Mitochondrial F0F1 H+-ATP synthase. Characterization of F0 components involved in H+ translocation

The membrane F0 sector of mitochondrial ATP synthase complex was rapidly isolated by direct extraction with CHAPS from F1-depleted submitochondrial particles. The preparation thus obtained is stable and can be reconstituted in artificial phospholipid membranes to result in oligomycin-sensitive proton conduction, or recombined with purified F1 to give the oligomycin-sensitive F0F1-ATPase complex. The F0 preparation and constituent polypeptides were characterized by SDS-polyacrylamide gel electrophoresis and immunoblot analysis. The functional role of F0 polypeptides was examined by means of trypsin digestion and reconstitution studies. It is shown that, in addition to the 8 kDa DCCD-binding protein, the nuclear encoded protein [(1987) J. Mol. Biol. 197, 89-100], characterized as an intrinsic component of F0 (F0I, PVP protein [(1988) FEBS Lett. 237,9-14]) [corrected] is involved in H+ translocation and the sensitivity of this process to the F0 inhibitors, DCCD and oligomycin.     

F Pilus as f+ Antigen

Specific aggregate formation of F pili was observed, by electron microscopy, in a mixture of male Escherichia coli (or of isolated F pili) and anti-f(+) serum. Cellular appendages other than F pili never showed such aggregation when mixed with anti-f(+) serum. The f(+) agglutinability of male cells, as well as F piliation, was sensitive to mechanical agitation. The f(+) agglutination was inhibited when appropriate numbers of phage M12, capable of attaching to F pili, were mixed with the male culture before the addition of anti-f(+) serum. Correlation between f(+) agglutinability and the extent of F piliation was observed. It was concluded that the F pilus is the structure of the f(+) antigen and is responsible for f(+) agglutination.     

F?/Cl? selectivity in CLCF-type F?/H+ antiporters

Many bacterial species protect themselves against environmental F⁻ toxicity by exporting this anion from the cytoplasm via CLC^F F⁻/H⁺ antiporters, a subclass of CLC superfamily anion transporters. Strong F⁻ over Cl⁻ selectivity is biologically essential for these membrane proteins because Cl⁻ is orders of magnitude more abundant in the biosphere than F⁻. Sequence comparisons reveal differences between CLC^Fs and canonical Cl⁻-transporting CLCs within regions that, in the canonical CLCs, coordinate Cl⁻ ion and govern anion transport. A phylogenetic split within the CLC^F clade, manifested in sequence divergence in the vicinity of this ion-binding center, raises the possibility that these two CLC^F subclades might exhibit differences in anion selectivity. Several CLC^F homologues from each subclade were examined for F⁻/Cl⁻ selectivity of anion transport and equilibrium binding. Differences in both of these anion-selectivity metrics correlate with sequence divergence among CLC^Fs. Chimeric constructs identify two residues in this region that largely account for the subclade differences in selectivity. In addition, these experiments serendipitously uncovered an unusually steep, Cl⁻-specific voltage dependence of transport that greatly enhances F⁻ selectivity at low voltage.     

Investigation of DNA complexes with histones F3 and F3+F2a2]

Characteristic viscosity, sedimentation constant and optical anisotropy were studied of the complexes formed between DNA and histone fractions F3 and F3+F2a2. The parameters mentioned continuously change with the increase of protein content within the complex. Analysis of experimental data shows that binding of a histone bads to a decrease of size and thermodynamic rigidity of the DNA molecule. On the basis of results obtained a model of F3 histone binding with DNA is suggested, amino acid sequence of this protein being taken into account. Comparison of behaviour of nucleohistones DNA+F3 and DNA+F1 studied previously testifies different way of binding of these histones to DNA.     

