变形链球菌F-ATPase启动子荧光蛋白载体的构建及表达

目的构建由质子移位膜ATP酶(membrane-bound proton-translocating ATPase,F-ATPase)启动子启动的绿色荧光蛋白报告基因穿梭表达载体,观察其在大肠埃希菌中的表达同时鉴定表达产物。方法以变形链球菌(UA159)基因组为模板,扩增F-ATPase启动子片段,构建由F-ATPase启动子启动的绿色荧光表达载体pFgfp,酶切F-ATPase启动子及绿色荧光蛋白编码基因,连接到穿梭质粒pDL276,构建重组载体pLFgfp。结果重组质粒pLFgfp酶切及基因序列分析证实目的片段成功插入,重组载体转化后的大肠埃希菌有绿色荧光蛋白的表达,并能随着细菌传代继续表达。结论 F-ATPase启动子启动的绿色荧光蛋白穿梭表达载体pLFgfp构建成功,为研究生物膜环境中耐酸菌F-ATPase毒力因子的表达奠定基础。     

Purification,characterization and crystallization of the F-ATPase from Paracoccus denitrificans

The structures of F-ATPases have been determined predominantly with mitochondrial enzymes, but hitherto no F-ATPase has been crystallized intact. A high-resolution model of the bovine enzyme built up from separate sub-structures determined by X-ray crystallography contains about 85% of the entire complex, but it lacks a crucial region that provides a transmembrane proton pathway involved in the generation of the rotary mechanism that drives the synthesis of ATP. Here the isolation, characterization and crystallization of an integral F-ATPase complex from the α-proteobacterium Paracoccus denitrificans are described. Unlike many eubacterial F-ATPases, which can both synthesize and hydrolyse ATP, the P. denitrificans enzyme can only carry out the synthetic reaction. The mechanism of inhibition of its ATP hydrolytic activity involves a ζ inhibitor protein, which binds to the catalytic F₁-domain of the enzyme. The complex that has been crystallized, and the crystals themselves, contain the nine core proteins of the complete F-ATPase complex plus the ζ inhibitor protein. The formation of crystals depends upon the presence of bound bacterial cardiolipin and phospholipid molecules; when they were removed, the complex failed to crystallize. The experiments open the way to an atomic structure of an F-ATPase complex.     

Stochastic proton pumping ATPases: from single molecules to diverse physiological roles

We discuss the most recent reports on two proton pumps, F-ATPase (ATP synthase) and V-ATPase (endomembrane proton pump). They are formed from similar extrinsic (F1 or V1) and intrinsic (Fo or Vo) membrane sectors, and couple chemistry and proton transport through subunit rotation for apparently different physiological roles. Emphasis is placed on the stochastic rotational catalysis of F-ATPase and isoforms of V-ATPase.     

ATP-synthesis in archaea: Structure-function relations of the halobacterial A-ATPase

The archaeal (A)-ATPase has been described as a chimeric energy converter with close relationship to both the vacuolar ATPase class in higher eukaryotes and the coupling factor (F)-ATPase class in eubacteria, mitochondria and chloroplasts. With respect to their structure and some inhibitor responses, A-ATPases are more closely related to the vacuolar ATPase type than to F-ATPase. Their function, ATP synthesis at the expense of an ion gradient, however, is a typical attribute of the F-ATPase class. V-type ATPases serve as generators of a proton gradient driving the accumulation of solutes within vesicles such as the vacuoles of plant cells. The three catalytic subunits (A) of the archaeal ATPases are the largest subunits of the A1-part and, like in V-ATPases, closer related to the F-ATPase -subunits, whereas B corresponds to F-ATPase . The catalytic subunits A of archaeal ATPases contain an insert of about 80 amino acids in their primary structures that may be aligned to comparable structures in V-ATPases. The location of this additional peptide in Haloferax volcanii is shown using the 2.8 Å X-ray resolution of the bovine mitochondrial F-ATPase [Abrahams et al. (1994) Nature 370: 621-628]. A three dimensional structure for the catalytic subunit of Haloferax volcanii ATPase is proposed using the Swiss-Model Automated Protein Modelling Server. The halobacterial ATPase is a halophilic protein; it contains about 20% negatively charged amino acid residues. A large portion of acidic residues is located on the outer surface of the protein as well as in the insert of subunit A. This result is discussed in terms of protein stability under high salt stress conditions.     

