FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2040563318> ?p ?o ?g. }

• W2040563318 endingPage "507" @default.
• W2040563318 startingPage "501" @default.
• W2040563318 abstract "The role of the C-domain of the ϵ subunit of ATP synthase was investigated by fusing either the 20-kDa flavodoxin (Fd) or the 5-kDa chitin binding domain (CBD) to the N termini of both full-length ϵ and a truncation mutant ϵ88-stop. All mutant ϵ proteins were stable in cells and supported F1F0 assembly. Cells expressing the Fd-ϵ or Fd-ϵ88-stop mutants were unable to grow on acetate minimal medium, indicating their inability to carry out oxidative phosphorylation because of steric blockage of rotation. The other forms of ϵ supported growth on acetate. Membrane vesicles containing Fd-ϵ showed 23% of the wild type ATPase activity but no proton pumping, suggesting that the ATP synthase is intrinsically partially uncoupled. Vesicles containing CBD-ϵ were indistinguishable from the wild type in ATPase activity and proton pumping, indicating that the N-terminal fusions alone do not promote uncoupling. Fd-ϵ88-stop caused higher rates of uncoupled ATP hydrolysis than Fd-ϵ, and ϵ88-stop showed an increased rate of membrane-bound ATP hydrolysis but decreased proton pumping relative to the wild type. Both results demonstrate the role of the C-domain in coupling. Analysis of the wild type and ϵ88-stop mutant membrane ATPase activities at concentrations of ATP from 50 μm to 8 mm showed no significant dependence of the ratio of bound/released ATPase activity on ATP concentration. These results support the hypothesis that the main function of the C-domain in the Escherichia coli ϵ subunit is to reduce uncoupled ATPase activity, rather than to regulate coupled activity. The role of the C-domain of the ϵ subunit of ATP synthase was investigated by fusing either the 20-kDa flavodoxin (Fd) or the 5-kDa chitin binding domain (CBD) to the N termini of both full-length ϵ and a truncation mutant ϵ88-stop. All mutant ϵ proteins were stable in cells and supported F1F0 assembly. Cells expressing the Fd-ϵ or Fd-ϵ88-stop mutants were unable to grow on acetate minimal medium, indicating their inability to carry out oxidative phosphorylation because of steric blockage of rotation. The other forms of ϵ supported growth on acetate. Membrane vesicles containing Fd-ϵ showed 23% of the wild type ATPase activity but no proton pumping, suggesting that the ATP synthase is intrinsically partially uncoupled. Vesicles containing CBD-ϵ were indistinguishable from the wild type in ATPase activity and proton pumping, indicating that the N-terminal fusions alone do not promote uncoupling. Fd-ϵ88-stop caused higher rates of uncoupled ATP hydrolysis than Fd-ϵ, and ϵ88-stop showed an increased rate of membrane-bound ATP hydrolysis but decreased proton pumping relative to the wild type. Both results demonstrate the role of the C-domain in coupling. Analysis of the wild type and ϵ88-stop mutant membrane ATPase activities at concentrations of ATP from 50 μm to 8 mm showed no significant dependence of the ratio of bound/released ATPase activity on ATP concentration. These results support the hypothesis that the main function of the C-domain in the Escherichia coli ϵ subunit is to reduce uncoupled ATPase activity, rather than to regulate coupled activity. ATP synthase is the enzyme responsible for the formation of ATP during oxidative phosphorylation. This enzyme, found on the inner membranes of mitochondria and bacteria, and on the thylakoid membranes in chloroplasts, uses the energy stored in a transmembrane proton gradient to produce ATP from the precursors ADP and Pi. The enzyme can be subdivided into two sectors. In Escherichia coli, the F1 sector is composed of 5 different polypeptides with a stoichiometry of α3β3γδϵ, and houses the three catalytic nucleotide binding sites. The F0 sector forms a proton-specific pore and is comprised of three different integral membrane proteins, showing a stoichiometry of ab2c10. During ATP synthesis, protons move through the F0 pore and drive the rotation of the c10γϵ oligomer. Movement of this “rotor” drives the sequential conformational changes in α3β3 that promote both the binding of substrates, ADP and Pi, and the formation and release of ATP as predicted by the binding change mechanism of Paul Boyer (1Boyer P.D. Annu. Rev. Biochem. 