A mutation in the Escherichia coli F0F1-ATP synthase rotor, gammaE208K, perturbs conformational coupling between transport and catalysis

Cross-linking studies on the Escherichia coli F0F1-ATP synthase indicated a site of interaction involving gamma and epsilon subunits in F1 and subunit c in F0 (Watts, S. D., Tang, C., and Capaldi, R. A. (1996) J. Biol. Chem. 271, 28341-28347). To assess the function of these interactions, we introduced random mutations in this region of the gamma subunit (gamma194-213). One mutation, gammaGlu-208 to Lys (gammaE208K), caused a temperature-sensitive defect in oxidative phosphorylation-dependent growth. ATP hydrolytic rates of the gammaE208K F0F1 enzyme became increasingly uncoupled from H+ pumping above 28 degreesC. In contrast, Arrhenius plot of steady-state ATP hydrolysis of the mutant enzyme was linear from 20 to 50 degreesC. Analysis of this plot revealed a significant increase in the activation energy of the catalytic transition state to a value very similar to soluble, epsilon subunit-inhibited F1 and suggested that the mutation blocked normal release of epsilon inhibition of ATP hydrolytic activity upon binding of F1 to F0. The difference in temperature dependence suggested that the gammaE208K mutation perturbed release of inhibition via a different mechanism than it did energy coupling. Suppressor mutations in the polar loop of subunit c restored ATP-dependent H+ pumping and transition state thermodynamic parameters close to wild-type values indicating that interactions between gamma and c subunits mediate release of epsilon inhibition and communication of coupling information.     

Unusual pKa of the carboxylate at the putative catalytic position of the thermophilic F1-ATPase beta subunit determined by 13C-NMR

Glutamic acid-190 in the beta subunit of F1-ATPase from thermophilic Bacillus PS-3 (TF1) was reported to be essential for the ATPase activity. The mutant TF1beta subunit in which Glu-190 had been substituted by cysteine was carboxymethylated with 13C-labeled monoiodoacetic acid. The pKa value of the carboxymethylene group at the 190 position was determined as 5.6 +/- 0.4 by 13C-NMR. On the basis of this value, the pKa of the carboxylate of Glu-190 of the TF1beta subunit was estimated to be 6.8 +/- 0.5. The unusually high pKa could play a role in the catalytic mechanism of F1-ATPase.     

Interaction of the delta and b subunits contributes to F1 and F0 interaction in the Escherichia coli F1F0-ATPase

Interactions of the F1F0-ATPase subunits between the cytoplasmic domain of the b subunit (residues 26-156, bcyt) and other membrane peripheral subunits including alpha, beta, gamma, delta, epsilon, and putative cytoplasmic domains of the a subunit were analyzed with the yeast two-hybrid system and in vitro reconstitution of ATPase from the purified subunits as well. Only the combination of bcyt fused to the activation domain of the yeast GAL-4, and delta subunit fused to the DNA binding domain resulted in the strong expression of the beta-galactosidase reporter gene, suggesting a specific interaction of these subunits. Expression of bcyt fused to glutathione S-transferase (GST) together with the delta 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 delta subunit from proteolytic attack in the cell. These results indicated that the membrane peripheral domain of b subunit stably interacted with the delta subunit in the cell. The affinity purified GST-bcyt did not contain significant amounts of delta, 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 bcyt together with alpha, beta, gamma, delta, and epsilon, or with the same combination except epsilon. 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 delta was stabilized by F1 subunits other than epsilon and also suggested that b-delta interaction was important for F1-F0 interaction.     