A Journey from Mammals to Yeast with Vacuolar H+-ATPase (V-ATPase)

The vacuolar H⁺-ATPase (V-ATPase) is one of the most fundamental enzymes in nature. It functions in almost every eukaryotic cell and energizes a wide variety of organelles and membranes. V-ATPase has a structure and mechanism of action similar to F-ATPase and several of their subunits probably evolved from common ancestors. In eukaryotic cells, F-ATPase is confined to the semiautonomous organelles, chloroplasts and mitochondria, which contain their own genes that encode some of the F-ATPase subunits. In contrast to F-ATPases, whose primary function in eukaryotic cells is to form ATP at the expense of the protonmotive force (pmf), V-ATPases function exclusively as ATP-dependent proton pumps. The pmf generated by V-ATPases in organelles and membranes of eukaryotic cells is utilized as a driving force for numerous secondary transport processes. It was the survival of the yeast mutant without the active enzyme and yeast genetics that allowed the identification of genuine subunits of the V-ATPase. It also revealed special properties of individual subunits, factors that are involved in the enzyme's biogenesis and assembly, as well as the involvement of V-ATPase in the secretory pathway, endocytosis, and respiration. It may be the insect V-ATPase that unconventionally resides in the plasma membrane of their midgut, that will give the first structure resolution of this complex.     

F-ATPase of Drosophila melanogaster Forms 53-Picosiemen (53-pS) Channels Responsible for Mitochondrial Ca2+-induced Ca2+ Release

Mitochondria of Drosophila melanogaster undergo Ca²⁺-induced Ca²⁺ release through a putative channel (mCrC) that has several regulatory features of the permeability transition pore (PTP). The PTP is an inner membrane channel that forms from F-ATPase, possessing a conductance of 500 picosiemens (pS) in mammals and of 300 pS in yeast. In contrast to the PTP, the mCrC of Drosophila is not permeable to sucrose and appears to be selective for Ca²⁺ and H⁺. We show (i) that like the PTP, the mCrC is affected by the sense of rotation of F-ATPase, by Bz-423, and by Mg²⁺/ADP; (ii) that expression of human cyclophilin D in mitochondria of Drosophila S₂R⁺ cells sensitizes the mCrC to Ca²⁺ but does not increase its apparent size; and (iii) that purified dimers of D. melanogaster F-ATPase reconstituted into lipid bilayers form 53-pS channels activated by Ca²⁺ and thiol oxidants and inhibited by Mg²⁺/γ-imino ATP. These findings indicate that the mCrC is the PTP of D. melanogaster and that the signature conductance of F-ATPase channels depends on unique structural features that may underscore specific roles in different species.     

Subunit rotation of vacuolar-type proton pumping ATPase: relative rotation of the G and C subunits

Vacuolar-type ATPases V1V0 (V-ATPases) are found ubiquitously in the endomembrane organelles of eukaryotic cells. In this study, we genetically introduced a His tag and a biotin tag onto the c and G subunits, respectively, of Saccharomyces cerevisiae V-ATPase. Using this engineered enzyme, we observed directly the continuous counter-clockwise rotation of an actin filament attached to the G subunit when the enzyme was immobilized on a glass surface through the c subunit. V-ATPase generated essentially the same torque as the F-ATPase (ATP synthase). The rotation was inhibited by concanamycin and nitrate but not by azide. These results demonstrated that the V- and F-ATPase carry out a common rotational catalysis.     

Crystal structure of yeast V-ATPase subunit C reveals its stator function

Vacuolar H(+)-ATPase (V-ATPase) has a crucial role in the vacuolar system of eukaryotic cells. It provides most of the energy required for transport systems that utilize the proton-motive force that is generated by ATP hydrolysis. Some, but not all, of the V-ATPase subunits are homologous to those of F-ATPase and the nonhomologous subunits determine the unique features of V-ATPase. We determined the crystal structure of V-ATPase subunit C (Vma5p), which does not show any homology with F-ATPase subunits, at 1.75 A resolution. The structural features suggest that subunit C functions as a flexible stator that holds together the catalytic and membrane sectors of the enzyme. A second crystal form that was solved at 2.9 A resolution supports the flexible nature of subunit C. These structures provide a framework for exploring the unique mechanistic features of V-ATPases.     