1997; 66: 717-749Crossref PubMed Scopus (1565) Google Scholar). The two b subunits, combined with the δ subunit, form a peripheral stalk that connects the F1 and F0 sectors, preventing their rotation relative to each other. In E. coli, ATP synthase is reversible and under anaerobic conditions can act as an ATP-driven proton pump energizing the inner membrane to power membrane transporters and the flagellar motor. For recent reviews see Refs. 2Walker J.E. Biochim. Biophys. Acta. 1458. 2000: 221-510Google Scholar, 3Peterson P.L. J. Bioenerg. Biomembr.32. 2000: 325-540Google Scholar, 4Futai M. Wada Y. Kaplan J.H. Handbook of ATPases. Wiley-VCH, Weinheim Germany2004: 237-336Google Scholar.The ϵ subunit, which lies at the interface of F1 and F0, has been suggested to function within the enzyme as an inhibitor, a regulator, or a coupling factor (5Kato-Yamada Y. Bald D. Koike M. Motohashi K. Hisabori T. Yoshida M. J. Biol. Chem. 1999; 274: 33991-33994Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 6Xiao Y. Metzl M. Mueller D.M. J. Biol. Chem. 2000; 275: 6963-6968Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 7Duvezin-Caubet S. Caron M. Giraud M.F. Velours J. di Rago J.P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13235-13240Crossref PubMed Scopus (44) Google Scholar, 8Tsunoda S.P. Rodgers A.J. Aggeler R. Wilce M.C. Yoshida M. Capaldi R.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6560-6564Crossref PubMed Scopus (160) Google Scholar, 9Suzuki T. Murakami T. Iino R. Suzuki J. Ono S. Shirakihara Y. Yoshida M. J. Biol. Chem. 2003; 278: 46840-46846Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). The high resolution structure of the isolated E. coli ϵ, solved by both x-ray crystallography (10Uhlin U. Cox G.B. Guss J.M. Structure. 1997; 5: 1219-1230Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar) and NMR spectroscopy (11Wilkens S. Dahlquist F.W. McIntosh L.P. Donaldson L.W. Capaldi R.A. Nat. Struct. Biol. 1995; 2: 961-967Crossref PubMed Scopus (155) Google Scholar, 12Wilkens S. Capaldi R.A. J. Biol. Chem. 1998; 273: 26645-26651Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), shows a two domain protein with an N-terminal 10-stranded β-barrel (residues 1–87, termed the N-domain) and a C-terminal helix-turn-helix domain (residues 88–138, termed the C-domain). The latter is not absolutely required for oxidative phosphorylation, or photophosphorylation in the case of the chloroplast enzyme, but is necessary for the inhibition of ATPase activity (5Kato-Yamada Y. Bald D. Koike M. Motohashi K. Hisabori T. Yoshida M. J. Biol. Chem. 1999; 274: 33991-33994Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 13Nowak K.F. McCarty R.E. Biochemistry. 2004; 43: 3273-3279Crossref PubMed Scopus (33) Google Scholar, 14Kuki M. Noumi T. Maeda M. Amemura A. Futai M. J. Biol. Chem. 1988; 263: 17437-17442Abstract Full Text PDF PubMed Google Scholar, 15Xiong H. Zhang D. Vik S.B. Biochemistry. 1998; 37: 16423-16429Crossref PubMed Scopus (42) Google Scholar). The structure of ϵ in the ATP synthase complex has been controversial as two different conformations of ϵ; the “up conformation” (16Rodgers A.J. Wilce M.C. Nat. Struct. Biol. 2000; 7: 1051-1054Crossref PubMed Scopus (152) Google Scholar, 17Hausrath A.C. Capaldi R.A. Matthews B.W. J. Biol. Chem. 2001; 276: 47227-47232Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 18Hausrath A.C. Gruber G. Matthews B.W. Capaldi R.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13697-13702Crossref PubMed Scopus (82) Google Scholar) and the “down conformation” (19Gibbons C. Montgomery M.G. Leslie A.G. Walker J.E. Nat. Struct. Biol. 2000; 7: 1055-1061Crossref PubMed Scopus (433) Google Scholar), have been seen in crystal structures. The N-domain is located in essentially the same position in both conformations, but in the up conformation the helices of the C-domain are extended and partially wrap around γ to make contact with α3β3, whereas in the down conformation the two α-helices fold on themselves and lie next to the N-domain on top of the c10 oligomer. It has been recently suggested (8Tsunoda S.P. Rodgers A.J. Aggeler R. Wilce M.C. Yoshida M. Capaldi R.