosis (6Azoulay-Zohar H. Israelson A. Abu-Hamad S. Shoshan-Barmatz V. Biochem. J. 2004; 377: 347-355Crossref PubMed Scopus (334) Google Scholar). HK-I interacts directly with VDAC to induce channel closure and prevent the release of cytochrome c. Moreover, HK-I overexpression in U-937 cells protected against apoptotic cell death induced by either staurosporine (6Azoulay-Zohar H. Israelson A. Abu-Hamad S. Shoshan-Barmatz V. Biochem. J. 2004; 377: 347-355Crossref PubMed Scopus (334) Google Scholar) or VDAC1 overexpression (17Zaid H. Abu-Hamad S. Israelson A. Nathan I. Shoshan-Barmatz V. Cell Death Differ. 2005; 12: 751-760Crossref PubMed Scopus (252) Google Scholar). It has also been shown that activation of glycogen synthase kinase 3β (GSK3β) induces the dissociation of HK-II from mitochondria via the phosphorylation of VDAC and that the cytotoxicity induced by chemotherapeutic drugs is increased when the binding of HK-II to mitochondria is disrupted (18Pastorino J.G. Hoek J.B. Shulga N. Cancer Res. 2005; 65: 10545-10554Crossref PubMed Scopus (345) Google Scholar). Interactions of VDAC with other proteins, including creatine kinase (19Schlattner U. Tokarska-Schlattner M. Wallimann T. Biochim. Biophys. Acta. 2006; 1762: 164-180Crossref PubMed Scopus (441) Google Scholar), cytochrome c (20Mannella C.A. J. Struct. Biol. 1998; 121: 207-218Crossref PubMed Scopus (128) Google Scholar), the benzodiazepine receptor (21McEnery M.W. J. Bioenerg. Biomembr. 1992; 24: 63-69Crossref PubMed Scopus (68) Google Scholar), the adenine nucleotide translocator (22Vyssokikh M.Y. Brdiczka D. Acta Biochim. Pol. 2003; 50: 389-404Crossref PubMed Scopus (146) Google Scholar), actin (23Roman I. Figys J. Steurs G. Zizi M. Biochim. Biophys. Acta. 2006; 1758: 479-486Crossref PubMed Scopus (35) Google Scholar), and the mtHSP70 heat shock protein have also been proposed. VDAC is proposed to be a critical component of the mitochondrial phase of apoptosis, with its interaction with Bcl-2 family proteins and controlling the rate of release of intermembrane space proteins that activate the execution phase of apoptosis (10Shoshan-Barmatz V. Israelson A. Brdiczka D. Sheu S.S. Curr. Pharm. Des. 2006; 12: 2249-2270Crossref PubMed Scopus (273) Google Scholar). For none of these proteins, however, has the interaction site(s) of VDAC been identified. The tertiary structure of VDAC has not yet been solved. Several lines of experimental evidence point to the proposal that VDAC structure comprising a transmembrane β-barrel formed by 13 (9Colombini M. Mol. Cell Biochem. 2004; 256-257: 107-115Crossref PubMed Google Scholar) or 16 (25De Pinto V. Messina A. Accardi R. Aiello R. Guarino F. Tomasello M.F. Tommasino M. Tasco G. Casadio R. Benz R. De Giorgi F. Ichas F. Baker M. Lawen A. Ital. J. Biochem. 2003; 52: 17-24PubMed Google Scholar) β-strands and an amphipathic N-terminal α-helix assigned by difference mapping as being exposed to the cytoplasm (25De Pinto V. Messina A. Accardi R. Aiello R. Guarino F. Tomasello M.F. Tommasino M. Tasco G. Casadio R. Benz R. De Giorgi F. Ichas F. Baker M. Lawen A. Ital. J. Biochem. 2003; 52: 17-24PubMed Google Scholar), crossing the membrane (9Colombini M. Mol. Cell Biochem. 2004; 256-257: 107-115Crossref PubMed Google Scholar), or lying on the membrane surface (26Reymann S. Florke H. Heiden M. Jakob C. Stadtmuller U. Steinacker P. Lalk V.E. Pardowitz I. Thinnes F.P. Biochem. Mol. Med. 1995; 54: 75-87Crossref PubMed Scopus (64) Google Scholar). Recently, we have demonstrated that a single mutation in VDAC1, i.e. glutamate 72 replaced by glutamine, inhibited HK-I interaction with VDAC1 and prevented HK-I-mediated protection against apoptotic cell death induced by overexpression of native VDAC1 (17Zaid H. Abu-Hamad S. Israelson A. Nathan I. Shoshan-Barmatz V. Cell Death Differ. 