Structural investigations of the membrane-embedded rotor ring of the F-ATPase from Clostridium paradoxum

The Na(+)-translocating F-ATPase of the thermoalkaliphilic bacterium Clostridium paradoxum harbors an oligomeric ring of c subunits that resists dissociation by sodium dodecyl sulfate. The c ring has been isolated and crystallized in two dimensions. From electron microscopy of these c-ring crystals, a projection map was calculated to 7 A resolution. In the projection map, each c ring consists of two concentric, slightly staggered, packed rings, each composed of 11 densities representing the alpha-helices. On the basis of these results, it was determined that the F-ATPase from C. paradoxum contains an undecameric c ring.     

Expression, purification and secondary structure analysis of Saccharomyces cerevisiae vacuolar membrane H+-ATPase subunit F (Vma7p).

The vacuolar H+-ATPase is an acid pump found in virtually all eukaryotic cells. It shares a common macromolecular organization with the F1F0-ATPase, and some V-ATPase subunits are structural and functional homologues of F-ATPase components. However, the vacuolar complex contains several subunits which do not resemble F-ATPase subunits at the sequence level, and which currently have no specific function assigned. One example is subunit F, the Vma7p polypeptide of Saccharomyces cerevisiae. A recombinant form of Vma7p was expressed in Escherichia coli and purified to homogeneity. Mass spectroscopy confirmed a mass of 13460 Da for Vma7p, and dynamic light scattering showed that the polypeptide was globular and monodisperse even at high concentrations. Analysis of secondary structure by circular dichroism and FTIR showed that Vma7p comprises 30% alpha-helix and 32-42% beta-sheet. The protein fold recognition programme 'Threader 2' produced highly significant matches between Vma7p and five alpha-beta sandwich folds. Relative proportions of secondary structure elements within these folds were broadly consistent with the spectroscopic data. Although Vma7p does not share sequence similarity with the F-ATPase epsilon subunit, the analysis suggests that the polypeptides not only have similar masses and assemble into homologous core complexes, but also share similar secondary structures. It is possible that the two polypeptides are homologous and perform similar functions within their respective ATPases. The production of high yields of homogeneous, folded, monodisperse protein will facilitate high resolution crystallography and NMR spectroscopy studies.     

Expression,purification and secondary structure analysis of Saccharomyces cerevisiae vacuolar membrane H + -ATPase subunit F (Vma7p)

The vacuolar H + -ATPase is an acid pump found in virtually all eukaryotic cells. It shares a common macromolecular organization with the F 1 F 0 -ATPase, and some V-ATPase subunits are structural and functional homologues of F-ATPase components. However, the vacuolar complex contains several subunits which do not resemble F-ATPase subunits at the sequence level, and which currently have no specific function assigned. One example is subunit F, the Vma7p polypeptide of Saccharomyces cerevisiae. A recombinant form of Vma7p was expressed in Escherichia coli and purified to homogeneity. Mass spectroscopy confirmed a mass of 13 460 Da for Vma7p, and dynamic light scattering showed that the polypeptide was globular and monodisperse even at high concentrations. Analysis of secondary structure by circular dichroism and FTIR showed that Vma7p comprises 30% &#102 -helix and 32-42% &#103 -sheet. The protein fold recognition programme 'Threader 2' produced highly significant matches between Vma7p and five &#102 - &#103 sandwich folds. Relative proportions of secondary structure elements within these folds were broadly consistent with the spectroscopic data. Although Vma7p does not share sequence similarity with the F-ATPase epsilon subunit, the analysis suggests that the polypeptides not only have similar masses and assemble into homologous core complexes, but also share similar secondary structures. It is possible that the two polypeptides are homologous and perform similar functions within their respective ATPases. The production of high yields of homogeneous, folded, monodisperse protein will facilitate high resolution crystallography and NMR spectroscopy studies.     

Rotational catalysis in proton pumping ATPases: From E. coli F-ATPase to mammalian V-ATPase

We focus on the rotational catalysis of Escherichia coli F-ATPase (ATP synthase, F(O)F(1)). Using a probe with low viscous drag, we found stochastic fluctuation of the rotation rates, a flat energy pathway, and contribution of an inhibited state to the overall behavior of the enzyme. Mutational analyses revealed the importance of the interactions among β and γ subunits and the β subunit catalytic domain. We also discuss the V-ATPase, which has different physiological roles from the F-ATPase, but is structurally and mechanistically similar. We review the rotation, diversity of subunits, and the regulatory mechanism of reversible subunit dissociation/assembly of Saccharomyces cerevisiae and mammalian complexes. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Enhanced acid resistance of oral streptococci at lethal pH values associated with acid-tolerant catabolism and with ATP synthase activity

