Functional F1-ATPase Essential in Maintaining Growth and Membrane Potential of Human Mitochondrial DNA-depleted ρ° Cells
1998; Elsevier BV; Volume: 273; Issue: 36 Linguagem: Inglês
10.1074/jbc.273.36.22983
ISSN1083-351X
Autores Tópico(s)Biochemical and Molecular Research
ResumoF1-ATPase assembly has been studied in human ρ° cells devoid of mitochondrial DNA (mtDNA). Since, in these cells, oxidative phosphorylation cannot provide ATP, their growth relies on glycolysis. Despite the absence of the mtDNA-coded F0 subunits 6 and 8, ρ° cells possessed normal levels of F1-ATPase α and β subunits. This F1-ATPase was functional and azide- or aurovertin-sensitive but oligomycin-insensitive. In addition, aurovertin decreased cell growth in ρ° cells and also reduced their mitochondrial membrane potential, as measured by rhodamine 123 fluorescence. Therefore, a functional F1-ATPase was important to maintain the mitochondrial membrane potential and the growth of these ρ° cells. Bongkrekic acid, a specific adenine nucleotide translocator (ANT) inhibitor, also reduced ρ° cell growth and mitochondrial membrane potential. In conclusion, ρ° cells need both a functional F1-ATPase and a functional ANT to maintain their mitochondrial membrane potential, which is necessary for their growth. ATP hydrolysis catalyzed by F1 must provide ADP3− at a sufficient rate to maintain a rapid exchange with the glycolytic ATP4− by ANT, this electrogenic exchange inducing a mitochondrial membrane potential efficient enough to sustain cell growth. However, since the effects of bongkrekic acid and of aurovertin were additive, other electrogenic pumps should cooperate with this pathway. F1-ATPase assembly has been studied in human ρ° cells devoid of mitochondrial DNA (mtDNA). Since, in these cells, oxidative phosphorylation cannot provide ATP, their growth relies on glycolysis. Despite the absence of the mtDNA-coded F0 subunits 6 and 8, ρ° cells possessed normal levels of F1-ATPase α and β subunits. This F1-ATPase was functional and azide- or aurovertin-sensitive but oligomycin-insensitive. In addition, aurovertin decreased cell growth in ρ° cells and also reduced their mitochondrial membrane potential, as measured by rhodamine 123 fluorescence. Therefore, a functional F1-ATPase was important to maintain the mitochondrial membrane potential and the growth of these ρ° cells. Bongkrekic acid, a specific adenine nucleotide translocator (ANT) inhibitor, also reduced ρ° cell growth and mitochondrial membrane potential. In conclusion, ρ° cells need both a functional F1-ATPase and a functional ANT to maintain their mitochondrial membrane potential, which is necessary for their growth. ATP hydrolysis catalyzed by F1 must provide ADP3− at a sufficient rate to maintain a rapid exchange with the glycolytic ATP4− by ANT, this electrogenic exchange inducing a mitochondrial membrane potential efficient enough to sustain cell growth. However, since the effects of bongkrekic acid and of aurovertin were additive, other electrogenic pumps should cooperate with this pathway. adenine nucleotide translocator 3-(cyclohexylamino)-1-propanesulfonic acid subunit II and IV of cytochrome oxidase dicyclohexylcarbodiimide carbonyl cyanidep-trifluoromethoxyphenylhydrazone nonyl acridine orange rhodamine 123 10 mm Tris-HCl, pH 7.6, 0.1 mm NaCl oligomycin sensitivity conferring protein polymerase chain reaction. The biogenesis of mitochondrial proteins is controlled by both nuclear and mitochondrial genomes. The proteins coded by the mtDNA are subunits of enzyme complexes involved in oxidative phosphorylation. Since all these complexes also contain proteins coded by the nuclear genome, mechanisms regulating the coordinated expression and assembly of the subunits of nuclear and mitochondrial origin must exist. In yeast cells, nuclear DNA-coded components continue to be synthesized and imported into mitochondria, even when the synthesis of mtDNA-coded subunits is blocked (cf. for review, Refs. 1Tzagoloff A. Myers A.M. Annu. Rev. Biochem. 