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6560-6564Crossref PubMed Scopus (160) Google Scholar, 9Suzuki T. Murakami T. Iino R. Suzuki J. Ono S. Shirakihara Y. Yoshida M. J. Biol. Chem. 2003; 278: 46840-46846Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar) that when the cellular concentration of ATP is low, ϵ adopts the up conformation and inhibits ATP hydrolysis by allowing for rotation only in the direction of synthesis. The ϵ subunit was suggested to adopt the down conformation when ATP is high, allowing ATP synthase to catalyze either hydrolysis or synthesis of ATP.Previously, we have provided in vivo evidence for rotation by fusing proteins of various size to the C terminus of ϵ (20Cipriano D.J. Bi Y. Dunn S.D. J. Biol. Chem. 2002; 277: 16782-16790Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). In addition to stopping rotation, the larger fusion proteins also generated an uncoupled ATPase activity that was sensitive to inhibition by DCCD, 3The abbreviations used are: DCCDdicyclohexylcarbodiimideCBDchitin binding domainFCCPp-(trifluoromethoxy)phenylhydrazoneFdflavodoxinPVDFpolyvinylidene difluoride.3The abbreviations used are: DCCDdicyclohexylcarbodiimideCBDchitin binding domainFCCPp-(trifluoromethoxy)phenylhydrazoneFdflavodoxinPVDFpolyvinylidene difluoride. implying that it required movement of the rotor. Because the full 360° rotation of the rotor is blocked by the fusion protein, any such movement must be in a partial, reciprocating fashion. We hypothesized that the C-domain of ϵ serves a coupling function in ATP synthase, keeping the enzyme efficient by preventing rotation in the wrong direction during ATP hydrolysis. Blocking the proper interactions of the C-domain with α3β3 by addition of the fusion protein would block this function and allow the rotor to slip backwards. Based on these findings, we suggested that ϵ is involved in keeping the ATP synthase efficient by preventing uncoupled ATP hydrolysis.To test this hypothesis, and to test for intrinsic uncoupling in ATP synthase, we have separated the effect of blocking rotation from the uncoupling effect of C-terminal modifications by creating a series of N-terminal fusion proteins, C-terminal truncations, and a combination of the two. Fusion of a large 20-kDa protein to the N terminus of ϵ blocked rotation and inhibited ATP hydrolysis, but did not eliminate ATPase activity. The residual ATPase activity of this “rotation-blocked” enzyme was doubled if the C-domain was truncated. Truncation of the C-domain of normal ϵ resulted in an increased rate of ATP hydrolysis but a decreased rate of proton pumping, again indicating the generation of uncoupled activity. These results support our hypothesis that the C-domain functions to reduce uncoupled ATP hydrolysis. The mechanism for the generation of the uncoupled activity is discussed.EXPERIMENTAL PROCEDURESGeneral Methods—Recombinant DNA techniques were performed as described by Sambrook et al. (21Sambrook J. Fritsh E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). All plasmid sequences produced by PCR and primed synthesis were confirmed by DNA sequencing. Membrane protein concentrations were determined by the method of Lowry et al. (22Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Membrane preparation, assays of ATP hydrolysis, ATP-dependent proton pumping assays, and growth assays were performed as described by Cipriano et al. (20Cipriano D.J. Bi Y. Dunn S.D. J. Biol. Chem. 2002; 277: 16782-16790Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). ATPase assays performed under different concentrations of ATP were performed with the following modifications: The hydrolysis reaction was started by the addition of 0.3 ml of reaction mix containing 50 mm Tris-HCl pH 8, 2.5 mm PEP, 0.2 mg/ml pyruvate kinase, and either 8 mm ATP/4 mm MgCl2, 4 mm ATP/2 mm MgCl2, 0.5 mm ATP/0.25 mm MgCl2, or 0.05 mm ATP/0.025 mm MgCl2. Aurovertin D was prepared as described previously (23Dunn S.D. Zadorozny V.D. Tozer R.G. Orr L.E. Biochemistry. 1987; 26: 4488-4493Crossref PubMed Scopus (63) Google Scholar). The purified ϵ subunit was expressed from plasmid pES2 (24Skakoon E.N. Dunn S.D. Arch. Biochem. Biophys. 