Interaction of the δ and b Subunits Contributes to F1 and F0 Interaction in the Escherichia coli F1F0-ATPase
1997; Elsevier BV; Volume: 272; Issue: 48 Linguagem: Inglês
10.1074/jbc.272.48.30047
ISSN1083-351X
AutoresKen Sawada, Nozomi Kuroda, Hikaru Watanabe, Chie Moritani-Otsuka, Hiroshi Kanazawa,
Tópico(s)Acute Lymphoblastic Leukemia research
ResumoInteractions of the F1F0-ATPase subunits between the cytoplasmic domain of the b subunit (residues 26–156, bcyt) and other membrane peripheral subunits including α, β, γ, δ, ε, and putative cytoplasmic domains of the a subunit were analyzed with the yeast two-hybrid system and in vitroreconstitution of ATPase from the purified subunits as well. Only the combination of bcyt fused to the activation domain of the yeast GAL-4, and δ subunit fused to the DNA binding domain resulted in the strong expression of the β-galactosidase reporter gene, suggesting a specific interaction of these subunits. Expression of bcyt fused to glutathione S-transferase (GST) together with the δ subunit in Escherichia coli resulted in the overproduction of these subunits in soluble form, whereas expression of the GST-bcyt fusion alone had no such effect, indicating that GST-bcyt was protected by the co-expressed δ subunit from proteolytic attack in the cell. These results indicated that the membrane peripheral domain of b subunit stably interacted with the δ subunit in the cell. The affinity purified GST-bcyt did not contain significant amounts of δ, suggesting that the interaction of these subunits was relatively weak. Binding of these subunits observed in a direct binding assay significantly supported the capability of binding of the subunits. The ATPase activity was reconstituted from the purified bcyttogether with α, β, γ, δ, and ε, or with the same combination except ε. Specific elution of the ATPase activity from glutathione affinity column with the addition of glutathione after reconstitution demonstrated that the reconstituted ATPase formed a complex. The result indicated that interaction of b and δ was stabilized by F1 subunits other than ε and also suggested that b-δ interaction was important for F1-F0interaction. Interactions of the F1F0-ATPase subunits between the cytoplasmic domain of the b subunit (residues 26–156, bcyt) and other membrane peripheral subunits including α, β, γ, δ, ε, and putative cytoplasmic domains of the a subunit were analyzed with the yeast two-hybrid system and in vitroreconstitution of ATPase from the purified subunits as well. Only the combination of bcyt fused to the activation domain of the yeast GAL-4, and δ subunit fused to the DNA binding domain resulted in the strong expression of the β-galactosidase reporter gene, suggesting a specific interaction of these subunits. Expression of bcyt fused to glutathione S-transferase (GST) together with the δ subunit in Escherichia coli resulted in the overproduction of these subunits in soluble form, whereas expression of the GST-bcyt fusion alone had no such effect, indicating that GST-bcyt was protected by the co-expressed δ subunit from proteolytic attack in the cell. These results indicated that the membrane peripheral domain of b subunit stably interacted with the δ subunit in the cell. The affinity purified GST-bcyt did not contain significant amounts of δ, suggesting that the interaction of these subunits was relatively weak. Binding of these subunits observed in a direct binding assay significantly supported the capability of binding of the subunits. The ATPase activity was reconstituted from the purified bcyttogether with α, β, γ, δ, and ε, or with the same combination except ε. Specific elution of the ATPase activity from glutathione affinity column with the addition of glutathione after reconstitution demonstrated that the reconstituted ATPase formed a complex. The result indicated that interaction of b and δ was stabilized by F1 subunits other than ε and also suggested that b-δ interaction was important for F1-F0interaction. Proton-translocating ATPase (F1F0-ATPase) expressed on the membranes of mitochondria, chloroplasts, and bacteria has a key role in energy transduction (1Futai M. Kanazawa H. Microbiol. Rev. 1983; 47: 285-312Crossref PubMed Google Scholar, 2Futai M. Noumi T. Maeda M. Annu. Rev. Biochem. 