1986; 55: 249-285Crossref PubMed Scopus (327) Google Scholar and 2Attardi G. Schatz G. Annu. Rev. Cell Biol. 1988; 4: 289-333Crossref PubMed Scopus (1113) Google Scholar). The groups of Schatz and co-workers (3Suzuki C.K. Rep M. Maarten van Dijl J. Suda K. Grivell L.A. Schatz G. Trends Biochem. Sci. 1997; 22: 118-123Abstract Full Text PDF PubMed Scopus (212) Google Scholar) and Neupert (4Neupert W. Annu. Rev. Biochem. 1997; 66: 863-917Crossref PubMed Scopus (984) Google Scholar) have shown that a mitochondrial membrane potential is a key requirement for protein import into mitochondria (5Gasser S.M. Daum G. Schatz G. J. Biol. Chem. 1982; 257: 13034-13041Abstract Full Text PDF PubMed Google Scholar). In normal cells, the mitochondrial membrane potential is mainly maintained by transmembrane proton pumping occurring during electron transfer or during ATP hydrolysis catalyzed by the ATPase-ATP synthase. In ρ° cells depleted of mtDNA, these complexes cannot be functional since all complexes involved in proton pumping contain mtDNA-coded subunits (6Anderson S. Banquier A.T. Barrel B.G. de Bruijn M.H.L. Coulson A.R. Drouin J. Eperon I.C. Nierlich D.P. Rose B.A. Sanger F. Schreier P.H. Smith A.J.H. Staden R. Young I.G. Nature. 1981; 290: 457-465Crossref PubMed Scopus (7841) Google Scholar). However, proteins of nuclear origin are imported into the ρ° cell mitochondria (7Herzberg N.H. Middelkoop E. Adorf M. Dekker H.L. Van Galen M.J. Van den Berg M. Bolhuis P.A. Van den Bogert C. Eur. J. Cell Biol. 1993; 61: 400-408PubMed Google Scholar). Therefore, the mitochondrial membrane potential must be maintained by other electrogenic pumps. In yeast, it has been suggested that the adenine nucleotide translocator (ANT)1 mediates an exchange of ATP4− synthesized in the cytosol during glycolysis for ADP3− to maintain this mitochondrial membrane potential (8Kolarov J. Klingenberg M. FEBS Lett. 1974; 45: 320-323Crossref PubMed Scopus (29) Google Scholar). However, the exact mechanism that maintains this potential has not been thoroughly investigated. Since the mtDNA-coded subunits are essential components for oxidative phosphorylation, ρ° cells lacking mtDNA rely on glycolysis for their energy demand. The NADH produced during glycolysis is then reoxidized by the lactate dehydrogenase (9Martinus R.D. Garth G.P. Webster T.L. Cartwright P. Naylor D.J. Hoj P.B. Hoogenraad N.J. Eur. J. Biochem. 1996; 240: 98-103Crossref PubMed Scopus (259) Google Scholar). In addition, several reports indicate that some nuclearly coded subunits of the respiratory chain complexes are imported and partly assembled into the mitochondria of the ρ° cells (10Nijtmans L.G.J. Spelbrink J.N. Van Galen M.J.M. Zwaan M. Klement P. Van den Bogert C. Biochim. Biophys. Acta. 1995; 1265: 117-126Crossref PubMed Scopus (46) Google Scholar). The role (if any) of these partly assembled complexes is unknown. The present study reports observations concerning the role and mechanism of assembly of the mitochondrial ATPase in human mtDNA-deficient ρ° cells. The ATPase-ATP synthase (F0F1) is made of a hydrophilic portion F1 required for enzyme catalysis and connected by a stalk to the hydrophobic sector F0, which is involved in proton translocation occurring during ATP synthesis or ATP hydrolysis. (cf. Ref. 11Boyer P.D. Annu. Rev. Biochem. 