Purification and reconstitution into proteoliposomes of the F1F0 ATP synthase from the obligately anaerobic gram-positive bacterium Clostridium thermoautotrophicum

The proton-translocating F1F0 ATP synthase from Clostridium thermoautotrophicum was solubilized from cholate-washed membranes with Zwittergent 3-14 at 58 degrees C and purified in the presence of octylglucoside by sucrose gradient centrifugation and ion-exchange chromatography on a DEAE-5PW column. The purified enzyme hydrolyzed ATP at a rate of 12.6 micromol min(-1) mg(-1) at 58 degrees C and pH 8.5. It was composed of six different polypeptides with molecular masses of 60, 50, 32, 19, 17, and 8 kDa. These were identified as alpha, beta, gamma, delta, epsilon, and c subunits, respectively, as their N-terminal amino acid sequences matched the deduced N-terminal amino acid sequences of the corresponding genes of the atp operon sequenced from Clostridium thermoaceticum (GenBank accession no. U64318), demonstrating the close similarity of the F1F0 complexes from C. thermoaceticum and C. thermoautotrophicum. Four of these subunits, alpha, beta, gamma, and epsilon, constituted the F1-ATPase purified from the latter bacterium. The delta subunit could not be found in the purified F1 although it was present in the F1F0 complex, indicating that the F0 moiety consisted of the delta and the c subunits and lacked the a and b subunits found in many aerobic bacteria. The c subunit was characterized as N,N'-dicyclohexylcarbodiimide reactive. The F1F0 complex of C. thermoautotrophicum consisting of subunits alpha, beta, gamma, delta, epsilon, and c was reconstituted with phospholipids into proteoliposomes which had ATP-Pi exchange, carbonylcyanide p-trifluoromethoxy-phenylhydrazone-stimulated ATPase, and ATP-dependent proton-pumping activities. Immunoblot analyses of the subunits of ATP synthases from C. thermoautotrophicum, C. thermoaceticum, and Escherichia coli revealed antigenic similarities among the F1 subunits from both clostridia and the beta subunit of F1 from E. coli.     

The formation or the reduction of a disulfide bridge on the gamma subunit of chloroplast ATP synthase affects the inhibitory effect of the epsilon subunit

We have studied the change of the catalytic activity of chimeric complexes that were formed by chloroplast coupling factor 1 (CF1) -gamma, alpha and beta subunits of thermophilic bacterial F1 after formation or reduction of the disulfide bridge of different gamma subunits modified by oligonucleotide-directed mutagenesis techniques. For this purpose, three mutant gamma subunits were produced: gamma Delta194-230, here 37 amino acids from Pro-194 to Ile-230 are deleted, gammaC199A, Cys-199 is changed to Ala, and gamma Delta200-204, amino acids from Asp-200 to Lys-204 are deleted. All of the chimeric subunit complexes produced from each of these mutant CF1-gamma subunits and alpha and beta subunits from thermophilic bacterial F1 lost the sensitivity against thiol reagents when compared with the complex containing wild-type CF1-gamma. The pH optimum (pH 8.5-9.0) and the concentration of methanol to stimulate ATPase activities were not affected by these mutations. These indicate that the introduction of the mutations did not change the main features of ATPase activity of the chimeric complex. However, the interaction between gamma subunit and epsilon subunit was strongly influenced by the type of gamma subunit itself. Although the ATPase activity of the chimeric complex that contained gamma Delta200-204 or gammaC199A was inhibited by the addition of recombinant epsilon subunit from CF1 similarly to complexes containing the reduced wild-type gamma subunit, the recombinant epsilon subunit did not inhibit the ATPase of the complex, which contained the oxidized form of gamma subunit. Therefore the affinity of the epsilon subunit to the gamma subunit may be dependent on the state of the gamma subunit or the epsilon subunit may bind to the oxidized form of gamma subunit in a mode that does not inhibit the activity. The ATPase activity of the complex that contains gamma Delta194-230 was not efficiently inhibited by epsilon subunit. These results show that the formation or reduction of the disulfide bond on the gamma subunit may induce a conformational change in the region that directly affects the interaction of this subunit with the adjacent epsilon subunit.     