1989; 58: 111-136Crossref PubMed Scopus (403) Google Scholar, 3Walker J.E. Saraste M. Gay N.J. Biochem. Biophys. Acta. 1984; 768: 164-200Crossref PubMed Scopus (371) Google Scholar, 4Senior A.E. Annu. Rev. Biophys. Biophys. Chem. 1990; 19: 7-41Crossref PubMed Scopus (329) Google Scholar, 5Fillingame R.H. Krulwich T.A. The Bacteria. XII. Academic Press, New York1990: 345-391Google Scholar, 6Pedersen P.L. Amzel L.M. J. Biol. Chem. 1993; 268: 9937-9940Abstract Full Text PDF PubMed Google Scholar, 7Weber J. Senior A. Biochim. Biophys. Acta. 1997; 1319: 19-58Crossref PubMed Scopus (395) Google Scholar). The complex subunit structure of this enzyme is basically similar among various species, but there are some differences in the membrane integral subcomplex (F0). Escherichia coli ATPase is composed of two different moieties, F1 and F0. F1,occupying the peripheral membrane portion, has the catalytic activity with five different subunits, α, β, γ, δ, and ε. F0 is the integral membrane portion with three different subunits, a, b, and c that forms a proton channel. The enzyme catalyzes ATP synthesis with a H+ gradient across the membrane formed by the respiratory chain in mitochondria and bacteria or photosynthetic electron transport in chloroplasts. In the reverse reaction, this enzyme pumps protons from the inside to the outside of bacterial cells and mitochondria coupled with hydrolysis of ATP. Based on the three-dimensional structure of αβγ complex of the bovine F1-ATPase (8Abrahams J.P. Leslie A.G.W. Lutter R. Walker J. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2751) Google Scholar) and the results of kinetic analyses of nucleotide binding and catalysis (9Penefsky H.S. Cross R.L. Adv. Enzyme Regul. 1991; 64: 173-214Google Scholar), rotation of α3β3 around γ was proposed to occur during catalysis (8Abrahams J.P. Leslie A.G.W. Lutter R. Walker J. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2751) Google Scholar). Data to support this hypothesis have been reported (10Duncan T.M. Bulygin V.V. Zhou Y. Hutcheon M.L. Cross R.L. Proc. Natl. Acad. Sci., U. S. A. 1995; 92: 10964-10968Crossref PubMed Scopus (459) Google Scholar, 11Sabbert D. Engelbrecht S. Junge W. Nature. 1996; 381: 623-625Crossref PubMed Scopus (464) Google Scholar, 12Gogol E.P. Johnston E. Aggeler R. Capaldi R.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9585-9589Crossref PubMed Scopus (100) Google Scholar), and recently direct visualization of this rotation of the γ subunit within the αβγ complex has been performed for thermophilic bacteria (13Noji H. Yasuda R. Yoshida M. Kinoshita Jr., K. Nature. 1997; 386: 299-302Crossref PubMed Scopus (1965) Google Scholar). Although evidence of the rotation within the F1 complex has been thus presented, its structural basis, especially in terms of the connecting portion between the αβγ complex and F0 is not well understood (14Capaldi R.A. Aggeler R. Turina P. Wilkens S. Trends Biochem. Sci. 1994; 19: 284-288Abstract Full Text PDF PubMed Scopus (132) Google Scholar). The δ and ε subunits were shown to be important for this interaction (1Futai M. Kanazawa H. Microbiol. Rev. 1983; 47: 285-312Crossref PubMed Google Scholar, 2Futai M. Noumi T. Maeda M. Annu. Rev. Biochem. 