1997; 66: 717-749Crossref PubMed Scopus (1639) Google Scholar, for review). In mammals, F1 contains five different subunits: α, β, γ, δ, and ε. F0 is made of the subunits a (or subunit 6), b, c (also named subunit 9 or DCCD-binding protein), d, e, f, g, and A6L (or subunit 8); the stalk contains OSCP, F6, and parts of F0 such as subunits b and d or of F1 such as γ (12Collinson I.R. van Raaj M.J. Runswick M.J. Fearnley I.M. Skehel J.M. Orris G.L. Miroux B. Walker J.E. J. Mol. Biol. 1994; 242: 408-421PubMed Google Scholar). The subunits a (or 6) and A6L (or 8) that are coded by the mtDNA are absent in human ρ° cells. Here, we show that F1 is assembled in human ρ° cells as an azide- or aurovertin-sensitive ATPase and that this ATPase activity is essential for the ρ° cell growth because it is involved in the maintenance of their mitochondrial membrane potential. Cell culture reagents were from Life Technology Inc. except uridine, which was from Sigma. The ρ° HeLa S3 cells devoid of mtDNA were prepared by ethidium bromide treatment of HeLa S3 cells (ρ+) (ATCC CCL2.2) by Dr. J. L. Vayssière. The ρ° 143B TK− cells were obtained from 143B TK− osteosarcoma (ρ+) (ATCC CRL8303) (13Morais R. Zinkewich-Péotti K. Parent M. Wang H. Babai F. Zollinger M. Cancer Res. 1994; 54: 3889-3896PubMed Google Scholar). The ρ+ and ρ° cells were kindly provided to us by Dr. Vayssière and Dr. Morais. The bongkrekic acid was a generous gift of Prof. P. V. Vignais. The peroxidase-labeled anti-mouse antibody was obtained from Biosys. The anti-F1-ATPase α and β subunits were prepared from the clones 20D6 and 14D5 (14Moradi-Amèli M. Godinot C. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 6167-6171Crossref PubMed Scopus (37) Google Scholar), and the anti-cytochrome oxidase subunit II and subunit IV were prepared from the clones: 12C4-F12 and 10G8-C12-D12, respectively, kindly provided by Dr. Taanman (15Taanman J.W. Burton M.D. Marusich M.F. Kennaway N.G. Capaldi R.A. Biochim. Biophys. Acta. 1996; 1315: 199-207Crossref PubMed Scopus (54) Google Scholar). The fluorescent molecular probe NAO was obtained from Molecular Probes. The nitrocellulose membranes were from Schleicher and Schuell. All other biochemical reagents were from Bœhringer Mannheim or Sigma. HeLa S3 cells were grown at 37 °C in a humidified atmosphere with 5% CO2 in Dulbecco's modified Eagle's medium, Ham's F-12 supplemented with 2% Ultroser G, antibiotics (200 units/ml penicillin and 200 μg/ml streptomycin), and 0.5 mm pyruvate. Osteosarcoma 143B cells were grown in the same atmosphere in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, antibiotics (200 units/ml penicillin plus 200 μg/ml streptomycin), and 1 mm pyruvate. Uridine was added in cell culture media for ρ° HeLa S3 cells (100 μg/ml) and ρ° 143B cells (50 μg/ml). Centrifugation of ρ° 143B cells, which are easily damaged, was avoided. ρ+ and ρ° cell pellets were treated for 1 h at 60 °C with 10 μg of proteinase K dissolved in PCR buffer, and quantitative PCR was directly performed on serial dilutions of cells to amplify mtDNA. Two couples of forward and reverse primers hybridized with the mtDNA sequence at positions 592 to 611 and 1344 to 1325 or 15437 to 15456 and 15777 to 15758, located at the level of the 12 S RNA and of the cytochromeb genes (6Anderson S. Banquier A.T. Barrel B.G. de Bruijn M.H.L. Coulson A.R. Drouin J. Eperon I.C. Nierlich D.P. Rose B.A. Sanger F. Schreier P.H. Smith A.J.H. Staden R. Young I.G. Nature. 1981; 290: 457-465Crossref PubMed Scopus (7841) Google Scholar), respectively. The PCR was performed as in Boudizi et al. (16Bouzidi M.F. Carrier H. Godinot C. Biochim. Biophys. Acta. 1996; 1317: 199-209Crossref PubMed Scopus (20) Google Scholar), using hybridization temperatures of 55 and 53 °C, respectively. A control to check the amount of DNA in the various cell extracts was made by amplification of the nuclear gene coding for the 18 S RNA from positions 1196 to 1806 using 17-mer oligomers and a hybridization temperature of 62 °C. Cellular RNA extraction and reverse transcription were performed on ρ+ and ρ° cells as described previously (17Enjolras N. Godinot C. Mol. Cell. Biochem. 