1993; 302: 272-278Crossref PubMed Scopus (30) Google Scholar) and purified as described (20Cipriano D.J. Bi Y. Dunn S.D. J. Biol. Chem. 2002; 277: 16782-16790Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Western blots were performed essentially as described by Cipriano et al. (20Cipriano D.J. Bi Y. Dunn S.D. J. Biol. Chem. 2002; 277: 16782-16790Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) using PVDF membranes and probed with antibodies raised against the α, β, and ϵ subunits. Because the N-terminal domain of ϵ by itself washes off of the PVDF membranes, a modified procedure for blotting the ϵ mutants was devised where 20 μg of membrane protein were blotted and then fixed to the membrane prior to the blocking step by soaking in transfer buffer containing 0.5% glutaraldehyde for 10 min at room temperature. The monoclonal antibody raised against α (α-II) (25Aggeler R. Capaldi R.A. Dunn S. Gogol E.P. Arch. Biochem. Biophys. 1992; 296: 685-690Crossref PubMed Scopus (10) Google Scholar) was a generous gift from Drs. Robert Aggeler and Rod Capaldi of the University of Oregon, and the monoclonal antibody raised against b (b-10–6 D1) was kindly provided by Drs. Gabriele Deckers-Hebestreit and Karlheinz Altendorf of Universität Osnabrück. The anti ϵ (ϵ-1) monoclonal antibody has been previously described (26Skakoon E.N. Dunn S.D. Arch. Biochem. Biophys. 1993; 302: 279-284Crossref PubMed Scopus (14) Google Scholar, 27Dunn S.D. Tozer R.G. Antczak D.F. Heppel L.A. J. Biol. Chem. 1985; 260: 10418-10425Abstract Full Text PDF PubMed Google Scholar). Final plasmids containing the appropriate ϵ mutants in the entire unc operon encoding ATP synthase were transformed and expressed in the unc deletion strain DK8 (28Klionsky D.J. Brusilow W.S. Simoni R.D. J. Bacteriol. 1984; 160: 1055-1060Crossref PubMed Google Scholar).Construction of C-terminal ϵ Truncations—Plasmid pDC1 (20Cipriano D.J. Bi Y. Dunn S.D. J. Biol. Chem. 2002; 277: 16782-16790Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), containing the uncC gene encoding ϵA137H with an AvrII site immediately following the stop codon, was the starting point for construction of the C-terminal ϵ deletions. Products of 3 PCR reactions using pDC1 as the template, the M13R universal primer as the 5′-primer, and primers 1, 2, or 3 (see Fig. 1B for primer sequences) as the 3′-primer, were cut with XmaI and AvrII and inserted into pDC1 cut with the same enzymes to make pDC41 (wild type), pDC42 (ϵ88-stop), and pDC43 (ϵ108-stop), respectively. pDC41, pDC42, and pDC43 were then cut with PsyI and AvrII, and the small fragments isolated and ligated into the 8830 bp PsyI/AvrII fragment of pSD135 (20Cipriano D.J. Bi Y. Dunn S.D. J. Biol. Chem. 2002; 277: 16782-16790Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) to produce pDC44, pDC45, and pDC46, respectively. These plasmids contain all genes encoding ATP synthase with wild-type ϵ (pDC44), ϵ88-stop (pDC45), and ϵ108-stop (pDC46).Construction of an N-terminal ϵ Fusion Cloning Vector—Plasmid pSD15 containing the 1182-bp PstI fragment of the unc operon cloned into the PstI site of pUC8 (29Vieira J. Messing J. Gene (Amst.). 1982; 19: 259-268Crossref PubMed Scopus (3770) Google Scholar) was the starting point for construction of the N-terminal ϵ fusions. To facilitate the construction, the single BamHI restriction site and one of two AatII sites were removed. The single BamHI site in pUC8 sequence was removed by digestion with BamHI, blunting the 5′ overhangs with the Klenow fragment of DNA polymerase I and religating to produce pDC39. The AatII site in pUC8 sequence was removed by partial digestion with AatII, isolation of singly cut linear plasmid by gel purification, treatment with T4 DNA polymerase to remove the 3′ overhangs, and ligation of the blunted ends. Transformants for which the AatII site within pUC8 vector sequence had been removed were screened by restriction mapping to identify pDC62. Two partially overlapping oligonucleotides (primers 4 and 5) were annealed and made double-stranded by treatment with Klenow fragment of DNA polymerase, digested with AatII, and inserted into the single AatII site in pDC62 to make pDC40. To facilitate future cloning steps the AvrII site just after the stop codon of the ϵ gene was moved into pDC40. The 465-bp BsaBI/HindIII fragment of pDC41 was inserted into pDC40 using those sites to produce pDC47. The sequence of pDC47 corresponding to