Structural characterization of an ATPase active F1-/V1 -ATPase (alpha3beta3EG) hybrid complex

Co-reconstitution of subunits E and G of the yeast V-ATPase and the alpha and beta subunits of the F(1)-ATPase from the thermophilic Bacillus PS3 (TF(1)) resulted in an alpha(3)beta(3)EG hybrid complex showing 53% of the ATPase activity of TF(1). The alpha(3)beta(3)EG oligomer was characterized by electron microscopy. By processing 40,000 single particle projections, averaged two-dimensional projections at 1.2-2.4-nm resolution were obtained showing the hybrid complex in various positions. Difference mapping of top and side views of this complex with projections of the atomic model of the alpha(3)beta(3) subcomplex from TF(1) (Shirakihara, Y., Leslie, A. G., Abrahams, J. P., Walker, J. E., Ueda, T., Sekimoto, Y., Kambara, M., Saika, K., Kagawa, Y., and Yoshida, M. (1997) Structure 5, 825-836) demonstrates that a seventh mass is located inside the shaft of the alpha(3)beta(3) barrel and extends out from the hexamer. Furthermore, difference mapping of the alpha(3)beta(3)EG oligomer with projections of the A(3)B(3)E and A(3)B(3)EC subcomplexes of the V(1) from Caloramator fervidus (Chaban, Y., Ubbink-Kok, T., Keegstra, W., Lolkema, J. S., and Boekema, E. J. (2002) EMBO Rep. 3, 982-987) shows that the mass inside the shaft is made up of subunit E, whereby subunit G was assigned to belong at least in part to the density of the protruding stalk. The formation of an active alpha(3)beta(3)EG hybrid complex indicates that the coupling subunit gamma inside the alpha(3)beta(3) oligomer of F(1) can be effectively replaced by subunit E of the V-ATPase. Our results have also demonstrated that the E and gamma subunits are structurally similar, despite the fact that their genes do not show significant homology.     

Interaction between delta and epsilon subunits of F1-ATPase from pig heart mitochondria. Circular dichroism and intrinsic fluorescence of purified and reconstituted delta epsilon complex

A delta epsilon complex has been purified as a molecular entity from pig heart mitochondrial F1-ATPase. This delta epsilon complex has also been reconstituted from purified delta and epsilon subunits. Both isolated and reconstituted delta epsilon complexes have delta 1 epsilon 1 stoichiometry and are indistinguishable by their chromatographic behavior, their circular dichroism spectra (CD spectra), and their intrinsic fluorescence features. The content of secondary structures deduced from CD spectra of the delta epsilon complex appears to be the sum of the respective contributions of purified delta and epsilon subunits. All intrinsic fluorescence studies carried out on isolated epsilon subunit and delta epsilon complex show that the single tryptophan residue located on epsilon is involved in the interaction between delta and epsilon subunits. Results obtained with F1-ATPase are in favor of the same delta epsilon interaction in the entire enzyme.     

The ATPase activity of the alpha 3 beta 3 complex of the F1-ATPase of the thermophilic bacterium PS3 is inactivated on modification of tyrosine 307 in a single beta subunit by 7-chloro-4-nitrobenzofurazan

The catalytically active alpha 3 beta 3 complex, assembled as described (Miwa, K., and Yoshida, M. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 6484-6487) from the isolated alpha and beta subunits of the F1-ATPase of the thermophilic bacterium PS3 (TF1), is inactivated by 7-chloro-4-nitrobenzofurazan (Nbf-Cl) with characteristics very similar to those observed when TF1, which has the subunit composition, alpha 3 beta 3 gamma delta epsilon, is inactivated by the reagent under the same conditions. Both native TF1 and the alpha 3 beta 3 complex are inactivated by 200 microM Nbf-Cl with a pseudo-first order rate constant of 3.7 x 10(-2) min-1 in the presence of 0.2 M Na2SO4 at pH 7.6 and 23 degrees C. The rate of increase in absorbance at 385 nm of reaction mixtures containing 200 microM [14C]Nbf-Cl and TF1, the wild-type alpha 3 beta 3 complex, or the mutant alpha 3(beta Y307----F)3 complex, each at 18 microM was also examined. Since the alpha 3(beta y307----F)3 complex is resistant to inactivation by Nbf-Cl, difference spectrophotometry revealed that inactivation of native TF1 and the wild-type alpha 3 beta 3 complex could be correlated with formation of about 1 mol of Nbf-O-Tyr/mol of enzyme or complex. Fractionation of peptic digests of the labeled enzyme and complexes by reversed-phase high performance liquid chromatography resolved a major radioactive peptide that was common to labeled TF1 and the labeled alpha 3 beta 3 complex but was absent in the digest of the labeled alpha 3(beta Y307----F)3 complex. This labeled peptide was shown to contain Tyr-beta 307 derivatized with [14C]Nbf-Cl by automatic amino acid sequence analyses. From these results, it is concluded that one-third of the sites' reactivity of Nbf-Cl with Tyr-beta 307 in TF1 or its equivalent in other F1-ATPases is not influenced by the presence of the gamma, delta, or epsilon subunits. It has also been shown that Tyr-307 is not modified to an appreciable extent when the isolated beta subunit is treated with [14C]Nbf-Cl under conditions in which this residue is nearly completely labeled in a single beta subunit when TF1 or the alpha 3 beta 3 complex is inactivated by the reagent.     