Electron cryomicroscopy reveals different F1+F2 protein States in intact parainfluenza virions

Electron cryomicrographs of intact parainfluenza virus 5 (PIV5) virions revealed two different surface structures, namely, a continuous layer and distinct individual spikes. The structure of these spikes reconstructed from intact virions was compared with known F ectodomain structures and was found to be different from the prefusion PIV5 F0 structure but, surprisingly, very similar to the human PIV3 F postfusion structure. Hence, we conclude that the individual F1+F2 spikes in intact PIV5 virions also correspond to the postfusion state. Since the observed fusion activity of PIV5 virions has to be associated with prefusion F1+F2 proteins, they have necessarily to be localized in the continuous surface structure. The data therefore strongly suggest that the prefusion state of the F1+F2 protein requires stabilization, most probably by the association with hemagglutinin-neuraminidase. The conversion of F1+F2 proteins from the prefusion toward the postfusion state while embedded in the virus membrane is topologically difficult to comprehend on the basis of established models and demands reconsideration of our current understanding.     

Membrane embedded location of Na+ or H+ binding sites on the rotor ring of F1F0 ATP synthases.

Recent crosslinking studies indicated the localization of the coupling ion binding site in the Na+-translocating F1F0 ATP synthase of Ilyobacter tartaricus within the hydrophobic part of the bilayer. Similarly, a membrane embedded H+-binding site is accepted for the H+-translocating F1F0 ATP synthase of Escherichia coli. For a more definite analysis, we performed parallax analysis of fluorescence quenching with ATP synthases from both I. tartaricus and E. coli. Both ATP synthases were specifically labelled at their c subunit sites with N-cyclohexyl-N'-(1-pyrenyl)carbodiimide, a fluorescent analogue of dicyclohexylcarbodiimide and the enzymes were reconstituted into proteoliposomes. Using either soluble quenchers or spinlabelled phospholipids, we observed a deeply membrane embedded binding site, which was quantitatively determined for I. tartaricus and E. coli to be 1.3 +/- 2.4 A and 1.8 +/- 2.8 A from the bilayer center apart, respectively. These data show a conserved topology among enzymes of different species. We further demonstrated the direct accessibility for Na+ ions to the binding sites in the reconstituted I. tartaricus c11 oligomer in the absence of any other subunits, pointing to intrinsic rotor channels. The common membrane embedded location of the binding site of ATP synthases suggest a common mechanism for ion transfer across the membrane.     

Postpartum subestrus in dairy cows: comparison of treatment with prostaglandin F2 alpha or GnRH + prostaglandin F2 alpha + GnRH

Two experiments (Experiment 1, 185 cows in 1996/97; Experiment 2, 168 cows in 1997/98) were conducted with Prim Holstein dairy cattle in the Mayenne region of France to investigate subestrus. Cows which had not been observed in estrus since calving were allocated alternately to treatment groups between 60 and 90 d post partum as follows: Experiment 1-Group 1: GnRH (Day 0, 100 micrograms i.m.), PGF2 alpha (Day 7, 25 mg i.m.), GnRH (Day 9, 100 micrograms i.m.) and AI (Day 10); Group 2: PGF2 alpha (Day 0, 25 mg i.m.), AI at estrus, or, if estrus was not observed, a second PGF2 alpha injection on Day 13, and AI on Day 16 and Day 17. Treatments in Experiment 2 were as follows: Group 1: as Experiment 1-Group 1 but AI at the observed estrus after Day 0, or at Day 10 if estrus was not observed; Group 2: as Experiment 1--Group 2, however, if a second PGF2 alpha injection was given on Day 13, AI at the observed estrus. Progesterone was measured in serum at Day 0 and in milk at AI. Pregnancy diagnosis was performed by measuring bovine pregnancy-specific protein B (bPSPB; Day 50 +/- 3) and confirmed by ultrasonography when the result was doubtful. In Experiment 1, farmers observed 47/101 (46.9%) Group 1 cows in estrus, 33/91 cows on Day 10 and 10 cows before Day 10. The progesterone concentrations were compatible with estrus in 69/86 (80%) cows on Day 10. In Group 2, 36/83 (43.4%) cows were inseminated after the first PGF2 alpha injection. After the second PGF2 alpha injection, only 29/43 (67%) cows had a low progesterone concentration at AI. Pregnancy rates were 36.1 and 32.5% for Groups 1 and 2, respectively. In Experiment 2, estrus was observed in 31/93 (33.7%) Group 1 cows. In Group 2, 51/75 (66%) cows were inseminated after the first injection of PGF2 alpha, 13/75 (17.3%) cows after the second injection, while 11/75 (14.7%) were not observed in estrus. Pregnancy rates were 53.7 and 53.3% in Groups 1 and 2, respectively. In conclusion, it is recommended that subestrus be treated with PGF2 alpha followed by AI at the observed estrus when estrus detection is good, while the use of GnRH + PGF2 alpha + GnRH is recommended when estrus detection is poor.     