1989; 58: 111-136Crossref PubMed Scopus (403) Google Scholar, 5Fillingame R.H. Krulwich T.A. The Bacteria. XII. Academic Press, New York1990: 345-391Google Scholar, 6Pedersen P.L. Amzel L.M. J. Biol. Chem. 1993; 268: 9937-9940Abstract Full Text PDF PubMed Google Scholar, 14Capaldi R.A. Aggeler R. Turina P. Wilkens S. Trends Biochem. Sci. 1994; 19: 284-288Abstract Full Text PDF PubMed Scopus (132) Google Scholar). The atomic structure of the ε subunit and a portion of the δ subunit was resolved by NMR analysis for E. coli(14Capaldi R.A. Aggeler R. Turina P. Wilkens S. Trends Biochem. Sci. 1994; 19: 284-288Abstract Full Text PDF PubMed Scopus (132) Google Scholar, 15Wilkens S. Dahlquist F.W. McIntosh L.P. Donaldson L.W. Capaldi R.A. Nature Struct. Biol. 1995; 2: 961-967Crossref PubMed Scopus (155) Google Scholar, 16Wilkens S. Dunn S.D. Chandler J. Dahlquist F.W. Capaldi R.A. Nature Struct. Biol. 1997; 4: 196-201Crossref Scopus (110) Google Scholar). The ε subunit interacts with the DELSEED region of the α and β subunits (14Capaldi R.A. Aggeler R. Turina P. Wilkens S. Trends Biochem. Sci. 1994; 19: 284-288Abstract Full Text PDF PubMed Scopus (132) Google Scholar, 19Dallmann H.G. Geoffrey-Flynn T. Dunn S.D. J. Biol. Chem. 1992; 267: 18953-18960Abstract Full Text PDF PubMed Google Scholar) and also binds to the cytoplasmic region of the c subunit (17Zhang Y. Fillingame R.H. J. Biol. Chem. 1995; 270: 24609-24614Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). However, the topological arrangement of the δ subunit in the F1F0 complex is less well known than that of the ε subunit. In an early study of the biochemical properties of the purified δ subunit from E. coli (18Sternweis P. J. Biol. Chem. 1978; 253: 3123-3128Abstract Full Text PDF PubMed Google Scholar), an elongated structure was estimated. This subunit is also known to open sealed proton channel activity of F0 during enzyme biosynthesis (20Monticello R.A. Brusilow W.S.A. J. Bacteriol. 1994; 176: 1383-1389Crossref PubMed Google Scholar). Based on the results of hydropathy analysis of the primary structure of the b subunit of F0 (2Futai M. Noumi T. Maeda M. Annu. Rev. Biochem. 1989; 58: 111-136Crossref PubMed Scopus (403) Google Scholar, 3Walker J.E. Saraste M. Gay N.J. Biochem. Biophys. Acta. 1984; 768: 164-200Crossref PubMed Scopus (371) Google Scholar), the majority of this subunit is estimated to be hydrophilic and possibly extruded into the cytoplasm, whereas the small amino-terminal portion is essential for its integration into membranes (22Perlin D.S. Senior A.E. Arch. Biochem. Biophys. 1985; 236: 603-611Crossref PubMed Scopus (31) Google Scholar, 23Hoppe J. Brunner J. Jorgensen B.B. Biochemistry. 1984; 23: 5610-5616Crossref PubMed Scopus (70) Google Scholar, 24Dunn S.D. J. Biol. Chem. 1992; 267: 7630-7636Abstract Full Text PDF PubMed Google Scholar). The region between residues 25 and 146 of this subunit was overproduced as a soluble form and was shown to be capable of binding to the F1 complex (24Dunn S.D. J. Biol. Chem. 1992; 267: 7630-7636Abstract Full Text PDF PubMed Google Scholar). Although these previous studies strongly suggested interactions between the b and δ or ε subunits, direct evidence of b-δ or b-ε interaction has not been reported. The a subunit of F0 has been shown by genetic analysis to be required for F1-F0 binding (21Eya S. Noumi T. Maeda M. Futai M. J. Biol. Chem. 1988; 263: 10056-10062Abstract Full Text PDF PubMed Google Scholar) and its interacting partners within F1 subunits have been also determined by chemical cross-linking experiments (25Aris J.P. Simoni R.D. J. Biol. Chem. 1983; 258: 14599-14609Abstract Full Text PDF PubMed Google Scholar). However, the precise regions involved in the binding of this subunit to a specific F1 subunit have not been elucidated. Here, we have found a specific interaction between the b and δ subunits with two approaches; a genetic approach with the yeast two-hybrid system (26Moritani C. Sawada K. Takemoto K. Shin Y. Nemoto S. Noumi T. Kanazawa H. Biochim. Biophys. Acta. 1996; 258: 14599-14609Google Scholar, 27Fields S. Song O. Nature. 