the N terminus of ϵ is shown in detail in Fig. 1A.Construction of N-terminal ϵ Fusions—PCR reactions using either primers 6 and 7 or else primers 8 and 9 were used to amplify DNA fragments carrying the CBD from the Bacillus circulans chitinase A1 (chiA1, Swiss-Prot P20533) and the E. coli flavodoxin (fldA, Swiss-Prot P23243) using plasmid pTYB1 (New England Biolabs) and E. coli chromosomal DNA as templates, respectively. Insertion of the PCR products into pDC47 using KpnI and BamHI yielded plasmids pDC48 and pDC49, which contain the N-terminal CBD-ϵ and flavodoxin-ϵ (Fd-ϵ) fusions, respectively. These mutants were then moved into the unc operon on plasmid pSD135 using the EagI and AvrII sites to produce pDC52 (CBD-ϵ) and pDC53 (Fd-ϵ).Construction of N-terminal Fusion/C-terminal Deletion ϵ Double Mutants—The deletion mutant ϵ88-stop was fused with the N-terminal fusions as follows: The 312-bp HindIII/BsaBI fragment of pDC42 was moved into pDC48 and pDC49, which had been cut with the same enzymes to produce pDC50 (CBD-ϵ88-stop) and pDC51 (flavodoxin-ϵ88-stop), respectively. These mutants were then moved into the unc operon on plasmid pSD135 using the EagI and AvrII sites to produce pDC54 (CBD-ϵ88-stop) and pDC55 (flavodoxin-ϵ88-stop).RESULTSConstruction of N-terminal ϵ Fusions and C-terminal ϵ Truncations—To investigate the effect of physically blocking rotation in ATP synthase and the role of the C-domain in energy coupling, a series of N-terminal fusion proteins were constructed both in the presence and absence of the C-domain. The strategy was: 1) to block rotation of the rotor with the N-terminal fusions while leaving the C terminus intact, allowing us to ask how tightly ATP hydrolysis is coupled to rotation; 2) to remove the C-domain from this rotation-blocked ATP synthase to study the uncoupling effect of a C-terminal truncation in the absence of rotation, and 3) to test the uncoupling effect of C-terminal ϵ truncations in rotation-allowed ATP synthase. Based on previous results with the small C-terminal ϵ fusion protein (20Cipriano D.J. Bi Y. Dunn S.D. J. Biol. Chem. 2002; 277: 16782-16790Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), the removal of the ϵ C-domain should result in increased rates of DCCD-sensitive ATP hydrolysis that are uncoupled from proton pumping.The N-terminal fusions were designed to avoid or minimize effects on the translation of the mRNA, the folding of the ϵ polypeptide, or the assembly of ATP synthase (Fig. 1, A and B). Because translation efficiency depends on nucleotide sequences both upstream and downstream of the start codon, no changes were made in either the upstream region or the sequence encoding the first 10 amino acid residues of ϵ. A doubled-stranded oligonucleotide prepared from primers 4 and 5 (Fig. 1B) was cloned into the AatII site. This oligonucleotide included KpnI and BamHI restriction enzyme sites for subsequent insertion of the fusions, a sequence encoding a 13-residue linker designed to form an α-helical segment bounded by flexible Gly-Ser hinges and a repeat of the sequence encoding the natural ϵ residues from Ala-1 to those residues encoded within the AatII site itself. The N-terminal methionine of ϵ is normally removed (30Dunn S.D. J. Biol. Chem. 1982; 257: 7354-7359Abstract Full Text PDF PubMed Google Scholar). The repeat of the N-terminal sequence provided the residues of the first strand of the flattened β-barrel of the N-domain with the normal downstream context. The construct (Figs. 1A and 2A) can be viewed as encoding the mature ϵ polypeptide preceded by a leader that provided normal levels of translation initiation, sites for N-terminal fusions, and the flexible linker.FIGURE 2Schematic representation of the ϵ mutants created for this study. A, the general structure of all N-terminal ϵ fusions created. The fused proteins were inserted into the KpnI and BamHI restriction sites fusing the proteins between a 9-residue leader sequence and a 13-residue flexible linker. The two domains of ϵ are shown in dark gray (N-domain) and hatched (C-domain). B, the domain structure of all ϵ mutants created. This is a schematic representation showing the overall structure of the mutants and is not necessarily drawn to scale.