Mg2+ -dependent inactivation / H+ -dependent activation equilibrium of chloroplast F1-ATPase

The catalytic part of chloroplast thylakoid ATPase, the chloroplast coupling factor CF1, is reversibly inactivated during incubation in the presence of Mg2+. The inactivation has two phases. Its fast phase occurs at basic pH of the incubation medium (k = 6 min-1), while the slow phase ( k = 0.1-0.2 min-1) depends on pH only slightly throughout the studied range (5.5-9.0). As followed from changes in the inactivation effect of magnesium ions, Mg2+ affinity for the enzyme decreases dramatically with decreasing medium pH. The pH-dependence of Mg2+ dissociation apparent constant suggests that the binding/dissociation equilibrium is determined by protonation/deprotonation of specific acid-base groups of the enzyme. The analysis of pH-dependence plots gives the equilibrium constant of magnesium dissociation (3-9 M) and the dissociation constant of the protonated groups pK 5.8-6.7). Sodium azide is known to stabilize the inactive CF1-MgADP complex; when added to the incubation medium it diminishes the Mg2+ dissociation constant and has no effect on the dissociation constant of the acid-base groups. At lower pH, Mg2+-inactivated CF1-ATPase reactivates. Octyl glucoside accelerates the reactivation, while Triton-100 affects it only slightly. The reactivation rate of membrane-bound CF1 (thylakoid ATPase) inactivated by preincubation with Mg2+ in the presence of gramicidin is a few times higher than that of isolated CF1. These results suggest that the reactivation of isolated and membrane-bound CF1-ATPase is determined by protonation of a limited number of acid-base groups buried in the enzyme molecule.     

Effect of prostaglandin F2 alpha on Na+-K+-ATPase activity in luteal membranes

Slices of rat corpora lutea (CL) incubated with prostaglandin F2 alpha (PGF2 alpha) in Krebs-Hensenleit (K-H) Ringer solution showed a decrease in Na+-K+-ATPase activity after 60 min of incubation. However, PGF2 alpha in vitro did not alter Na+-K+-ATPase activity of isolated luteal membrane fractions. Following PGF2 alpha-induced in vivo luteal regression, reduction of Vmax and elevation of the activation energy above transition temperature of the lipid phase of the membrane occurred without changes in Km, optimum pH and transition temperature. These results suggest that reduction of Na+-K+-ATPase activity after PGF2 alpha treatment may be due to reduction in the number of enzyme molecules or to masking of the active site of the enzyme without any change in enzyme characteristics. In addition, a change in membrane-bound enzyme activity may be an early step in PGF2 alpha-induced luteolysis.     

Role of the carboxyl-terminal region of the PVP protein (F0I subunit) in the H+ conduction of F0F1 H+-ATP synthase of bovine heart mitochondria

By means of protein sequencing, labelling with thiol reagents and reconstitution studies it is shown that the carboxyl-terminal region of the PVP protein (F0I subunit, nuclear-encoded protein of Mr 25,000) of mitochondrial F0 promotes transmembrane proton conduction by F0 and the sensitivity of this process to oligomycin.     