1989; 340: 245-246Crossref PubMed Scopus (4860) Google Scholar) and a biochemical approach with overproduction, purification, and in vitro binding of the subunits (28Shin Y. Sawada K. Nagakura T. Miyanaga M. Moritani C. Noumi T. Tsuchiya T. Kanazawa H. Biochim. Biophys. Acta. 1996; 1273: 62-70Crossref PubMed Scopus (12) Google Scholar). The results directly indicate that the interaction of b and δ has an important role for F1 and F0interaction. E. colistrains BL21 and JM109 were used (28Shin Y. Sawada K. Nagakura T. Miyanaga M. Moritani C. Noumi T. Tsuchiya T. Kanazawa H. Biochim. Biophys. Acta. 1996; 1273: 62-70Crossref PubMed Scopus (12) Google Scholar) for overproduction of various peptides and genetic manipulations including preparation of various plasmids, respectively. E. coli cells were cultured in a minimal medium (Tanaka medium) (29Kanazawa H. Tamura F. Mabuchi K. Miki T. Futai M. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 7005-7009Crossref PubMed Scopus (51) Google Scholar) supplemented with glucose or glycerol at 37 °C. For the selection of transformants with plasmids, appropriate antibiotics were added to these media. For the two-hybrid system, plasmids pGAD424 carrying the activation domain of GAL4, and pGBT9 carrying the binding domain of GAL4 (27Fields S. Song O. Nature. 1989; 340: 245-246Crossref PubMed Scopus (4860) Google Scholar) were used. A DNA fragment corresponding to the cytoplasmic domain (residues 26–156) of the b subunit (bcyt) was amplified by the polymerase chain reaction (30Saiki R.K. Gelfand D.H. Stoffel S. Scharf S.J. Higuchi R. Horn G.T. Mullis K.B. Erlich H.A. Science. 1988; 239: 487-491Crossref PubMed Scopus (13487) Google Scholar) with primer oligonucleotides (TableI), bT26-F (forward primer) and bT156-R (reverse primer), and genomic DNA from the wild-type DNA or plasmid pKM02 carrying the entire unc operon. The nucleotide sequence and deduced primary structure of the b subunit were based on the published results (31Gay N.J. Walker J.E. Nucleic Acid Res. 1981; 9: 3919-3926Crossref PubMed Scopus (135) Google Scholar, 32Mabuchi K. Kanazawa H. Kayano T. Futai M. Biochem. Biophys. Res. Commun. 1981; 102: 172-179Crossref PubMed Scopus (38) Google Scholar, 33Nielsen J. Hansen F.G. Hoppe J. Friedle P von Meyenberg Mol. Gen. Genet. 1981; 184: 33-39Crossref PubMed Scopus (69) Google Scholar). The amplified DNA was digested withEcoRI and BamHI, and the fragment subsequently recovered from agarose gels after electrophoresis was ligated intoEcoRI and BamHI digested pGAD424 or pGBT9. The chimeric plasmids thus constructed were named pGAD-bcyt and pGBT-bcyt, respectively. To prepare bcytpeptides, DNA corresponding to the same region (residues 26–156) or this region plus the δ subunit gene was amplified by polymerase chain reaction with primers bG26-F and bT156-R or bG26-F and DER-C1. The amplified DNA was integrated into the BamHI site of the expression vector pGEX-2T (28Shin Y. Sawada K. Nagakura T. Miyanaga M. Moritani C. Noumi T. Tsuchiya T. Kanazawa H. Biochim. Biophys. Acta. 1996; 1273: 62-70Crossref PubMed Scopus (12) Google Scholar) and resultant chimeric plasmids were named pGEX-bcyt and pGEX-bcyt and δ. Following essentially the same steps, portions of the a subunit between residues 60 and 100, and 160 and 200 were amplified by polymerase chain reaction with the oligonucleotide primers aT60-F and aT100-R, and aT160-F and aT200-R, respectively, and