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The ϵ mutants constructed for this study are illustrated in Fig. 2B. Either the 20-kDa E. coli flavodoxin or the 5-kDa CBD from the B. circulans chitinase A1 protein were inserted as N-terminal fusions. Truncations of ϵ after residue Gln-87 or Ser-107 were also constructed, removing the entire C-domain or just the C-terminal helix, respectively. N-terminal fusions were constructed for ϵ88-stop. These forms of ϵ were expressed from the unc operon in a derivative of plasmid pACWU1.2 (31Kuo P.H. Ketchum C.J. Nakamoto R.K. FEBS Lett. 1998; 426: 217-220Crossref PubMed Scopus (48) Google Scholar) in the unc-deficient strain DK8 (28Klionsky D.J. Brusilow W.S. Simoni R.D. J. Bacteriol. 1984; 160: 1055-1060Crossref PubMed Google Scholar).Growth Characteristics of N-terminal Fusions/C-terminal Truncations—To address the ability of these mutant ϵ subunits to provide normal ϵ function in vivo, transformants of DK8 (28Klionsky D.J. Brusilow W.S. Simoni R.D. J. Bacteriol. 1984; 160: 1055-1060Crossref PubMed Google Scholar) carrying plasmids expressing F1F0 were tested for the ability to grow on agar plates containing acetate as the sole carbon and energy source (Table 1). Growth on acetate tests for the function of ATP synthase because no substrate level phosphorylation is possible, and the cells rely solely on oxidative phosphorylation to produce ATP. Fusion of the relatively small CBD to the N terminus of ϵ had no effect on the essential function of ϵ, because cells bearing this mutation gave rise to colonies of the same size as the wild type. Cells bearing the C-terminal truncations of either normal ϵ or CBD-ϵ also grew. However, the larger flavodoxin-ϵ and flavodoxin-ϵ88-stop mutants did not grow on acetate indicating that the larger N-terminal fusion blocks the normal function of ATP synthase. We interpret this as the prevention of full 360° rotation because of steric restraints imposed on the rotor by the proximity of the b2 stator.TABLE 1Growth properties of ϵ-fusion strains All cells were grown at 37 °C using M9 minimal medium and the appropriate carbon source. Acetate growth tests were performed using agar plates containing 0.2% sodium acetate. Growth was assessed after incubation for 3 days. Growth yields were obtained by measuring (in duplicates) the A600 of stationary phase liquid cultures containing 0.04% glucose and expressed as a percentage of the wild-type.Plasmid/strainATP synthase typeGrowth on acetateGrowth yield% wild typepDC14/DK7Δϵ-37pDC14/DK8Δαβγδϵabc-54pDC44/DK8Wild-type+100pDC53/DK8Flavodoxin-ϵ-57pDC55/DK8Flavodoxin-ϵ88-stop-63pDC52/DK8CBD-ϵ+91pDC54/DK8CBD-ϵ88-stop+83pDC45/DK8ϵ88-stop+82pDC46/DK8ϵ108-stop+90 Open table in a new tab Western Blot Analysis of Expressed ϵ Mutants—Two potential concerns when conducting work of this nature is that the fusion protein may prevent the normal assembly of the F1F0 complex, or that the fusion protein could be undergoing cleavage. To address these concerns membrane vesicles were prepared and Western blots performed probing with antibodies raised against the α, ϵ, and b subunits (Fig. 3). There was a low but detectable level of α in membranes prepared from the Δϵ strain likely attributed to a small amount of F1 that was trapped in the pellet during the preparation. Membranes prepared from strains carrying the various ϵ deletions and fusions all had a relatively normal amount of α in the membranes indicating that the ϵ mutants do not interfere with the proper assembly of F1F0.FIGURE 3Effect of ϵ mutation on F1F0 assembly and ϵ stability. Samples of membrane protein (2 μg) were subjected to SDS-PAGE analysis followed by Western blotting to PVDF membranes. Blots were probed with 125I-labeled antibodies raised against α (A), b (B), and ϵ (C). Anti-ϵ blots failed to show ϵ88-stop unless 20 μg of protein were blotted and fixed to the membrane by soaking in transfer buffer with 0.5% glutaraldehyde before the blocking step.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The ϵ88-stop construct was washed off the PVDF membranes using our standard blotting procedure (32Dunn S.D. Anal. Biochem. 