AutoCyte Interactive Screening System. Experience at a university hospital cytology laboratory

The F1-ATPase is a multimeric enzyme (alpha3 beta3 gamma delta epsilon) primarily responsible for the synthesis of ATP under aerobic conditions. The entire coding region of each of the genes was deleted separately in yeast, providing five null mutant strains. Strains with a deletion in the genes encoding alpha-, beta-, gamma- or delta-subunits were unable to grow, while the strain with a null mutation in epsilon was able to grow slowly on medium containing glycerol as the carbon source. In addition, strains with a null mutation in gamma or delta became 100% rho0/rho- and the strain with the null mutation in gamma grew much more slowly on medium containing glucose. These additional phenotypes were not observed in strains with the double mutations: Delta alpha Delta gamma, Delta beta Delta gamma, Deltaatp11 Delta gamma, Delta alpha Delta delta, Delta beta Delta delta or Deltaatp11 Delta delta. These results indicate that epsilon is not an essential component of the ATP synthase and that mutations in the genes encoding the alpha- and beta-subunits and in ATP11 are epistatic to null mutations in the genes encoding the gamma- and delta-subunits. These data suggest that the propensity to form rho0/rho- mutations in the gamma and delta null deletion mutant stains and the slow growing phenotypes of the null gamma mutant strain are due to the assembly of F1 deficient in the corresponding subunit. These results have profound implications for the physiology of normal cells.     

The stalk region of the Escherichia coli ATP synthase. Tyrosine 205 of the gamma subunit is in the interface between the F1 and F0 parts and can interact with both the epsilon and c oligomer

The soluble portion of the Escherichia coli F1F0 ATP synthase (ECF1) and E. coli F1F0 ATP synthase (ECF1F0) have been isolated from a novel mutant gammaY205C. ECF1 isolated from this mutant had an ATPase activity 3.5-fold higher than that of wild-type enzyme and could be activated further by maleimide modification of the introduced cysteine. This effect was not seen in ECF1F0. The mutation partly disrupts the F1 to F0 interaction, as indicated by a reduced efficiency of proton pumping. ECF1 containing the mutation gammaY205C was bound to the membrane-bound portion of the E. coli F1F0 ATP synthase (ECF0) isolated from mutants cA39C, cQ42C, cP43C, and cD44C to reconstitute hybrid enzymes. Cu2+ treatment or reaction with 5,5'-dithio-bis(2-nitro-benzoic acid) induced disulfide bond formation between the Cys at gamma position 205 and a Cys residue at positions 42, 43, or 44 in the c subunit but not at position 39. Using Cu2+ treatment, this covalent cross-linking was obtained in yields as high as 95% in the hybrid ECF1 gammaY205C/cQ42C and in ECF1F0 isolated from the double mutant of the same composition. The covalent linkage of the gamma to a c subunit had little effect on ATPase activity. However, ATP hydrolysis-linked proton translocation was lost, by modification of both gamma Cys-205 and c Cys-42 by bulky reagents such as 5,5'-dithio-bis (2-nitro-benzoic acid) or benzophenone-4-maleimide. In both ECF1 and ECF1F0 containing a Cys at gamma 205 and a Cys in the epsilon subunit (at position 38 or 43), cross-linking of the gamma to the epsilon subunit was induced in high yield by Cu2+. No cross-linking was observed in hybrid enzymes in which the Cys was at position 10, 65, or 108 of the epsilon subunit. Cross-linking of gamma to epsilon had only a minimal effect on ATP hydrolysis. The reactivity of the Cys at gamma 205 showed a nucleotide dependence of reactivity to maleimides in both ECF1 and ECF1F0, which was lost in ECF1 when the epsilon subunit was removed. Our results show that there is close interaction of the gamma and epsilon subunits for the full-length of the stalk region in ECF1F0. We argue that this interaction controls the coupling between nucleotide binding sites and the proton channel in ECF1F0.     