the wild-type sequences of the subunit on plasmid pKM02 as a template. The amplified DNAs were digested with the appropriate restriction enzymes shown in Fig. 1 and ligated to pGAD424 or pGBT9. The resultant chimeric plasmids were named pGAD-a60–100, pGBT-a60–100, pGAD-a160–200, and pGBT-a160–200. The nucleic acid and deduced amino acid sequence of the a subunit were cited from (34Kanazawa H. Futai M. Ann. N. Y. Acad. Sci. 1982; 402: 45-64Crossref PubMed Scopus (49) Google Scholar). Polymerase chain reaction was performed using the conditions as described previously (35Miki J. Matsuda T. Kariya H. Ohmori H. Tsuchiya T. Futai M. Kanazawa H. Arch. Biochem. Biophys. 1992; 294: 373-381Crossref PubMed Scopus (13) Google Scholar).Table IOligonucleotide primers used for polymerase chain reactionPrimerSequencePositionbT25-F5′-GCATGAATTC,TGGCCGCCATTAATGGCAGC-3′1239bT156-R5′-GCATGGATCCTTA,CAGTTCAGCGACAAGTT-3′1631aT60-F5′-GCATGAATTC,TTCCGTAGCGTAGCCAAAAA-3′178aT100-R5′-GCATGGATCC,TTACAGCTTGCTTTTGCCAT-3′300aT160-F5′-GCATGAATTC,CTGATTCTGTTCTACAGCAT-3′478aT200-R5′-GCATGGATCCTTA,CAGGCTTACCCCTTCAAGGA-3′600Position 1 corresponds to the first base (A) of the initiation codon of the a subunit gene in the unc operon. Sequences of the top two lines and the other lines correspond to the sequences of the b and a subunits, respectively. The first 5′ bases of the given reading frame (sequence) are shown next to a comma, and the residue number is shown in the left column (position). The first four nucleotides in all oligonucleotides were random and the subsequent GGATTC and GAATTC sequences are recognition sites for the restriction endonucleasesBamHI and EcoRI, respectively. Open table in a new tab Position 1 corresponds to the first base (A) of the initiation codon of the a subunit gene in the unc operon. Sequences of the top two lines and the other lines correspond to the sequences of the b and a subunits, respectively. The first 5′ bases of the given reading frame (sequence) are shown next to a comma, and the residue number is shown in the left column (position). The first four nucleotides in all oligonucleotides were random and the subsequent GGATTC and GAATTC sequences are recognition sites for the restriction endonucleasesBamHI and EcoRI, respectively. The chimeric plasmids of pGAD424 and pGBT9 derivatives constructed as described above or as previously reported for F1 subunits (26Moritani C. Sawada K. Takemoto K. Shin Y. Nemoto S. Noumi T. Kanazawa H. Biochim. Biophys. Acta. 1996; 258: 14599-14609Google Scholar) were introduced into yeast SFY526 carrying the β-galactosidase reporter gene. Expression of β-galactosidase was measured as described previously (26Moritani C. Sawada K. Takemoto K. Shin Y. Nemoto S. Noumi T. Kanazawa H. Biochim. Biophys. Acta. 1996; 258: 14599-14609Google Scholar). The optical density at 420 nm ofo-nitrophenol released from the substrateo-nitrophenyl β-galactoside was normalized by the cell density of yeast measured photometrically at 600 nm and was expressed in Miller units (26Moritani C. Sawada K. Takemoto K. Shin Y. Nemoto S. Noumi T. Kanazawa H. Biochim. Biophys. Acta. 1996; 258: 14599-14609Google Scholar). The expression plasmid pGEX-bcyt was introduced into E. coli BL21 (28Shin Y. Sawada K. Nagakura T. Miyanaga M. Moritani C. Noumi T. Tsuchiya T. Kanazawa H. Biochim. Biophys. Acta. 1996; 1273: 62-70Crossref PubMed Scopus (12) Google Scholar). Transformed E. coli was cultured in 500 ml of M9ZB medium supplemented with 0.2% glucose with vigorous shaking at 37 °C. At 0.6 A 600, isopropyl-1-thio-β-d-galactopyranoside was added to the culture (0.4 mm) and then the incubation was continued for another 2 h. Cells were harvested, washed, suspended in 6 ml of phosphate-buffered saline with 1% Triton X-100, and disrupted by sonication. The disrupted materials were subjected to low (10,000 − g for 10 min) and high speed (100,000 −g for 60 min) centrifugation to fractionate proteins into soluble (supernatant) and membrane (precipitate) fractions as described previously (35Miki J. Matsuda T. Kariya H. Ohmori H. Tsuchiya T. Futai M. Kanazawa H. Arch. Biochem. Biophys. 