The H+-translocating ATP synthase in Halobacterium halobium differs from F0F1-ATPase/synthase

Cell envelope vesicles of Halobacterium halobium synthesize ATP by utilizing base-acid transition (an outside acidic pH jump) under optimal conditions (1 M NaCl, 80 mM MgCl2, pH 6.8) even in the presence of azide (a specific inhibitor of F0F1-ATPase) (Mukohata & Yoshida (1987) J. Biochem. 101, 311-318). An azide-insensitive ATPase was isolated from the inner face of the vesicle membrane, and shown to hydrolyze ATP under very specific conditions (1.5 M Na2SO4, 10 mM MnCl2, pH 5.8) (Nanba & Mukohata (1987) J. Biochem. 102, 591-598). This ATPase activity could also be detected when the vesicle components were solubilized by detergent. The relationship between ATP synthesis and the membrane-bound ATPase was investigated by modification of the vesicles with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) or N-ethylmaleimide (NEM). The inhibition pattern of ATP synthesis in the modified vesicles and that of ATP hydrolysis of the solubilized modified vesicles were compared under the individual optimum conditions. The inhibition patterns were almost identical, suggesting that the ATP synthesis and hydrolysis are catalyzed by a single enzyme complex. The ATP synthase includes the above ATPase (300-320 kDa), which is composed of two pairs of 86 and 64 kDa subunits. This is a novel H+-translocating ATP synthase functioning in the extremely halophilic archaebacterium. This "archae-ATP-synthase" differs from F0F1-ATPase/synthase, which had been thought to be ubiquitous among all respiring organisms on our biosphere.     

Intersubunit bridging by Na+ ions as a rationale for the unusual stability of the c-rings of Na+-translocating F1F0 ATP synthases

The oligomeric c-rings of Na⁺-translocating F₁F₀ ATP synthases exhibit unusual stability, resisting even boiling in SDS. Here, we show that the molecular basis for this remarkable property is intersubunit crossbridging by Na⁺ or Li⁺ ions. The heat stability of c₁₁ was dependent on the presence of Na⁺ or Li⁺ ions. For equal stability, 10 times higher Li⁺ than Na⁺ concentrations were required, reflecting the 10 times lower binding affinity for Li⁺ than for Na⁺. In a recent structural model of c₁₁, the Na⁺ or Li⁺ binding ligands are located on neighboring c-subunits, which thus become crossbridged by the binding of either alkali ion with a concomitant increase in the stability of the ring. Site-directed mutagenesis strengthens the essential role of glutamate 65 in the crossbridging of the subunits and also corroborates the proposed stabilizing effect of an ion bridge including aspartate 2.     

Membrane topography of the coupling ion binding site in Na+-translocating F1F0 ATP synthase.

A carbodiimide with a photoactivatable diazirine substituent was synthesized and incubated with the Na(+)-translocating F(1)F(0) ATP synthase from both Propionigenium modestum and Ilyobacter tartaricus. This caused severe inhibition of ATP hydrolysis activity in the absence of Na(+) ions but not in its presence, indicating the specific reaction with the Na(+) binding c-Glu(65) residue. Photocross-linking was investigated with the substituted ATP synthase from both bacteria in reconstituted 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC)-containing proteoliposomes. A subunit c/POPC conjugate was found in the illuminated samples but no a-c cross-links were observed, not even after ATP-induced rotation of the c-ring. Our substituted diazirine moiety on c-Glu(65) was therefore in close contact with phospholipid but does not contact subunit a. Na(+)in/(22)Na(+)out exchange activity of the ATP synthase was not affected by modifying the c-Glu(65) sites with the carbodiimide, but upon photoinduced cross-linking, this activity was abolished. Cross-linking the rotor to lipids apparently arrested rotational mobility required for moving Na(+) ions back and forth across the membrane. The site of cross-linking was analyzed by digestions of the substituted POPC using phospholipases C and A(2) and by mass spectroscopy. The substitutions were found exclusively at the fatty acid side chains, which indicates that c-Glu(65) is located within the core of the membrane.     