2005; 12: 751-760Crossref PubMed Scopus (252) Google Scholar). In this study, further analysis of those VDAC1 domains interacting with HK-I was carried out by site-directed mutagenesis of murine VDAC1. In doing so, we have localized two cytoplasmic domains in the VDAC1 protein that are required for interaction with HK-I and for HK-I-mediated protection against cell death via inhibiting release of cytochrome c. Materials—Carboxymethyl (CM)-cellulose, cis-diammine-dichloroplatinum (II) (cisplatin), n-decane, glucose 6-phosphate, glucose-6-phosphate dehydrogenase, leupeptin, mannitol, phenylmethylsulfonyl fluoride, soybean asolectin, staurosporine, poly-d-lysine, and propidium iodide (PI) were purchased from Sigma. Cibacron blue-agarose was purchased from Amersham Biosciences (Uppsala, Sweden). n-Octyl-β-d-glucopyranoside was obtained from Bachem AG (Bubendorf, Switzerland). Mito Tracker red dye CMXPos was purchased from Molecular Probes. Lauryl-(dimethyl)-amine oxide was obtained from Fluka (Buchs, Switzerland). Hydroxyapatite (Bio-Gel HTP) was purchased from Bio-Rad, and celite from Merck (Darmstadt, Germany). Monoclonal anti-VDAC antibodies came from Calbiochem-Novobiochem (Nottingham, UK). Monoclonal antibodies against actin and green fluorescence protein (GFP) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-cytochrome c antibodies were obtained from BD Biosciences Pharmingen. Horseradish peroxidase-conjugated anti-mouse antibodies were obtained from Promega (Madison, WI). Metafectene was purchased from Biotex (Munich, Germany). Cell growth medium RPMI 1640 and Dulbecco's modified Eagle's medium and the supplements fetal calf serum (FCS), l-glutamine, and penicillin-streptomycin were purchased from Biological Industries (Kibbutz Beit Haemek, Israel). Blasticidin and zeocin were purchased from InvivoGen (San Diego). Puromycin was purchased from ICN Biomedicals (Eschwege, Germany). Plasmids and Site-directed Mutagenesis—mVDAC1 (obtained from W. J. Craigen, University of Houston) was cloned into the plasmid pEGFP-N1 (Clontech) for construction of mVDAC1-GFP. Site-directed mutagenesis of mVDAC1 was carried out in vitro by overlapping PCR amplification. Mutant mVDAC1 genes were constructed using the T7 and SP6 universal primers and those primers described in Table 1. Plasmid pEGFP-N1, carrying the wild-type mVDAC1 gene, served as the template for amplification of mutant mVDAC1 genes. Native or mutated mVDAC1 coding sequences were cloned into the BamH1 and EcoRV restriction sites of the pcDNA4/TO vector (Invitrogen) containing the zeocin resistance gene and two tetracycline operator sites within the human cytomegalovirus immediate-early promoter to allow for tetracycline-regulated expression of mVDAC1 in transfected cells. The HK-I-GFP fusion protein in which GFP was connected to the HK-I C-terminal (pEGFP-HK-I), was constructed using an EcoR1 restriction site to introduce GFP into the 3′ (at the stop codon) of HK1 in plasmid pcDNA3.1 (provided by J. E. Wilson, Michigan State University) by site-directed mutagenesis with overlapping PCR amplification, using the following primers: 5′-CCCTTCGATCGCCGGAATTCCAGGATCCTCCCAGCC-3′ (forward) and 5′-GGCTGGGAGGATCCTGGAATTCCGGCGATCGAAGGG-3′ (reverse). HK1 was excised from plasmid pCDNA3.1 by EcoR1 and subcloned into pEGFP-N1. All constructs were confirmed by sequencing.TABLE 1List of the primers used for site-directed mutagenesis in VDAC1MutantPrimer