1992; 294: 373-381Crossref PubMed Scopus (13) Google Scholar). The amounts of protein recovered were 92.4 mg and 14.3 mg for the supernatant and membrane fractions, respectively, after high speed centrifugation. The supernatant fraction was subjected to glutathione-Sepharose (Pharmacia Biotech Inc.) (2.0 ml) column chromatography as described previously (28Shin Y. Sawada K. Nagakura T. Miyanaga M. Moritani C. Noumi T. Tsuchiya T. Kanazawa H. Biochim. Biophys. Acta. 1996; 1273: 62-70Crossref PubMed Scopus (12) Google Scholar). The fraction eluted with 10 mm glutathione contained 26.6 mg of protein in which the major band stained by Coomassie Brilliant Blue was shown to be GST-bcyt. Purified GST-δ and GST-ε were prepared as described previously (28Shin Y. Sawada K. Nagakura T. Miyanaga M. Moritani C. Noumi T. Tsuchiya T. Kanazawa H. Biochim. Biophys. Acta. 1996; 1273: 62-70Crossref PubMed Scopus (12) Google Scholar). GST-δ (7.0 mg), GST-ε (6.0 mg), and GST-bcyt (8.2 mg) were digested with thrombin (11.5 units for 1 mg of fusion protein, Sigma T3010) for 20 h. Subsequently, the digested materials were subjected to glutathione-Sepharose affinity chromatography, and 2.2, 2.0, and 2.7 mg of δ, ε, and bcyt, respectively, were obtained as practically homogeneous subunits. The F1-ATPase was reconstituted from purified subunits by dialyzing the subunit mixture against reconstitution buffer (50 mm succinate-Tris, pH 6.0, 10% glycerol, 0.1 mm dithiothreitol, 0.1 mm EDTA, 2 mm ATP, and 2 mmMgCl2) for 8 h at 26 °C (36Dunn S.D. Futai M. J. Biol. Chem. 1980; 255: 113-118Abstract Full Text PDF PubMed Google Scholar). The α, β, and γ subunits were purified as described previously (28Shin Y. Sawada K. Nagakura T. Miyanaga M. Moritani C. Noumi T. Tsuchiya T. Kanazawa H. Biochim. Biophys. Acta. 1996; 1273: 62-70Crossref PubMed Scopus (12) Google Scholar). For a typical reconstitution experiment, α (180 μg), β (165 μg), γ (100 μg), δ (20.7 μg), ε (16.4 μg), and GST-bcyt (89.5 μg) were mixed in 2.4 ml of reconstitution buffer. After dialysis, the reconstituted materials that contained the subunits in 3.0 ml were applied to glutathione-Sepharose (0.3 ml). After washing the column with 7.0 ml of reconstitution buffer, the ATPase was eluted with 10 mm glutathione in reconstitution buffer. Aliquots of the eluted materials were used for the ATPase assay (37Futai M. Sternweis P.C. Heppel L.A. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 2725-2729Crossref PubMed Scopus (210) Google Scholar), protein measurement by the published procedure (29Kanazawa H. Tamura F. Mabuchi K. Miki T. Futai M. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 7005-7009Crossref PubMed Scopus (51) Google Scholar, 30Saiki R.K. Gelfand D.H. Stoffel S. Scharf S.J. Higuchi R. Horn G.T. Mullis K.B. Erlich H.A. Science. 1988; 239: 487-491Crossref PubMed Scopus (13487) Google Scholar), and SDS-gel electrophoretic analysis as described previously (21Eya S. Noumi T. Maeda M. Futai M. J. Biol. Chem. 1988; 263: 10056-10062Abstract Full Text PDF PubMed Google Scholar). Aliquots of 2 or 4 μg of bcyt or GST were fixed on Millipore GVHP filters (39Shin Y. Sawada K. Moritani C. Kanazawa H. Arch. Biochem. Biophys. 