The alpha 3(beta Y341W)3 gamma subcomplex of the F1-ATPase from the thermophilic Bacillus PS3 fails to dissociate ADP when MgATP is hydrolyzed at a single catalytic site and attains maximal velocity when three catalytic sites are saturated with MgATP

The hydrolytic properties of the alpha3beta3gamma and mutant alpha3(betaY341W)3gamma subcomplexes of the TF1-ATPase have been compared. ATPase activity of the mutant is less sensitive to turnover-dependent inhibition by azide, less suppressed by increasing concentrations of Mg2+ during assay, and less stimulated by lauryl dimethylamine oxide (LDAO). Therefore, it has much lower propensity than wild-type to entrap inhibitory MgADP in a catalytic site during turnover. The fluorescence of the introduced tryptophans in the alpha3(betaY341W)3gamma subcomplex is completely quenched when catalytic sites are saturated with ATP or ADP with or without Mg2+ present. As reported for the betaY331W mutant of Escherichia coli F1 (Weber, J., Wilke-Mounts, S., Lee, R. S.-F., Grell, E., Senior, A. E. (1993) J. Biol. Chem. 268, 20126-20133), this provides a direct probe of nucleotide binding to catalytic sites. Addition of stoichiometric MgATP to the mutant subcomplex quenched one-third the tryptophan fluorescence which did not recover after 60 min. This was caused by entrapment of MgADP in a single catalytic site. Titration of catalytic sites of the alpha3(betaY341W)3gamma subcomplex with MgADP or MgATP revealed Kd's of < 50 nM, about 0.25 microM and about 35 microM. Titrations were not affected by azide, whereas LDAO lowered the affinities of catalytic sites 2 and 3 for MgADP by 5-fold and 2-fold, respectively. During titration with MgATP, LDAO slightly lowered affinity at ATP concentrations below 30 microM and had no effect at ATP concentrations above 30 microM. Maximal velocity was attained when the third catalytic site was titrated with MgATP in the presence or absence of LDAO. The same Kd's for binding MgATP to the (alphaA396C)3beta3(gammaA22C) mutant were observed before and after inactivating it by cross-linking alpha to gamma. This implies that the different affinities of catalytic sites for MgATP do not represent negative cooperativity, but rather represent heterogeneous affinities of catalytic sites dictated by the position of the coiled-coil of the gamma subunit within the central cavity of the (alpha beta)3 hexamer.     

Subunit epsilon of the Escherichia coli ATP synthase: novel insights into structure and function by analysis of thirteen mutant forms

Structural models of subunit epsilon of the ATP synthase from Escherichia coli have been determined recently by NMR [Wilkens et al. (1995) Nat. Struct. Biol. 2, 961-967] and by X-ray crystallography [Uhlin et al. (1997) Structure 5, 1219-1230], revealing a two-domain protein. In this study, six new epsilon mutants were constructed and analyzed: Y63A, D81A, T82A, and three truncated mutants, tr80(S), tr94(LAS), and tr117(AS). Seven mutants constructed previously were also analyzed: E31A, E59A, S65A, E70A, T77A, R58A, and D81A/R85A. Subunits were purified by isoelectric focusing from extracts of cells that overproduced these 13 mutants. F1 was prepared lacking subunit epsilon by immobilized-Ni affinity chromatography. Three mutants, E70A, S65A, and E31A, showed somewhat higher affinities and extents of inhibition than the wild type. Three mutants, T82A, R85A, and tr94(LAS), showed both lower affinities and extents of inhibition, over the concentration range tested. Two showed no inhibition, D81A and tr80(S). The others, T77A, Y63A, E59A, and tr117(AS), showed lower affinities than wild type, but the extents of inhibition were nearly normal. Results indicate that the C-terminal domain of subunit epsilon contributes to inhibition of ATP hydrolysis, but it is not necessary for ATP-driven proton translocation. Interactions with subunit gamma are likely to involve a surface containing residues S65, E70, T77, D81, and T82, while residues R85 and Y63 are likely to be important in the conformation of subunit epsilon.     