sequenceE72Q-mVDAC1Forward5′-GACGTTTACACAGAAGTGGAAC-3′Reverse5′-GTTCCACTTCTGTGTAAACGTC-3′E65Q-mVDAC1Forward5′-TGGACTCAGTATGGGCTGACG-3′Reverse5′-GCCCATACTGAGTCCATCTG-3′K73L-mVDAC1Forward5′-ACAGAGCTGTGGAACACAGAC-3′Reverse5′-GTTCCACAGCTCTGTAAACGTC-3′D77N-mVDAC1Forward5′-GAACACACAGAACACCCTGGG-3′Reverse5′-GTGTTCTGTGTGTTCCACTTCTC-3′E202Q-mVDAC1Forward5′-GAAGTTGCAGACTGCTGTCAATCTC-3′Reverse5′-GCGAGATTGACAGCAGTCTGCAAC-3′N75A-mVDAC1Forward5′-GAACACAGACGCCACCC-3′Reverse5′-GTGGCGTCTGTGTTCC-3′G67A-mVDAC1Forward5′-GATGGACTGAGTATGCCCTGACG-3′Reverse5′-GTCAGGGCATACTCAGTCCATCTG-3′ Open table in a new tab Tissue Culture—The U-937 human monocytic cells were grown under an atmosphere of 95% air and 5% CO2 in RPMI 1640 supplemented with 10% FCS, 2 mm l-glutamine, 1000 units/ml penicillin, and 1 mg/ml streptomycin. Cells were plated at a density of 5.4 × 104 cells/cm2 in 24-well plates, washed once with PBS, and placed in serum-free medium. T-REx-293 cells, a transformed primary human embryonal kidney cell line (Invitrogen), were grown under an atmosphere of 95% air and 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 10% FCS, 2 mm l-glutamine, 1000 units/ml penicillin, 1 mg/ml streptomycin, and 5 μg/ml blasticidin. Other cell lines used are stably transfected derivatives of T-REx-293 expressing the tetracycline repressor. hVDAC1-shRNA-T-REx-293 cells are T-REx-293 cells stably transfected with the pSUPERretro plasmid encoding shRNA targeting hVDAC1 and were grown with 0.5 μg/ml puromycin and 5 μg/ml blasticidin (27Abu-Hamad S. Sivan S. Shoshan-Barmatz V. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 5787-5792Crossref PubMed Scopus (187) Google Scholar). hVDAC1-shRNA-T-REx-293 cells were also transfected with plasmid pcDNA4/TO encoding native, G67A-, E72Q-, N75A-, or E202Q-mVDAC1 under the control of tetracycline. Cells were grown with 200 μg/ml zeocin, 0.5 μg/ml puromycin, and 5 μg/ml blasticidin. MCF7 (breast cancer) cells were grown as U-937 cells except that RPMI 1640 was replaced with Dulbecco's modified Eagle's medium. Cell Transfection—Logarithmically growing U-937 cells were resuspended in RPMI 1640 supplemented with 10% FCS, 1000 units/ml penicillin, and 1 mg/ml streptomycin at a concentration of 2.5 × 107 cells/ml. Cells were transfected with plasmids pEGFP, pEGFP-mVDAC1, pEGFP-mutated-mVDAC1, or with plasmid pcDNA4/TO containing native or mutated mVDAC1. Transfection was performed by electroporation with a single pulse of a Bio-Rad micropulser II with a capacitance extender unit (200 V, 950 μF). Cells were incubated on ice for 10 min before and after transfection, and then resuspended in 20 ml of RPMI 1640 supplemented with 10% FCS, 2 mml-glutamine, 1000 units/ml penicillin, and 1 mg/ml streptomycin. Transfection efficiencies were 68-72%, as estimated by GFP expression. For transfection with metafectene, T-REx-293 cells or hVDAC1-shRNA-expressing T-REx-293 cells (∼3 × 105) were cultured in plates at 37 °C in a CO2 incubator to 30-50% confluence 24 h before transfection. In some cases, U-937 cells were transfected with plasmids pcDNA3.1 or pcDNA3.1-HK-I by electroporation and grown in complete RPMI 1640 with neomycin (400 μg/ml). The selected cells were transfected with plasmids pEGFP, pEGFP-mVDAC1, or pEGFP-mutated-mVDAC1 and grown in the presence of neomycin. The viability of doubletransfected cells was analyzed 48-76 h later. Acridine Orange/Ethidium Bromide Staining of Cells—To determine cell viability, cells were subjected to staining with acridine orange (AcOr) and ethidium bromide (EtBr) in PBS as described previously (27Abu-Hamad S. Sivan S. Shoshan-Barmatz V. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 5787-5792Crossref PubMed Scopus (187) Google Scholar). The AcOr/EtBr-stained cells were visualized by fluorescence microscopy (Olympus IX51), and images were recorded with an Olympus DP70 camera, using a SWB filter. In each independent experiment, in which early and late apoptotic cells were counted, ∼300 cells were counted for each treatment. Cytochrome c Release Induced by STS or Cisplatin—Control T-REx 293 cells and VDAC1-shRNA T-REx 293 cells expressing native or E72Q-mVDAC1 under the control of tetracycline (1 μg/ml) were grown on 60-mm dishes for 72 h and then were transfected with pcDNA3.1-HK-I. Following 72 h, cells were exposed to STS (1.25 μm) for 5 h. When MCF7 cells were used, cells were transfected with pcDNA3.1-HK-I for 102 h where after 72 and 97 h, cisplatin (50 μm) and STS (1.25 μm) were added, respectively. Cells were harvested, washed twice with PBS, incubated for 1 h on ice in extraction buffer (10 mm Hepes, pH 7.4, 200 mm manitol, 70 mm sucrose, 1 mm EGTA, 1 mm EDTA, 10 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride, and 1 mg/ml bovine serum albumin), and then lysed by a Dounce homogenizer (25 strokes). Homogenates were centrifuged (2500 × g) at 4 °C for 5 min, and the supernatants were re-centrifuged at 15,000 × g for 15 min at 4 °C. Aliquots (20 μl) of the resultant supernatants, designated as cytosolic fractions, were immediately boiled in SDS-PAGE sample buffer and resolved by SDS-PAGE on Tris-Tricine gels (12% polyacrylamide) and immunoblotted using monoclonal anti-cytochrome c antibodies (1:1000) followed by incubation with horseradish peroxidase-conjugated anti-mouse IgG and detection by chemiluminescence using a kit (Santa Cruz Biotechnology). Although supernatants from the differentially treated cells were derived from equal numbers of cells, protein concentration in the total cell lysates was estimated, and actin and HK levels were analyzed by immunoblotting using anti-actin and anti-HK antibodies, respectively. Flow Cytometry—T-REx-293 cells (5 × 105) were transfected with pcDNA3.1-HK-I and 72 h later with plasmids encoding native-, E72Q-, or L201A-mVDAC1-GFP. 48-h later, the cells were exposed to STS (0.6 μm) for 30 h. The cells and their growth medium were collected (5000 × g for 10 min), with the cells washed twice with PBS, and resuspended in 60 μl of PBS to which 390 μl of cold 80% ethanol was added followed by incubation overnight at -20 °C. One day prior to FACS analysis, the samples were centrifuged (5000 × g for 10 min) and washed twice with PBS. The pellet was resuspended in 0.5 ml of RNase buffer containing 30 μg/ml RNase and 0.1% Triton X-100 in PBS and incubated overnight at 4 °C. The DNA-labeling fluorochrome, propidium iodide, was added to a final concentration of 15 μg/ml, and after 15 min of incubation, DNA content was analyzed using a BD FACSVantage SE flow cytometer (BD Biosciences). Confocal Microscopy—T-REx-293 cells or hVDAC1-shRNA T-REx-293 cells stably expressing native mVDAC1 or E72Q-mVDAC1 under the control of tetracycline (1 μg/ml) (5 × 104) were grown on poly-d-lysine (PDL)-coated coverslips, and transfected with pEGFP-HK-I. After 48 h, cells were treated with MitoTracker red dye CMXPos (Molecular Probes) (25 nm), upon incubation for 15 min in an incubator (37 °C, 5% CO2) after which cells were washed with PBS for 30 min. Cells were fixed for 15 min using 3.7% paraformaldehyde prepared in PBS. After fixation, the cells were rinsed for 30 min in PBS. Cell imaging was carried out by confocal microscopy (Olympus 1X81). VDAC Purification, Channel Recording, and Analysis—Native and mutant mVDAC1 was extracted with LDAO from mitochondria isolated from yeast expressing these proteins and purified by chromatography on hydroxyapatite followed by carboxymethyl (CM)-cellulose, when LDAO was replaced by β-OG (11Shoshan-Barmatz V. Gincel D. Cell Biochem. Biophys. 