1997; 167: 113-125Crossref PubMed Scopus (6) Google Scholar). The PCR amplification of cDNAs corresponding to mitochondrial RNAs was made as above using additional forward and reverse primers hybridizing with the mtDNA sequence at positions 798 to 817 and 1344 to 1325 or 4875 to 4895 and 5117 to 5097, corresponding to 12 S RNA or ND2 fragments, respectively (6Anderson S. Banquier A.T. Barrel B.G. de Bruijn M.H.L. Coulson A.R. Drouin J. Eperon I.C. Nierlich D.P. Rose B.A. Sanger F. Schreier P.H. Smith A.J.H. Staden R. Young I.G. Nature. 1981; 290: 457-465Crossref PubMed Scopus (7841) Google Scholar). The hybridization temperatures were 61 and 55 °C, respectively. Each cell type was cultured in an 80-cm2 flask until 70% confluence. They were treated with trypsin and washed in phosphate saline buffer (10 mm sodium phosphate, 150 mm NaCl, pH 7.2). After centrifugation at 100 × g for 5 min, the cell pellets, homogenized at 0–4 °C in 0.3 ml of buffer containing 10 mm Tris-HCl, 0.2 mm dithiothreitol, 1 mm EDTA, and 2 mm ε-aminocaproate, pH 7.5, were sonicated by five bursts of 5 s separated by 1 s of cooling using a Vibra cell™ 72405 equipped with a 0.12-inch probe. Protein concentrations of the homogenate were determined with the bicinchoninic acid assay according to manufacturer's instructions (Pierce). Equivalent amounts of proteins of each cell type (50 μg) were separated on a 15% polyacrylamide-SDS gel and transferred to a nitrocellulose membrane at a constant current of 0.8 mA/cm2for 1.5 h in a semi-dry apparatus (Hoefer) using 10 mmCAPS, 10% methanol, pH 11, as a transfer buffer. The nitrocellulose membranes were then saturated for 1 h in TBS containing 10% skimmed milk. To detect the different proteins, the membranes were first incubated for 1 h with a primary monoclonal antibody diluted in TBS containing 0.1% Tween 20 (v/v) and then for 1 h with peroxidase-labeled anti-mouse antibody (Biosys) diluted at 1:2000 in TBS containing 0.1% Tween 20 (v/v). The peroxidase activity was finally revealed with the ECL™ reagent according to the manufacturer's instructions (Amersham Pharmacia Biotech). Between each incubation, the membranes were washed once for 10 min and twice for 5 min in TBS containing 0.1% Tween 20 (v/v). The primary antibodies used were anti-F1-ATPase α subunit, 20D6, (14Moradi-Amèli M. Godinot C. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 6167-6171Crossref PubMed Scopus (37) Google Scholar) diluted 1:5000; anti-F1-ATPase β subunit, 14D5, (14Moradi-Amèli M. Godinot C. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 6167-6171Crossref PubMed Scopus (37) Google Scholar) diluted 1:4000; anti-COX II (15Taanman J.W. Burton M.D. Marusich M.F. Kennaway N.G. Capaldi R.A. Biochim. Biophys. Acta. 1996; 1315: 199-207Crossref PubMed Scopus (54) Google Scholar) diluted 1:5000, and anti-COX IV (15Taanman J.W. Burton M.D. Marusich M.F. Kennaway N.G. Capaldi R.A. Biochim. Biophys. Acta. 1996; 1315: 199-207Crossref PubMed Scopus (54) Google Scholar) diluted 1:2000. Cells were sonicated, and the protein concentrations were measured as described for immunological studies. The rate of ATP hydrolysis was measured at 37 °C according to Pullman et al. (18Pullman M.E. Penefsky H.S. Datta A. Racker E. J. Biol. Chem. 1960; 235: 3322-3329Abstract Full Text PDF PubMed Google Scholar) by adding the cell homogenate (25 to 100 μg of proteins) to 660 μl of reaction buffer containing 50 mm Tris-Hepes, pH 8.0, 3.3 mmMgSO4, 4 mm phosphoenolpyruvate, 0.33 mm NADH, 3.3 mm ATP, 10 μg of lactate dehydrogenase, 50 μg of pyruvate kinase, and 1 μg of rotenone (to