Caries-causing oral bacteria such as Streptococcus mutans are protected by the actions of F-ATPases against acid damage in dental plaque acidified by glycolytic acid production or ingestion of acids foods and beverages. Catabolites such as glucose and sucrose were found to enhance the protection of S. mutans and also other oral lactic-acid bacteria against acid killing at lethal pH values as low as 2.5. Protection involved glycolysis with the production of lactate and ATP, which is a substrate for F-ATPases. ATP could also be produced by starved cells apparently through synthase activity of the F-ATPase associated with acid decline. Fluoride and the organic weak-acid indomethacin acted to diminish this protection, as did F-ATPase inhibitors such as dicyclohexylcarbodi-imide. Protection against acid killing involving catabolism and synthase activity is likely to be important for plaque cariogenicity.     

Subunit a of the yeast V-ATPase participates in binding of bafilomycin

Bafilomycin and concanamycin are potent and highly specific inhibitors of the vacuolar (H(+))-ATPases (V-ATPases), typically inhibiting at nanomolar concentrations. Previous studies have shown that subunit c of the integral V(0) domain participates in bafilomycin binding, and that this site resembles the oligomycin binding site of the F-ATPase (Bowman, B. J., and Bowman, E. J. (2002) J. Biol. Chem. 277, 3965-3972). Because mutations in F-ATPase subunit a also confer resistance to oligomycin, we investigated whether the a subunit of the V-ATPase might participate in binding bafilomycin. Twenty-eight subunit a mutations were constructed just N-terminal to the critical Arg(735) residue in transmembrane 7 required for proton transport, a region similar to that shown to participate in oligomycin binding by the F-ATPase. The mutants appeared to assemble normally and all but two showed normal growth at pH 7.5, whereas all but three had at least 25% of wild-type levels of proton transport and ATPase activity. Of the functional mutants, three displayed K(i) values for bafilomycin significantly different from wild-type (0.22 +/- 0.03 nm). These included E721K (K(i) 0.38 +/- 0.03 nm), L724A (0.40 +/- 0.02 nm), and N725F (0.54 +/- 0.06 nm). Only the N725F mutation displayed a K(i) for concanamycin (0.84 +/- 0.04 nm) that was slightly higher than wild-type (0.60 +/- 0.07 nm). These results suggest that subunit a of V-ATPase participates along with subunit c in binding bafilomycin.     

Acid tolerance of Streptococcus macedonicus as assessed by flow cytometry and single-cell sorting

An in situ flow cytometric viability assay employing carboxyfluorescein diacetate and propidium iodide was used to identify Streptococcus macedonicus acid tolerance phenotypes. The logarithmic-phase acid tolerance response (L-ATR) was evident when cells were (i) left to autoacidify unbuffered medium, (ii) transiently exposed to nonlethal acidic pH, or (iii) systematically grown under suboptimal acidic conditions (acid habituation). Stationary-phase ATR was also detected; this phenotype was gradually degenerated while cells resided at this phase. Single-cell analysis of S. macedonicus during induction of L-ATR revealed heterogeneity in both the ability and the rate of tolerance acquisition within clonal populations. L-ATR was found to be partially dependent on de novo protein synthesis and compositional changes of the cell envelope. Interestingly, acid-habituated cells were interlaced in lengthier chains and exhibited an irregular pattern of active peptidoglycan biosynthesis sites when probed with BODIPY FL vancomycin. L-ATR caused cells to retain their membrane potential after lethal challenge, as judged by ratiometric analysis with oxonol [DiBAC(4)(3)]. Furthermore, F-ATPase was important during the induction of L-ATR, but in the case of a fully launched response, inhibition of F-ATPase affected acid resistance only partially. Activities of both F-ATPase and the glucose-specific phosphoenolpyruvate-dependent phosphotransferase system were increased after L-ATR induction, distinguishing S. macedonicus from oral streptococci. Finally, the in situ viability assessment was compared to medium-based recovery after single-cell sorting, revealing that the culturability of subpopulations with identical fluorescence characteristics is dependent on the treatments imposed to the cells prior to acid challenge.     