1997; 340: 36-42Crossref PubMed Scopus (2) Google Scholar), which were activated with methanol and equilibrated with buffer (100 mm Tris-HCl, pH 7.4, 190 mmglycine, 5% MeOH) prior to fixation. After blocking nonspecific binding of proteins by soaking the filters in skimmed milk, the filter with bcyt and GST was washed with reconstitution buffer and then soaked again in 2 ml of reconstitution buffer containing 100 μg of the δ subunit for 8 h at 26 °C for binding. Then, the filter was washed with reconstitution buffer to remove unbound δ subunits and nonspecific protein binding was blocked with 10% skimmed milk solution. Bound δ was detected by anti-δ mouse IgG raised against the purified E. coli δ and visualized with an ABC Vectastain kit as described previously (35Miki J. Matsuda T. Kariya H. Ohmori H. Tsuchiya T. Futai M. Kanazawa H. Arch. Biochem. Biophys. 1992; 294: 373-381Crossref PubMed Scopus (13) Google Scholar). Preparation of plasmids, digestion and ligation of the DNA fragments, and other techniques related to handling DNA were performed according to the published procedures (40Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York1982Google Scholar). Nucleotide sequences of the amplified DNA in the expression plasmids were verified by the dideoxy method with appropriate primers and35S-α-deoxy CTP (37 TBq/mmol, Amersham) (41Sanger F. Coulson A.R. Barrell B.G. Smith A.J.H. Roe B.A. J. Mol. Biol. 1980; 143: 161-178Crossref PubMed Scopus (2194) Google Scholar) or with a DNA sequencer (Pharmacia, Alfexpress DNA sequencer). Aliquots of eluted materials after affinity chromatography with glutathione-Sepharose were subjected to SDS-polyacrylamide gel electrophoresis (12.5% acrylamide) (29Kanazawa H. Tamura F. Mabuchi K. Miki T. Futai M. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 7005-7009Crossref PubMed Scopus (51) Google Scholar) and separated peptides were blotted onto GVHP filters (Millipore) (35Miki J. Matsuda T. Kariya H. Ohmori H. Tsuchiya T. Futai M. Kanazawa H. Arch. Biochem. Biophys. 1992; 294: 373-381Crossref PubMed Scopus (13) Google Scholar). The membrane filters were soaked in a solution of poly- or monoclonal antibodies raised against the purified E. coli α, β,γ, δ, or GST, and the reacted bands were visualized with an ABC Vectastain kit as described previously (35Miki J. Matsuda T. Kariya H. Ohmori H. Tsuchiya T. Futai M. Kanazawa H. Arch. Biochem. Biophys. 1992; 294: 373-381Crossref PubMed Scopus (13) Google Scholar). Restriction endonucleases, T4 DNA ligase, Tth and Pfu DNA polymerase, and T7 DNA polymerase were purchased from Bethesda Research Labs, Toyobo Co., New England Biolabs, and Takara Co. Oligonucleotides used as primers were synthesized by DNAgency (Malvern, PA). Other materials were of the highest grade commercially available. To identify subunits capable of binding the b subunit, we took two approaches; a genetic approach with the yeast two-hybrid system (27Fields S. Song O. Nature. 1989; 340: 245-246Crossref PubMed Scopus (4860) Google Scholar) and a biochemical approach with in vitro binding assay of subunits. For these approaches, we constructed two types of chimeric plasmids (Fig.1). For biochemical approaches, DNA fragment spanning the membrane peripheral portion of the b subunit (residues 26–156, bcyt) that was deduced from hydropathy analyses was fused to the GST gene to create a fusion peptide, and for genetic analysis with the two-hybrid system, the same portion of the b subunit was fused to the activation or DNA binding domain of GAL4 gene. The bcyt region of the b subunit gene was amplified by the polymerase chain reaction and fused to the GST or GAL4 gene. Based on the previous model of membrane topology in the a subunit of F0 (3Walker J.E. Saraste M. Gay N.J. Biochem. Biophys. Acta. 1984; 768: 164-200Crossref PubMed Scopus (371) Google Scholar,42Hoppe J. Sebald W. Biochim. Biophys. Acta. 1984; 768: 1-27Crossref PubMed Scopus (162) Google