1986; 157: 144-153Crossref PubMed Scopus (418) Google Scholar). To correct for this the blot was fixed prior to the blocking step by soaking in transfer buffer containing 0.5% glutaraldehyde for 10 min. This allowed us to detect the ϵ88-stop construct, although it could not be quantified. With this limitation, there was no detectable degradation of the ϵ fusion proteins (even when blots were overexposed) indicating that the fusion proteins were stable (Fig. 3C).Analysis of ATPase Activity of Membrane-bound F1F0—To further analyze the effect of the N-terminal fusions and C-terminal truncations, the membranes described above were assayed for ATPase activity (Table 2). As noted previously with this expression system (20Cipriano D.J. Bi Y. Dunn S.D. J. Biol. Chem. 2002; 277: 16782-16790Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), there is significant variability in the amount of F1F0 present in different membrane preparations. This was true not only for preparations from cells with different plasmids but also for different preparations from the same plasmid/strain. For this reason, membranes were assayed for the ATPase activity of both membrane-bound F1F0 and for the activity of F1 after dissociation from F0 and ϵ, the latter providing a measure of the amount of ATP synthase present in the membranes. The membrane-bound ATPase activities were then normalized to the activity of soluble F1 released from these preparations, and expressed as the fraction of units bound per unit released, a unitless ratio that we call normalized activity. This treatment of the data gave reproducible results independent of the level of F1F0 in different preparations from the same strain. Previously we have shown that membrane-bound F1F0 typically shows a normalized activity of ∼0.4 (20Cipriano D.J. Bi Y. Dunn S.D. J. Biol. Chem. 2002; 277: 16782-16790Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Here the membrane-bound wild-type ATP synthase showed a normalized activity of 0.44 and was 85% inhibited by treatment with the F0-specific inhibitor, DCCD (Table 2). It should be noted that in all cases DCCD treatment showed less than 12% inhibition of the released activity (data not shown), indicating the specific labeling of F0 and not F1. The fusion of the 5-kDa CBD to the N terminus of ϵ had no effect on either the normalized hydrolysis activity (0.42), or the DCCD sensitivity (83%), of the membrane-bound enzyme implying that the N-terminal modification itself had no effect on expression of ϵ or assembly of the ATP synthase.TABLE 2ATPase activity of membrane vesicles Membranes were suspended to 2 mg/ml in 50 mm Tris-HCl, pH 8, 5 mm MgCl2, 300 mm KCl in the presence or absence of 50 μm DCCD, and incubated for 15 min at room temperature. Membranes were then diluted into ATPase assays to test for coupled activity as described under “Experimental Procedures.” Data shown are the average of triplicate assays ± S.D. 1 unit of activity is defined as 1 μmol of product formed per min.ATP synthase typeMembrane-bound ATPaseReleased ATPase activityaActivity values were corrected for background activity of 0.05 units/mg seen in the Δαβγδϵabc control strainNormalized activitybMembrane-bound activities were normalized to the activity released from membranes and expressed as the fraction of units bound per unit releasedActivityaActivity values were corrected for background activity of 0.05 units/mg seen in the Δαβγδϵabc control strainDCCD sensitivityunits/mg%units/mgΔαβγδϵabc0.00 ± 0.02N/AcN/A, not applicable0.00 ± 0.05N/AΔϵ0.03 ± 0.01N/A0.05 ± 0.01N/AWild-type ϵ0.80 ± 0.00851.83 ± 0.040.44 ± 0.01Flavodoxin-ϵ0.15 ± 0.01471.48 ± 0.000.10 ± 0.01Flavodoxin-ϵ88-stop0.41 ± 0.01541.95 ± 0.040.21 ±" @default.
• W2040563318 created "2016-06-24" @default.
• W2040563318 creator A5018513716 @default.
• W2040563318 creator A5024613818 @default.
• W2040563318 date "2006-01-01" @default.
• W2040563318 modified "2023-09-26" @default.
• W2040563318 title "The Role of the ϵ Subunit in the Escherichia coli ATP Synthase" @default.
• W2040563318 cites W145875931 @default.
• W2040563318 cites W1526178557 @default.
• W2040563318 cites W1579578057 @default.
• W2040563318 cites W1598184974 @default.
• W2040563318 cites W1665555949 @default.
• W2040563318 cites W1775749144 @default.
• W2040563318 cites W1825400051 @default.
• W2040563318 cites W1932376175 @default.
• W2040563318 cites W1964976203 @default.
• W2040563318 cites W1970662834 @default.
• W2040563318 cites W1970666814 @default.