inhibit NADH oxidation by the mitochondrial NADH-ubiquinone oxidoreductase). The assay was performed in the presence or absence of one of the mitochondrial ATPase inhibitors, 3.5 μmoligomycin, 60 μm aurovertin B, or 2 mmsodium azide, to estimate the percentage of ATPase activity that was related to the F1 or F0F1 complex in the cell homogenate. To determine the effects of aurovertin and bongkrekic acid on cellular growth, the cells were incubated into 24-well culture plates at a density varying between 2.5 × 103 to 104 cells/well. After 24 or 48 h, the culture medium was changed, and the tested drugs were added: 3 nm to 60 μm aurovertin and/or 2–10 μm bongkrekic acid or 0.1–0.5 μm FCCP. The cells were grown for 5 days with a culture medium change after 3 days, when indicated. The cells released by trypsin treatment were counted by trypan blue exclusion. The culture medium was collected after 5 days of cell treatment with bongkrekic acid. Proteins were precipitated with 7.5% trichloracetic acid. The assays were centrifuged for 7 min at 1,000 ×g. The supernatant fraction was extracted four times with an equal volume of diethyl ether. The lactate concentration in the deproteinized samples was estimated spectrophotometrically at 340 nm by using lactate dehydrogenase (19Bergmeyer H.U. Methods of Enzymatic Analysis. Springer Verlag, Berlin1973: 1464-1468Google Scholar). The fluorescence of NAO, which is reputed to be proportional to the amount of cardiolipin and independent of the mitochondrial membrane potential, was tested to compare the amount of mitochondrial membranes in ρ° and ρ+ cells (20Petit J.M. Huet O. Gallet P.F. Maftah A. Ratinaud M.H. Julien R. Eur. J. Biochem. 1994; 220: 871-879Crossref PubMed Scopus (92) Google Scholar). R123 fluorescence was used to estimate the mitochondrial membrane potential (21Chen L.B. Methods Cell Biol. 1989; 29: 103-123Crossref PubMed Scopus (195) Google Scholar). Each cell type was distributed into 96-well culture plates at a density of 1 to 5 × 104 cells/well and incubated 24 to 48 h before fluorescence measurement. Triplicates of each cell dilution were treated with or without 30 μm aurovertin and/or 10 μm bongkrekic acid for 30 min to 24 h before R123 fluorescence measurement. FCCP (0.1 μm) was used in some experiments to determine the residual fluorescence intensity when the mitochondrial membrane potential was collapsed. The cells were washed once with Hanks' balanced salt solution and incubated for 30 min at 37 °C (without CO2) with 0.1 ml of 6.3 μm NAO or at 37 °C in a humidified atmosphere containing 5% CO2 with 1 or 10 μm R123. The cells were then washed twice with Hanks' balanced salt solution, and the medium was removed. NAO and R123 fluorescence were rapidly measured in a microplate fluorescence reader (Victor) with excitation at 485 nm and emission at 535 nm (21Chen L.B. Methods Cell Biol. 1989; 29: 103-123Crossref PubMed Scopus (195) Google Scholar). It was checked that, under the tested conditions, R123 fluorescence was not modified by the presence of aurovertin, bongkrekic acid, or FCCP. A linear correlation made between the number of cells counted by trypan blue exclusion and crystal violet staining (22Saito K. Oku T. Ata N. Miyashiro H. Hattori M. Saiki I. Biol. Pharm. Bull. 1997; 20: 345-348Crossref PubMed Scopus (111) Google Scholar) permitted an estimate of the number of cells in all wells. ρ° HeLa S3 cell growth was dependent on the presence of pyruvate and uridine, as shown previously for 143B cells (13Morais R. Zinkewich-Péotti K. Parent M. Wang H. Babai F. Zollinger M. Cancer Res. 1994; 54: 3889-3896PubMed Google Scholar). The doubling times for ρ° and ρ+ HeLa S3 cells were 51 and 25 h, respectively, and that of ρ° and ρ+ 143B cells, 29 and 20 h. In the ρ° cells, no