Functional and idling rotatory motion within F1-ATPase

ATP synthase mediates proton flow through its membrane portion, F0, which drives the synthesis of ATP in its headpiece, F1. The F1-portion contains a hexagonal array of three subunits alpha and three beta encircling a central subunit gamma, that in turn interacts with a smaller epsilon and with F0. Recently we reported that the application of polarized absorption recovery after photobleaching showed the ATP-driven rotation of gamma over at least two, if not three, beta. Here we extend probes of such rotation aided by a new theory for assessing continuous versus stepped, Brownian versus unidirectional molecular motion. The observed relaxation of the absorption anisotropy is fully compatible with a unidirectional and stepping rotation of gamma over three equidistantly spaced angular positions in the hexagon formed by the alternating subunits alpha and beta. The results strongly support a rotational catalysis with equal participation of all three catalytic sites. In addition we report a limited rotation of gamma without added nucleotides, perhaps idling and of Brownian nature, that covers only a narrow angular domain.     

Asymmetry of the alpha subunit of the chloroplast ATP synthase as probed by the binding of Lucifer Yellow vinyl sulfone

The catalytic portion of the chloroplast ATP synthase (CF1) is structurally asymmetric. Asymmetry of the otherwise symmetrical alpha3beta3 heterohexamer is induced by the presence of tightly bound nucleotides and interactions with the single-copy, smaller subunits. Lucifer Yellow vinyl sulfone (4-amino-N-[3-(vinylsulfonyl)phenyl]naphthalimide-3,6-disulfonic acid) rapidly and covalently binds to lysine 378 on one alpha subunit [Nalin, C. M., Snyder, B., and McCarty, R. E., (1985) Biochemistry 24, 2318-2324] [Shapiro, A. B. (1991) Ph.D. Thesis, Cornell University, Ithaca, NY). The asymmetrical binding of Lucifer Yellow to CF1 provides a method to investigate the cause of asymmetry in the alpha subunits. The reaction of CF1 with Lucifer Yellow was monitored by total fluorescence of bound Lucifer Yellow as well as by quantitative determination of Lucifer Yellow bound to the tryptic peptide that contains lysine 378 of the alpha subunit. The total binding of Lucifer Yellow to CF1 was not affected by the presence of tightly bound nucleotides or nucleotide in the medium. Neither the total binding of Lucifer Yellow to CF1 nor the reaction of alpha-lysine 378 with Lucifer Yellow was changed by the removal of the epsilon subunit, the delta subunit, or both subunits. The extent of incorporation of Lucifer Yellow into lysine 378 of the alpha subunit in (alphabeta)n was about three times that of Lucifer Yellow incorporation into CF1. Reconstitution of (alphabeta)n with gamma restored the binding of one Lucifer Yellow per alpha3beta3gamma. Therefore, the interactions between gamma and the alphabeta heterohexamer are important in conferring asymmetry to the alpha subunits of CF1.     

Percent cholesterol absorption in normal women and men quantified with dual stable isotopic tracers and negative ion mass spectrometry

Amino acid substitutions at many positions in the a subunit of F1F0 ATP synthase result in impaired proton translocation and altered catalytic activity. In this work, we demonstrate that amino acid substitutions in the a subunit affect the epsilon subunit. In mutant F1F0 ATP synthases, the epsilon subunit was studied by determining its sensitivity to proteolysis and by chemical crosslinking under conditions of active turnover and in quiescent enzyme. Like native F1F0 ATP synthase, the epsilon subunit in enzymes carrying either the aarg-210-->ile or agly-218-->asp substitutions proved resistant to trypsin digestion during ATP hydrolysis. In each case, the epsilon subunit was rapidly digested in the presence of a nonhydrolyzable ligand, but this did not result in the activation of hydrolytic activity typically seen in wild-type enzyme. In enzyme carrying the aala-217-->arg substitution, the trypsin digestion of the epsilon subunit occurred regardless of ligand and was accompanied by a limited hydrolytic activation. Relative to the native F1F0 ATP synthase, the aala-217-->arg substitution resulted in reduced efficiency of crosslinking between the epsilon and beta subunits using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. These observations indicate that the structural changes resulting from amino acid substitutions in the a subunit are propagated to the epsilon subunit and are specific to the individual substitutions.     