Scholar, 43Vik S.B. Cain B.D. Chun K.T. Simoni R.D. J. Biol. Chem. 1988; 263: 6599-6605Abstract Full Text PDF PubMed Google Scholar, 44Yamada H. Moriyama Y. Maeda M. Futai M. FEBS Lett. 1996; 390: 34-38Crossref PubMed Scopus (30) Google Scholar), we amplified DNA of this gene corresponding to the putative peripheral membrane domain between residues 60 and 100, and 160 and 200, and also fused it to the binding or activation domain of theGAL4. To determine the binding partners of bcytand portions of the a subunit, we used chimeric plasmids of F1 subunits (α, β, γ, δ, and ε) fused to the activation or binding domain of the GAL4 gene for the yeast two-hybrid system constructed in the previous study (26Moritani C. Sawada K. Takemoto K. Shin Y. Nemoto S. Noumi T. Kanazawa H. Biochim. Biophys. Acta. 1996; 258: 14599-14609Google Scholar). First, we examined the interaction between bcyt and one of the F1 subunits, and then we analyzed bcyt-bcyt and also bcyt-a interactions with the putative peripheral membrane domains of the a subunit. The pair of α and β subunits gave strong expression of the reporter gene as we described previously (26Moritani C. Sawada K. Takemoto K. Shin Y. Nemoto S. Noumi T. Kanazawa H. Biochim. Biophys. Acta. 1996; 258: 14599-14609Google Scholar), whereas the vectors alone did not (Table II). Among the various combinations tested, the combination of bcyt fused to GAL4-ad (activation domain of GAL4) and δ fused to GAL4-bd (binding domain of GAL4) alone resulted in strong expression of the reporter gene, whereas all other combinations showed no significant expression (Table II). These results demonstrated that the interaction between bcyt and δ occurred under in vivoconditions and other combinations might not cause interaction in those pairs. Although the purified soluble fraction of b (residues 25–146 or 26–156) was reported to form a dimer in vitro (24Dunn S.D. J. Biol. Chem. 1992; 267: 7630-7636Abstract Full Text PDF PubMed Google Scholar, 45Howitt S.M. Rodgers A.J.W. Jeffrey P.D. Cox G.B. J. Biol. Chem. 1996; 271: 7038-7042Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), we did not observe this interaction in the two-hybrid system. The putative membrane peripheral region of the a subunit also did not show reporter gene expression, suggesting that these portions may not interact stably with the δ subunit.Table IIInteraction of the b cyt and the F 1 F 0 ATPase subunits in the yeast two-hybrid systemFusion proteinβ-Galactosidase activity(c)GAL4-adGAL4-bdbα0.09β0.12γ0.13δ15.0ε0.08b0.17αb0.10β0.08γ0.07δ0.08ε0.08αβ18.0−−0.08The expression plasmids carrying the genes for the indicated fusion proteins were cotransfected into yeast SFY526 by the lithium acetate method as described under “Materials and Methods.” Transformants were selected by plating onto SD medium lacking Trp and Leu. Transformants that contained both plasmids were grown to mid-log phase of cell growth in SD medium lacking Trp and Leu and then assayed for β-galactosidase activity, shown here in Miller units (c). The pairs in the top six lines indicate combinations bcyt fused to the activation domain of GAL4 (GAL4-ad) and various F1 F0subunits fused to the binding domain of GAL4 (GAL4-bd). Similarly, the next five lines indicate combinations of bcyt fused to the binding domain of GAL4 and various subunits fused to the activation domain of GAL4. In the bottom two lines, the pair of α and β subunits fused to the activation and binding domains, respectively, and the combination of vectors alone (−) are shown as positive and negative controls, respectively. Open table in a new tab The expression plasmids carrying the genes for the indicated fusion proteins were cotransfected i
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