full-size mtDNA could be detected by Southern blotting. However, although the cytochrome b fragment could not be amplified by PCR, a fragment corresponding to the 12 S RNA could be amplified in the two ρ° cell types. This came from sequences integrated into the nuclear DNA, as shown previously (23Tsuzuki T. Nomiyama H. Setoyama C. Maeda S. Shimada K. Gene. 1983; 25: 223-229Crossref PubMed Scopus (90) Google Scholar). The ρ° cells were devoid of functional mtDNA since no mitochondrial mRNA could be revealed by reverse transcription-PCR (data not shown). The immunoblot obtained after SDS-polyacrylamide gel electrophoresis of cellular proteins transferred to nitrocellulose, incubated with antibodies, and stained (Fig. 1 A) demonstrates that, as expected, the mitochondrially encoded COX II was absent from the two types of ρ° cells and present in ρ+ cells. On the contrary, the nuclearly encoded COX IV and F1-ATPase β subunits were expressed both in ρ+ and ρ° cells. The expression of COX IV was slightly lower in ρ° than in ρ+ cells, whereas that of the F1-ATPase β subunit was similar in both types of ρ+ and ρ° cells. In a parallel experiment, it was shown that the F1-ATPase α subunit was also expressed similarly in all cell types (data not shown). The ATPase activity was tested in ρ° and ρ+ cell homogenates. To differentiate the part of this activity that was due to the mitochondria from that originated from other cellular ATPases, inhibitors specific to the mitochondrial ATPase were added. The difference between the total activity and that obtained in the presence of the inhibitors corresponds to the mitochondrial F0F1 activity. Fig. 1 B shows that oligomycin, which binds to F0 (24Lardy H.A. Annu. Rev. Biochem. 1969; 38: 991-1034Crossref PubMed Scopus (125) Google Scholar), did not inhibit the ATPase activity of ρ° cells devoid of the mitochondrially coded F0 subunits 6 and 8. However, oligomycin inhibited the ATPase activity of ρ+ HeLa S3 cells by 35% and that of ρ+ 143B cells by 50%. On the contrary, inhibitors such as azide (25Linnett P.E. Beechey R.B. Methods Enzymol. 1979; 55: 472-518Crossref PubMed Scopus (207) Google Scholar) and aurovertin (26Chang T. Penefsky H.S. J. Biol. Chem. 1973; 248: 2746-2754Abstract Full Text PDF PubMed Google Scholar), which bind to F1, decreased the rate of ATP hydrolysis both in ρ° and ρ+ cell extracts. Aurovertin was the most efficient inhibitor, decreasing ATP hydrolysis by 55% in ρ+ and ρ° HeLa S3 cells and by 65% in ρ+ and ρ° 143B cells. In addition, to test whether this aurovertin-sensitive F1-ATPase activity depended on soluble F1 or on partly assembled F0F1 complex, its cold sensitivity was studied. Fig. 1, C and D, show that the ρ° cell ATPase activity decreased much more rapidly at 4 °C than that of ρ+ cells and than that studied after incubation of ρ° or ρ+ cells at 30 °C. Within 1 h at 4 °C, 50 or 60% of this activity was lost for the ρ° HeLa S3 or ρ° 143B cells, respectively, whereas at 30 °C, it decreased by about 10% for the ρ° HeLa S3 cells and was almost stable for the ρ° 143B cells. The ATPase activity was also stable for the ρ+ 143B cells at both temperatures, whereas it decreased for the first h by 25 and 10% in ρ+ HeLa S3 cells at 4 and 30 °C, respectively. To determine whether the functional F1-ATPase activity could play a role in the survival of ρ° cells, the effects of 3 nm–30 μm aurovertin B were tested on the growth of ρ° and ρ+ cells. The percentage of cell growth after 5 days of aurovertin treatment was compared with control cells. The sensitivity to aurovertin was higher in ρ+than in ρ° cells, since 50% of cell survival was obtained at about 50 nm aurovertin for ρ+ HeLa S3 cells and about 30 μm for ρ° HeLa S3 cells or between 5 and 10 μm for ρ+ 143B cells and