Nucleotide-dependent movement of the epsilon subunit between alpha and beta subunits in the Escherichia coli F1F0-type ATPase

Mutants of ECF1-ATPase were generated, containing cysteine residues in one or more of the following positions: alphaSer-411, betaGlu-381, and epsilonSer-108, after which disulfide bridges could be created by CuCl2 induced oxidation in high yield between alpha and epsilon, beta and epsilon, alpha and gamma, beta and gamma (endogenous Cys-87), and alpha and beta. All of these cross-links lead to inhibition of ATP hydrolysis activity. In the two double mutants, containing a cysteine in epsilonSer-108 along with either the DELSEED region of beta (Glu-381) or the homologous region in alpha (Ser-411), there was a clear nucleotide dependence of the cross-link formation with the epsilon subunit. In betaE381C/epsilonS108C the beta-epsilon cross-link was obtained preferentially when Mg2+ and ADP + Pi (addition of MgCl2 + ATP) was present, while the alpha-epsilon cross-link product was strongly favored in the alphaS411C/epsilonS108C mutant in the Mg2+ ATP state (addition of MgCl2 + 5'-adenylyl-beta,gamma-imidodiphosphate). In the triple mutant alphaS411C/betaE381C/epsilonS108C, the epsilon subunit bound to the beta subunit in Mg2+-ADP and to the alpha subunit in Mg2+-ATP, indicating a significant movement of this subunit. The gamma subunit cross-linked to the beta subunit in higher yield in Mg2+-ATP than in Mg2+-ADP, and when possible, i.e. in the triple mutant, always preferred the interaction with the beta over the alpha subunit.     

Intersubunit rotation in active F-ATPase

The enzyme ATP synthase, or F-ATPase, is present in the membranes of bacteria, chloroplasts and mitochondria. Its structure is bipartite, with a proton-conducting, integral membrane portion, F0, and a peripheral portion, F1. Solubilized F1 is composed of five different subunits, (alpha beta)3 gamma delta epsilon, and is active as an ATPase. The function of F-ATPase is to couple proton translocation through F0 with ATP synthesis in F1 (ref.3). Several lines of evidence support the spontaneous formation of ATP on F1 (refs 4,5) and its endergonic release at cooperative and rotating (or at least alternating) sites. The release of ATP at the expense of protonmotive force might involve mechanical energy transduction from F0 into F1 by rotation of the smaller subunits (mainly gamma) within (alpha beta)3, the catalytic hexagon of F1 as suggested by electron microscopy, by X-ray crystal structure analysis and by the use of cleavable crosslinkers. Here we record an intersubunit rotation in real time in the functional enzyme by applying polarized absorption relaxation after photobleaching to immobilized F1 with eosin-labelled gamma. We observe the rotation of gamma relative to immobilized (alpha beta)3 in a timespan of 100 ms, compatible with the rate of ATP hydrolysis by immobilized F1. Its angular range, which is of at least 200 degrees, favours a triple-site mechanism of catalysis, with gamma acting as a crankshaft in (alpha beta)3. The rotation of gamma is blocked when ATP is substituted with its non-hydrolysable analogue AMP-PNP.     

Photoinactivation of the F1-ATPase from spinach chloroplasts by dequalinium is accompanied by derivatization of methionine beta183