Acid Tolerance of Streptococcus macedonicus as Assessed by Flow Cytometry and Single-Cell Sorting

An in situ flow cytometric viability assay employing carboxyfluorescein diacetate and propidium iodide was used to identify Streptococcus macedonicus acid tolerance phenotypes. The logarithmic-phase acid tolerance response (L-ATR) was evident when cells were (i) left to autoacidify unbuffered medium, (ii) transiently exposed to nonlethal acidic pH, or (iii) systematically grown under suboptimal acidic conditions (acid habituation). Stationary-phase ATR was also detected; this phenotype was gradually degenerated while cells resided at this phase. Single-cell analysis of S. macedonicus during induction of L-ATR revealed heterogeneity in both the ability and the rate of tolerance acquisition within clonal populations. L-ATR was found to be partially dependent on de novo protein synthesis and compositional changes of the cell envelope. Interestingly, acid-habituated cells were interlaced in lengthier chains and exhibited an irregular pattern of active peptidoglycan biosynthesis sites when probed with BODIPY FL vancomycin. L-ATR caused cells to retain their membrane potential after lethal challenge, as judged by ratiometric analysis with oxonol [DiBAC₄(3)]. Furthermore, F-ATPase was important during the induction of L-ATR, but in the case of a fully launched response, inhibition of F-ATPase affected acid resistance only partially. Activities of both F-ATPase and the glucose-specific phosphoenolpyruvate-dependent phosphotransferase system were increased after L-ATR induction, distinguishing S. macedonicus from oral streptococci. Finally, the in situ viability assessment was compared to medium-based recovery after single-cell sorting, revealing that the culturability of subpopulations with identical fluorescence characteristics is dependent on the treatments imposed to the cells prior to acid challenge.     

Genetic and biochemical characterization of the F-ATPase operon from Streptococcus sanguis 10904

Oral streptococci utilize an F-ATPase to regulate cytoplasmic pH. Previous studies have shown that this enzyme is a principal determinant of aciduricity in the oral streptococcal species Streptococcus sanguis and Streptococcus mutans. Differences in the pH optima of the respective ATPases appears to be the main reason that S. mutans is more tolerant of low pH values than S. sanguis and hence pathogenic. We have recently reported the genetic arrangement for the S. mutans operon. For purposes of comparative structural biology we have also investigated the F-ATPase from S. sanguis. Here, we report the genetic characterization and expression in Escherichia coli of the S. sanguis ATPase operon. Sequence analysis showed a gene order of atpEBFHAGDC and that a large intergenic space existed upstream of the structural genes. Activity data demonstrate that ATPase activity is induced under acidic conditions in both S. sanguis and S. mutans; however, it is not induced to the same extent in the nonpathogenic S. sanguis. Expression studies with an atpD deletion strain of E. coli showed that S. sanguis-E. coli hybrid enzymes were able to degrade ATP but were not sufficiently functional to permit growth on succinate minimal media. Hybrid enzymes were found to be relatively insensitive to inhibition by dicyclohexylcarbodiimide, indicating loss of productive coupling between the membrane and catalytic subunits.     

The complex architecture of oxygenic photosynthesis

Oxygenic photosynthesis is the principal producer of both oxygen and organic matter on earth. The primary step in this process - the conversion of sunlight into chemical energy - is driven by four, multisubunit, membrane-protein complexes that are known as photosystem I, photosystem II, cytochrome b(6)f and F-ATPase. Structural insights into these complexes are now providing a framework for the exploration not only of energy and electron transfer, but also of the evolutionary forces that shaped the photosynthetic apparatus.     

On the structure of the stator of the mitochondrial ATP synthase

The structure of most of the peripheral stalk, or stator, of the F-ATPase from bovine mitochondria, determined at 2.8 A resolution, contains residues 79-183, 3-123 and 5-70 of subunits b, d and F6, respectively. It consists of a continuous curved alpha-helix about 160 A long in the single b-subunit, augmented by the predominantly alpha-helical d- and F6-subunits. The structure occupies most of the peripheral stalk in a low-resolution structure of the F-ATPase. The long helix in subunit b extends from near to the top of the F1 domain to the surface of the membrane domain, and it probably continues unbroken across the membrane. Its uppermost region interacts with the oligomycin sensitivity conferral protein, bound to the N-terminal region of one alpha-subunit in the F1 domain. Various features suggest that the peripheral stalk is probably rigid rather than resembling a flexible rope. It remains unclear whether the transient storage of energy required by the rotary mechanism takes place in the central stalk or in the peripheral stalk or in both domains.     

Discovery and Study of Transmembrane Rotary Ion-Translocating Nano-Motors: F-ATPase/Synthase of Mitochondria/Bacteria and V-ATPase of Eukaryotic Cells

Biochemistry (Moscow) - This review discusses the history of discovery and study of the operation of the two rotary ion-translocating ATPase nano-motors: (i) F-ATPase/synthase (holocomplex F1FO) of...