above 30 μm for ρ° 143B cells (not shown). Therefore, the aurovertin-induced inhibition of the mitochondrial F1-ATPase activity decreased cell growth in ρ° cells as well as in the parental ρ+ cells, although the aurovertin concentration exhibiting the same effect was higher in ρ° than in ρ+ cells. In addition, the aurovertin concentration necessary to inhibit the 143B cell growth was higher than that inhibiting the HeLa S3 cells. The green fluorescence of R123 was measured on both ρ° and ρ+ cell types to compare their mitochondrial membrane potential. The green fluorescence of NAO was measured in parallel assays to estimate the amount of mitochondrial membranes. Fig. 2 shows that the NAO fluorescence was similar in ρ° and ρ+ HeLa S3 cells (Fig. 2 A) and was slightly lower in ρ° 143B than in ρ+ 143B cells (Fig. 2 B). R123 fluorescence intensity shows that the mitochondrial membrane potential was of the same order of magnitude in ρ° HeLa S3 and ρ+ HeLa S3 cells (Fig. 2 A). In ρ° 143B cells, it was much lower than in ρ+ 143B cells, but the difference was much more important than that for NAO (Fig. 2 B). In the case of HeLa S3 cells, a 30-min treatment with 0.1 μm FCCP strongly decreased the mitochondrial membrane potential of both ρ° and ρ+ cells to a similar basal level. In the case of 143B cells, FCCP strongly decreased ρ+ mitochondrial membrane potential, but its effect was less important with ρ° 143B cells. However, the R123 fluorescence observed in the presence of FCCP reached a similar low level in ρ° and ρ+ 143B cells. Therefore, in the absence of mitochondrial membrane potential, the fluorescence intensity obtained with the uncoupler FCCP seems to correspond to the basal fluorescence of R123 in the cells. Increasing the R123 concentration from 1 to 10 μm did not change the relative fluorescence intensity observed in any cell type. In conclusion, the mitochondrial membrane potential, as estimated by R123 fluorescence, is similar in ρ° and ρ+ HeLa cells, whereas that observed in ρ° 143B cells is only 10 to 20% that of the ρ+ 143B cells. Because of the low mitochondrial membrane potential of ρ° 143B cells, the following studies involving mitochondrial membrane potential were conducted with HeLa S3 cells. The effects of 30 μm aurovertin were tested on the mitochondrial membrane potential of ρ° and ρ+ HeLa S3 cells, as measured by R123 fluorescence. Fig. 3 shows that in ρ+ and ρ° HeLa S3, a significant decrease of mitochondrial membrane potential was observed after a 30-min treatment. Similar results were obtained with a 6 h-treatment (not shown). Since the cell number was not modified after a 30-min or a 6-h aurovertin treatment, the aurovertin effect on mitochondrial membrane potential is not due to a lower cell number. Since the respiratory chain is not functional in ρ° cells, the membrane potential must be set up by a mechanism independent of this chain. The ANT, which exchanges ADP3−against ATP4− between the two faces of the inner membrane, is likely to participate in the maintenance of this potential, as suggested for ρ° yeast cells (8Kolarov J. Klingenberg M. FEBS Lett. 1974; 45: 320-323Crossref PubMed Scopus (29) Google Scholar). To determine whether the role played by the F1-ATPase could be mediated via the ANT, the effect of bongkrekic acid was tested on the growth and the mitochondrial membrane potential of both ρ° and ρ+ cells. Fig. 4 A shows that bongkrekic acid modified neither the ρ+ 143B cells growth nor that of the ρ° 143B cells after 5 days of treatment even at the highest tested concentration (10 μm). On the contrary, an inhibition of about 50% ρ° Hela S3 cell growth was observed after 5 days of treatment with 10 μm bongkrekic acid. Moreover, the inhibition could already be detected after a 3-day treatment (data not shown). In the case of the ρ+ HeLa S3 cells, no effect was observed before 5 days. At that time, 10 μmbongkrekic acid inhibited the cell growth by about 30%, but a culture medium acidification was observed. If this medium was replaced after 3 days, the growth inhibition was limited to 15%. Since, in the presence of bongkrekic acid, the ATP produced by oxidative phosphorylation cannot reach the cytoplasm, the ρ+ cell must then reconstitute its ATP pool via glycolysis. In this case, the NADH produced during glycolysis must be re-oxidized, and lactate should be accumulated. To verify this hypothesis, the lactate concentration was measured in ρ+ and ρ° HeLa S3 cells (Fig. 4 B). In the absence of bongkrekic acid, the lactate concentration (calculated per cell) was 6 times higher in ρ° than in ρ+ cells, as expected since in ρ° cells the ATP production completely relies on glycolysis. The lactate concentration strongly increased in ρ+ HeLa S3 cells treated with 10 μm bongkrekic acid, even if the number of cells was reduced. On the contrary, the lactate concentration decreased in ρ° HeLa S3 cells treated with 10 μm bongkrekic acid. However, the number of ρ° cells was simultaneously reduced. If the lactate amount was compared with the number of cells, it increased about 300% in ρ+ cells and only 30% in ρ° cells. Bongkrekic acid (10 μm) did not modify the mitochondrial membrane potential of ρ+ HeLa S3 cells either after a 30-min treatment (Fig. 5) or after a 22-h treatment (data not shown). On the contrary, in ρ° HeLa S3 cells, the mitochondrial membrane potential was reduced after a 30-min treatment as well as after a 22-h treatment. To verify whether F1-ATPase and ANT act together or in two independent pathways to maintain the ρ° HeLa S3 cell growth and mitochondrial membrane potential, the effects of aurovertin and bongkrekic acid were tested simultaneously (Fig. 6). The presence of either 30 μm aurovertin or 10 μm bongkrekic acid decreased the cell growth by about 50% after a 5-day treatment, whereas the simultaneous addition of bongkrekic acid and aurovertin decreased it by up to 85% that of the control value. The ρ° cell growth decrease observed with aurovertin or bongkrekic acid was less extensive than that observed with 0.1 or 0.5 μm FCCP (decrease of 92% of the control value). Similar to the effects observed on cell growth, the mitochondrial membrane potential was decreased more extensively in the presence of aurovertin and bongkrekic acid than when each drug was tested separately. When aurovertin concentration was increased up to 60 μm, no additional effect was observed either on cell growth or on mitochondrial membrane potential. First, our data demonstrate that ρ° cells devoid of the F0F1-ATPase subunits 6 and 8 contain an active F1-ATPase essential for their growth. The size and amount of F1 α and β subunits were similar in ρ° and ρ+ cells. Therefore, the α and β subunit precursors were imported in ρ° cells, and their signal sequences were normally processed in the mitochondria. Previous studies have already shown that the ρ° 143B cells contained the same amount of F1 β subunit mRNA as that of the parent ρ+cells, whereas the relative abundance of some other transcripts encoding mitochondrial proteins such as cytochrome c, cytochrome oxidase subunit IV and VIaL, and ANT 2 and ANT 3 were slightly more abundant (27Li K. Neufer P.D. Williams R.S. Am. J. Physiol. 1995; 269: C1265-C1270Crossref PubMed Google Scholar). Doxycline-induced inhibition of mitochondrial protein synthesis in human leukemia cells also decreased the contents of complex III or complex IV subunits of nuclear origin without modifying that of the F1-ATPase α and β subunits during two culture
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