In contrast to the F1-ATPases from bovine mitochondria and the thermophilic Bacillus PS3, which are reversibly inhibited by dequalinium in the absence of irradiation, the Mg2+-ATPase activity of heat- or dithiothreitol-activated chloroplast F1 (CF1) from spinach chloroplasts is slightly stimulated by dequalinium. Conversely, dequalinium is a partial inhibitor (maximal inhibition is 85-90%) of the Ca2+-ATPase of CF1 activated by heat, dithiothreitol, or octylglucoside. The Mg2+- and Ca2+-ATPase activities of CF1 respond differently in the presence of lauryl dimethylamine oxide (LDAO) in the assay medium. Whereas the Mg2+-ATPase activity of heat- or dithiothreitol-activated CF1 is stimulated up to 14-fold by increasing concentrations of LDAO, the Ca2+-ATPase is inhibited in a biphasic manner by increasing concentrations of LDAO. In the presence of LDAO, dequalinium does not stimulate the heat-activated Mg2+-ATPase over that promoted by LDAO alone. That dequalinium slightly stimulates Mg2+-ATPase activity although it inhibits Ca2+-ATPase activity can be reconciled by assuming that dequalinium binds to two sites in CF1, a stimulatory site that also binds LDAO and an inhibitory site. By acting as a partial inhibitor of the Mg2+-ATPase activity that it activates, the combined effect of dequalinium is modest stimulation. Irradiation of heat- or dithiothreitol-activated CF1 or the alpha3beta3gamma subcomplex of CF1 in the presence of 12 microM dequalinium led to rapid photoinactivation. ATP and ADP, separately or in combination with Mg2+, protect against photoinactivation. After photoinactivating the alpha3beta3gamma subcomplex of CF1 with [14C]dequalinium, tryptic and peptic digests of the isolated, derivatized beta subunit were fractionated by high performance liquid chromatography. Sequencing of the isolated, radioactive tryptic and peptic peptides revealed that Metbeta183, which is at or near the catalytic site, is derivatized in a single beta subunit when CF1 is photoinactivated with [14C]dequalinium.     

Tissue-specific distribution and modulatory role of the gamma subunit of the Na,K-ATPase

The Na,K-ATPase comprises a catalytic alpha subunit and a glycosylated beta subunit. Another membrane polypeptide, gamma, first described by Forbush et al. (Forbush, B., III, Kaplan, J. H., and Hoffman, J. F. (1978) Biochemistry 17, 3667-3676) associates with alpha and beta in purified kidney enzyme preparations. In this study, we have used a polyclonal anti-gamma antiserum to define the tissue specificity and topology of gamma and to address the question of whether gamma has a functional role. The trypsin sensitivity of the amino terminus of the gamma subunit in intact right-side-out pig kidney microsomes has confirmed that it is a type I membrane protein with an extracellular amino terminus. Western blot analysis shows that gamma subunit protein is present only in membranes from kidney tubules (rat, dog, pig) and not those from axolemma, heart, red blood cells, kidney glomeruli, cultured glomerular cells, alpha1-transfected HeLa cells, all derived from the same (rat) species, nor from three cultured cell lines derived from tubules of the kidney, namely NRK-52E (rat), LLC-PK (pig), or MDCK (dog). To gain insight into gamma function, the effects of the anti-gamma serum on the kinetic behavior of rat kidney sodium pumps was examined. The following evidence suggests that gamma stabilizes E1 conformation(s) of the enzyme and that anti-gamma counteracts this effect: (i) anti-gamma inhibits Na,K-ATPase, and the inhibition increases at acidic pH under which condition the E2(K) --> E1 phase of the reaction sequence becomes more rate-limiting, (ii) the oligomycin-stimulated increase in the level of phosphoenzyme was greater in the presence of anti-gamma indicating that the antibody shifts the E1 left and right arrow left and right arrow E2P equilibria toward E2P, and (iii) when the Na+-ATPase reaction is assayed with the Na+ concentration reduced to levels ( --> E2P transition, anti-gamma is stimulatory. These observations taken together with evidence that the pig gamma subunit, which migrates as a doublet on polyacrylamide gels, is sensitive to digestion by trypsin, and that Rb+ ions partially protect it against this effect, indicate that the gamma subunit is a tissue-specific regulator which shifts the steady-state equilibria toward E1. Accordingly, binding of anti-gamma disrupts alphabeta-gamma interactions and counteracts these modulatory effects of the gamma subunit.     