2003; 39: 279-292Crossref PubMed Scopus (168) Google Scholar). Reconstitution of purified VDAC into a planar lipid bilayer (PLB), current recording, and data analyses were all carried out as previously described (28Gincel D. Zaid H. Shoshan-Barmatz V. Biochem. J. 2001; 358: 147-155Crossref PubMed Scopus (319) Google Scholar). Briefly, PLB were prepared from soybean asolectin dissolved in n-decane (50 mg/ml). Only PLB with a resistance greater than 100 GΩ, were used. Purified protein (about 1 ng) was added to the cis chamber. After one or a few channels were inserted into the PLB, the excess protein was removed by perfusion of the cis chamber with 20 volumes of a solution to prevent further incorporation. Currents were recorded under voltage-clamp using a Bilayer Clamp BC-525B amplifier (Warner Instrument). The currents were measured with respect to the trans side of the membrane (ground). The currents were low-pass, filtered at 1 kHz (-3dB), using a Bessel filter (Frequency Devices 902), and digitized online using a Digidata 1200 interface board and pCLAMP 6 software (Axon Instruments). Sigma Plot 6.0 scientific software (Jandel Scientific) was used for curve fitting. All experiments were performed at 21-25 °C. The interaction of HK-I with VDAC and protection against cell death induced by STS or VDAC1 overexpression has been demonstrated (3Gottlob K. Majewski N. Kennedy S. Kandel E. Robey R.B. Hay N. Genes Dev. 2001; 15: 1406-1418Crossref PubMed Scopus (766) Google Scholar, 6Azoulay-Zohar H. Israelson A. Abu-Hamad S. Shoshan-Barmatz V. Biochem. J. 2004; 377: 347-355Crossref PubMed Scopus (334) Google Scholar, 17Zaid H. Abu-Hamad S. Israelson A. Nathan I. Shoshan-Barmatz V. Cell Death Differ. 2005; 12: 751-760Crossref PubMed Scopus (252) Google Scholar). We showed that E72Q-mVDAC1 no longer retains its capacity to bind HK-I (17Zaid H. Abu-Hamad S. Israelson A. Nathan I. Shoshan-Barmatz V. Cell Death Differ. 2005; 12: 751-760Crossref PubMed Scopus (252) Google Scholar). To define the domains and additional amino acid residues involved in the interaction of VDAC1 with HK-I, further mutagenesis of mVDAC1 was carried out. The effects of these mVDAC1 mutations on HK-I protection against cell death were then analyzed by expression of native or mutated mVDAC1 in either U-937 cells, in hVDAC1-shRNA T-REx cells, where the endogenous VDAC1 level was suppressed (by ∼85%) (27Abu-Hamad S. Sivan S. Shoshan-Barmatz V. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 5787-5792Crossref PubMed Scopus (187) Google Scholar) or in the same cells overexpressing HK-I. The amino acids to be modified were selected based on sequences predicted to be exposed to the cytosol according to most VDAC1 membrane topology proposed models (9Colombini M. Mol. Cell Biochem. 2004; 256-257: 107-115Crossref PubMed Google Scholar, 26Reymann S. Florke H. Heiden M. Jakob C. Stadtmuller U. Steinacker P. Lalk V.E. Pardowitz I. Thinnes F.P. Biochem. Mol. Med. 1995; 54: 75-87Crossref PubMed Scopus (64) Google Scholar). In addition, they were selected by a comparison to the Saccharomyces cerevisiae VDAC sequence known for its inability to bind HK-I (30Wilson J.E. J. Bioenerg. Biomembr. 1997; 29: 97-102Crossref PubMed Scopus (29) Google Scholar). Charged but Not Neutral Amino Acids in the Glu-72-containing Loop Are Essential for mVDAC1 Interaction with HK-I—The mVDAC1 residue Glu-72, implicated in HK-I binding (17Zaid H. Abu-Hamad S. Israelson A. Nathan I. Shoshan-Barmatz V. Cell Death Differ. 