Quinine specifically inhibits the proteolipid subunit of the F0F1 H+ -ATPase of Streptococcus pneumoniae.

Streptococcus pneumoniae is uniquely sensitive to quinine and its derivatives, but only those alkaloids having antimalarial properties, i.e., those in the erythro configuration, also possess antipneumococcal activity. Quinine and related compounds inhibit the pneumococcal H+ -ATPase. Quinine- and optochin-resistant pneumococci showed mutations that change amino acid residues located in one of the two transmembrane alpha-helices of the c subunit of the F0F1, H+ -ATPase.     

F+ RNA coliphage typing for microbial source tracking in surface waters

AIMS: The utility of coliphages to detect and track faecal pollution was evaluated using South Carolina surface waters that exceeded State faecal coliform standards. METHODS AND RESULTS: Coliphages were isolated from 117 surface water samples by single agar layer (SAL) and enrichment presence/absence (EP/A) methods. Confirmed F+ RNA coliphages were typed for microbial source tracking using a library-independent approach. Concentrations of somatic coliphages using 37 and 44.5 degrees C incubation temperatures were found to be significantly different and the higher temperature may be more specific for faecal contamination. The EP/A technique detected coliphages infecting Escherichia coli Famp in 38 (66%) of the 58 surface water samples negative for F+ coliphages by the SAL method. However, coliphages isolated by EP/A were found to be less representative of coliphage diversity within a sample. Among the 2939 coliphage isolates tested from surface water and known source samples, 813 (28%) were found to be F+ RNA. The majority (94%) of surface water F+ RNA coliphage isolates typed as group I. Group II and/or III viruses were identified from 14 surface water stations, the majority of which were downstream of wastewater discharges. These sites were likely contaminated by human-source faecal pollution. CONCLUSIONS: The results suggest that faecal contamination in surface waters can be detected and source identifications aided by coliphage analyses. SIGNIFICANCE AND IMPACT OF THE STUDY: This study supports the premise that coliphage typing can provide useful, but not absolute, information to distinguish human from animal sources of faecal pollution. Furthermore, the comparison of coliphage isolation methods detailed in this study should provide valuable information to those wishing to incorporate coliphage detection into water quality assessments.     

pH dependence of H+ conduction through the membrane moiety of the H+-ATPase (F0 . F1) and effects of tyrosyl residue modification

A convenient and reliable method to measure passive H+-translocating activity (H+ conductivity) was developed; vesicles reconstituted from the membrane moiety (F0) of H+-ATPase (F0 . F1) and soybean phospholipids were loaded with KCl by a freeze-thaw-sonication procedure and the rate of H+ uptake caused by the K+ diffusion potential upon addition of valinomycin was followed with a pH meter. Of the methods tested, a dialysis method using cholate plus deoxycholate gave the best results for reconstitution. Using this method, H+ conductivity of the membrane moiety of H+-ATPase from a thermophilic bacterium PS3 (TF0) was analyzed. Dependence of H+ conductivity of TF0 on H+ concentration fitted a Michaelis-Menten equation showing a Vmax of 31.3 microgram ion/min . mg of TF0 and a Km of 0.095 microgram ion/liter. Upon modification of a tyrosyl residue of TF0 with iodine, the Km value shifted to 0.71 microgram ion/liter, while the Vmax remained constant. These results were interpreted as indicating that a single tyrosyl residue in N,N'-dicyclohexylcarbodiimide-binding proteolipid of TF0 plays an important role as an H+ donor in the the rate-limiting step of H+ permeation through TF0. TF1, the catalytic moiety of H+-ATPase from the thermophilic bacterium PS3, blocked H+ conduction through TF0. A 1:1 stoichiometry of TF1 and TF0 was found in ATP-dependent membrane potential generation as well as H+ conduction.