High prevalence of mutations in the microtubule-associated protein tau in a population study of frontotemporal dementia in the Netherlands

We report here the large-scale purification of vacuolar (V0V1)-type Na+-ATPase from Enterococcus hirae achieved using column anion-exchange and gel filtration chromatographies; 32 mg of purified enzyme comprising nine subunits, A, B, C, D, E, F, G, I, and K, was obtained from 20 liter culture. This amount is 500-fold larger than that reported in the previous paper [Murata, T., Takase, K., Yamato, I., Igarashi, K., and Kakinuma, Y. (1997) J. Biol. Chem. 272, 24885-24890]. The purified enzyme shows a high specific activity of ATP hydrolysis (35.7 micromol Pi released/min/mg protein). ATP-driven 22Na+ uptake by reconstituted V0V1-proteoliposomes exhibited an apparent Kt value for Na+ of 40 microM, which is near the Km value (20 microM) for Na+ of the ATP hydrolytic activity. Denatured gel electrophoresis revealed that six subunits, A, B, C, D, E, and F, are releasable as the V1 subunit from the V0V1 complex by incubation with ethylenediaminetetraacetic acid; subunit G was not identified. The remaining V0-liposomes containing I and K subunits catalyzed Na+ uptake in response to potassium diffusion potential (Deltapsi, inside negative); the Kt value for Na+ of this reaction was estimated to be about 2 mM. Inhibition by N,N'-dicyclohexylcarbodiimide (DCCD) of the Na+-ATPase activity and Deltapsi-driven Na+ uptake by the V0-liposomes was prevented by the presence of Na+, suggesting that the Na+ binding site overlaps with the DCCD-reactive site.     

Functional properties of recombinant GABA(A) receptors composed of single or multiple beta subunit subtypes

GABA(A) receptor (GABAR) isoforms in the central nervous system are composed of combinations of alpha(1-6), beta(1-4), gamma(1-4), delta(1) and epsilon(1) subunit subtypes arranged in a pentamer. Many regions of the brain express high levels of mRNA encoding several different subunits and even multiple subunit subtypes. The stoichiometry of GABAR isoforms is unclear, and the number and identity of individual subunit subtypes that are coassembled remain uncertain. To examine the role of beta subunit subtypes in the functional properties of GABARS and to determine whether multiple beta subtypes can be coassembled in functional GABARs, plasmids containing cDNAs encoding rat beta1 and/or beta3, alpha5 and gamma2L subtypes were cotransfected into L929 fibroblasts. The properties of the expressed receptor populations were determined using whole-cell and single-channel recording techniques. The alpha5beta1gamma2L isoform was less sensitive to GABA than the alpha5beta3gamma2L isoform. alpha5beta1gamma2L isoform currents were also insensitive to the allosteric modulator loreclezole, while alpha5beta3gamma2L isoform currents were strongly potentiated by loreclezole. Fibroblasts transfected with plasmids containing cDNAs for both beta1 and beta3 subtypes along with alpha5 and gamma2L subtypes produced a receptor population with an intermediate sensitivity to GABA which was insensitive to loreclezole. These results suggest that functional GABARs can be formed that contain two different beta1 subunit subtypes with properties different from receptors that contain only a single beta1 subtype and that the beta1 subunit subtypes influence the response of GABARs to GABA and to the allosteric modulator loreclezole.     

Replicon typing characterization of plasmids encoding resistance to gentamicin and apramycin in Escherichia coli and Salmonella typhimurium isolated from human and animal sources in Belgium

cDNA sequences encompassing the full coding region for the human muscle acetylcholine receptor (AChR) epsilon and gamma subunits have been isolated. The deduced amino-acid sequences indicate that the mature epsilon subunit contains 473 amino acids and is preceded by a 20-amino-acid signal peptide. As predicted from genomic clones, the gamma subunit contains 495 amino acids preceded by a 22-amino-acid signal peptide. In common with the human alpha, beta, gamma and delta subunits the epsilon subunit is highly conserved between mammalian species. The epsilon subunit gene is not closely linked to the gamma and delta subunits on chromosome 2 but rather is located with the beta subunit on chromosome 17. Expression of the alpha-, beta-, gamma-, delta- and epsilon-subunit cRNAs in rabbit-reticulocyte lysates followed by analysis on SDS/PAGE show glycosylated proteins with apparent molecular masses of 44-60 kDa.