• W2040563318 cites W1980634926 @default.
• W2040563318 cites W1981183182 @default.
• W2040563318 cites W1984859812 @default.
• W2040563318 cites W1999087143 @default.
• W2040563318 cites W2017757947 @default.
• W2040563318 cites W2020315495 @default.
• W2040563318 cites W2021970848 @default.
• W2040563318 cites W2029050174 @default.
• W2040563318 cites W2032550955 @default.
• W2040563318 cites W2037917982 @default.
• W2040563318 cites W2048400201 @default.
• W2040563318 cites W2058021348 @default.
• W2040563318 cites W2060938758 @default.
• W2040563318 cites W2067389562 @default.
• W2040563318 cites W2068257821 @default.
• W2040563318 cites W2068974050 @default.
• W2040563318 cites W2076613543 @default.
• W2040563318 cites W2078000997 @default.
• W2040563318 cites W2080779717 @default.
• W2040563318 cites W2080902097 @default.
• W2040563318 cites W2082687020 @default.
• W2040563318 cites W2095644929 @default.
• W2040563318 cites W2101174691 @default.
• W2040563318 cites W2127545076 @default.
• W2040563318 cites W2130451367 @default.
• W2040563318 cites W2131671284 @default.
• W2040563318 cites W2157227092 @default.
• W2040563318 cites W2161524167 @default.
• W2040563318 cites W2164999384 @default.
• W2040563318 cites W2172029110 @default.
• W2040563318 cites W2287549554 @default.
• W2040563318 doi "https://doi.org/10.1074/jbc.m509986200" @default.
• W2040563318 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16267041" @default.
• W2040563318 hasPublicationYear "2006" @default.
• W2040563318 type Work @default.
• W2040563318 sameAs 2040563318 @default.
• W2040563318 citedByCount "53" @default.
• W2040563318 countsByYear W20405633182012 @default.
• W2040563318 countsByYear W20405633182013 @default.
• W2040563318 countsByYear W20405633182014 @default.
• W2040563318 countsByYear W20405633182015 @default.
• W2040563318 countsByYear W20405633182016 @default.
• W2040563318 countsByYear W20405633182017 @default.
• W2040563318 countsByYear W20405633182018 @default.
• W2040563318 countsByYear W20405633182019 @default.
• W2040563318 countsByYear W20405633182020 @default.
• W2040563318 countsByYear W20405633182021 @default.
• W2040563318 countsByYear W20405633182022 @default.
• W2040563318 crossrefType "journal-article" @default.
• W2040563318 hasAuthorship W2040563318A5018513716 @default.
• W2040563318 hasAuthorship W2040563318A5024613818 @default.
• W2040563318 hasBestOaLocation W20405633181 @default.
• W2040563318 hasConcept C104292427 @default.
• W2040563318 hasConcept C104317684 @default.
• W2040563318 hasConcept C112243037 @default.
• W2040563318 hasConcept C119120687 @default.
• W2040563318 hasConcept C141315368 @default.
• W2040563318 hasConcept C153911025 @default.
• W2040563318 hasConcept C181199279 @default.
• W2040563318 hasConcept C185592680 @default.
• W2040563318 hasConcept C23265538 @default.
• W2040563318 hasConcept C547475151 @default.
• W2040563318 hasConcept C55493867 @default.
• W2040563318 hasConcept C86803240 @default.
• W2040563318 hasConceptScore W2040563318C104292427 @default.
• W2040563318 hasConceptScore W2040563318C104317684 @default.
• W2040563318 hasConceptScore W2040563318C112243037 @default.
• W2040563318 hasConceptScore W2040563318C119120687 @default.
• W2040563318 hasConceptScore W2040563318C141315368 @default.
• W2040563318 hasConceptScore W2040563318C153911025 @default.
• W2040563318 hasConceptScore W2040563318C181199279 @default.
• W2040563318 hasConceptScore W2040563318C185592680 @default.
• W2040563318 hasConceptScore W2040563318C23265538 @default.
• W2040563318 hasConceptScore W2040563318C547475151 @default.
• W2040563318 hasConceptScore W2040563318C55493867 @default.
• W2040563318 hasConceptScore W2040563318C86803240 @default.
• W2040563318 hasIssue "1" @default.
• W2040563318 hasLocation W20405633181 @default.
• W2040563318 hasOpenAccess W2040563318 @default.
• W2040563318 hasPrimaryLocation W20405633181 @default.

• next