2005; 12: 751-760Crossref PubMed Scopus (252) Google Scholar), is not conserved in yeast VDAC1 as well as in Neurospora crassa VDAC, which is also unable to bind HK-I (30Wilson J.E. J. Bioenerg. Biomembr. 1997; 29: 97-102Crossref PubMed Scopus (29) Google Scholar). Comparison of the amino acid sequences of the Glu-72-containing loop of human or murine VDAC1 with the same regions of S. cerevisiae or N. crassa VDAC reveals that in addition to Glu-72, the charged amino acids Glu-65, Lys-73, and Asp-77 are not conserved in the fungal proteins (Fig. 1). To verify whether these amino acids participate in the mVDAC1-HK-I interaction, they were, respectively, replaced by Gln, Leu, and Asn. Because VDAC1 overexpression induces cell death (17Zaid H. Abu-Hamad S. Israelson A. Nathan I. Shoshan-Barmatz V. Cell Death Differ. 2005; 12: 751-760Crossref PubMed Scopus (252) Google Scholar), U-937 cells were first transformed to overexpress HK-I and only then transfected with plasmids encoding native, E65Q, E72Q-, K73L-, or D77N-mVDAC1-GFP (Fig. 2, A and B). Apoptotic cell death induced by overexpression of native mVDAC1 or mVDAC1-GFP was dramatically reduced in cells overexpressing HK-I, dropping from about 75-80% to ∼10% (Fig. 2B). On the other hand, as observed with E72Q-mVDAC1-expressing cells, no protective effect of HK-I against cell death was obtained in cells overexpressing E65Q-, K73L-, or D77N-mVDAC1-GFP (Fig. 2B). These results show that although the U-937 cells express endogenous hVDAC1, the presence of mVDAC1 mutants nonetheless completely prevented the anti-apoptotic effect of HK-I. The anti-apoptotic effect of HK-I was also observed in other cell lines such as T-REx-293 cells. In this case, we used hVDAC1-shRNA-T-REx 293 cells, in which the endogenous human VDAC1 level was suppressed (by ∼85%) using VDAC1-shRNA that specifically suppress the expression of human but not murine VDAC1 (27Abu-Hamad S. Sivan S. Shoshan-Barmatz V. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 5787-5792Crossref PubMed Scopus (187) Google Scholar). Thus, hVDAC1-shRNA-T-REx 293 cells were transfected to stably express native, G67A-, E72Q-, or N75A-mVDAC1 under the control of tetracycline. The inhibited growth of the hVDAC1-shRNA-expressing cells (27Abu-Hamad S. Sivan S. Shoshan-Barmatz V. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 5787-5792Crossref PubMed Scopus (187) Google Scholar) was restored by expression of G67A-, E72Q-, or N75A-mVDAC1, as induced by tetracycline (1 μg/ml) (data not shown). To induce cell death by mVDAC1 overexpression, cells stably expressing native and various mutants were exposed to high tetracycline concentrations (2.5 μg/ml) as well as to transiently expressed HK-I (Fig. 2C). In cells overexpressing HK-I, protection against apoptosis was observed only in cells overexpressing native, G67A-, or N75A-mVDAC1, but not in cells overexpressing E72Q-mVDAC1 (Fig. 2C). Immunoblot analyses using anti-HK antibodies of cell extracts confirmed the overexpression of HK-I in the transfected cells (Fig. 2D). These results indicate that HK-I interacts with the proposed VDAC1 cytosolic loop 1 (amino acids 62-80), the E72Q-containing loop, and point to the amino acid residues involved in this interaction. mVDAC1 Cytosolic Loop 2 Interaction with HK-I—Because mutating charged amino acid residues in the proposed cytosolic loop 1 modified HK-I interaction with VDAC1, charged amino acids in the proposed cytosolic loop 2 (amino acids 107-122), Lys-109 and Lys-112, were replaced by Leu to verify their importance in VDAC1-HK-I interaction. In this case, the effects of HK-I overexp