Comparison of the effects of prostaglandins F1alpha, F2alpha, F1beta, and F2beta on the canine pulmonary vascular bed.

The effects of four F series prostaglandins on the pulmonary vascular bed were compared under conditions of controlled pulmonary blood flow in the intact spontaneously breathing dog. PGF1alpha and PGF2alpha increased lobar arterial pressure whereas PGF1beta and PGF2beta had little if any effect when infused into the lobar artery. The increase in lobar arterial pressure in response to PGF1alpha and PGF2alpha was associated with a significant increase in lobar venous pressure but no change in left atrial pressure. These data indicate that PGF1alpha and PGF2alpha increase pulmonary vascular resistance by constricting lobar veins and vessels upstream to small veins, presumed to be small arteries. It is concluded that in the pulmonary vascular bed the configuration of the hydroxyl group at carbon 9 is an important determinant of pressor activity.     

Monoclonal Antibodies to the [alpha]- and [beta]-Subunits of the Plant Mitochondrial F1-ATPase

We have generated nine monoclonal antibodies against subunits of the maize (Zea mays L.) mitochondrial F1-ATPase. These monoclonal antibodies were generated by immunizing mice against maize mitochondrial fractions and randomly collecting useful hybridomas. To prove that these monoclonal antibodies were directed against ATPase subunits, we tested their cross-reactivity with purified F1-ATPase from pea cotyledon mitochondria. One of the antibodies ([alpha]-ATPaseD) cross-reacted with the pea F1-ATPase [alpha]-subunit and two ([beta]-ATPaseD and [beta]-ATPaseE) cross-reacted with the pea F1-ATPase [beta]-subunit. This established that, of the nine antibodies, four react with the maize [alpha]-ATPase subunit and the other five react with the maize [beta]-ATPase subunit. Most of the monoclonal antibodies cross-react with the F1-ATPase from a wide range of plant species. Each of the four monoclonal antibodies raised against the [alpha]-subunit recognizes a different epitope. Of the five [beta]-subunit antibodies, at least three different epitopes are recognized. Direct incubation of the monoclonal antibodies with the F1-ATPase failed to inhibit the ATPase activity. The monoclonal antibodies [alpha]-ATPaseD and [beta]-ATPaseD were bound to epoxide-glass QuantAffinity beads and incubated with a purified preparation of pea F1-ATPase. The ATPase activity was not inhibited when the antibodies bound the ATPase. The antibodies were used to help map the pea F1-ATPase subunits on a two-dimensional map of whole pea cotyledon mitochondrial protein. In addition, the antibodies have revealed antigenic similarities between various isoforms observed for the [alpha]- and [beta]-subunits of the purified F1-ATPase. The specificity of these monoclonal antibodies, along with their cross-species recognition and their ability to bind the F1-ATPase without inhibiting enzymic function, makes these antibodies useful and invaluable tools for the further purification and characterization of plant mitochondrial F1-ATPases.     

The role of the DELSEED motif of the beta subunit in rotation of F1-ATPase

F(1)-ATPase is a rotary motor protein, and ATP hydrolysis generates torque at the interface between the gamma subunit, a rotor shaft, and the alpha(3)beta(3) substructure, a stator ring. The region of conserved acidic "DELSEED" motif of the beta subunit has a contact with gamma subunit and has been assumed to be involved in torque generation. Using the thermophilic alpha(3)beta(3)gamma complex in which the corresponding sequence is DELSDED, we replaced each residue and all five acidic residues in this sequence with alanine. In addition, each of two conserved residues at the counterpart contact position of gamma subunit was also replaced. Surprisingly, all of these mutants rotated with as much torque as the wild-type. We conclude that side chains of the DELSEED motif of the beta subunit do not have a direct role in torque generation.     

The (alpha F(357)C)(3)(beta R(372)C)(3)gamma subcomplex of the F(1)-ATPase from the thermophilic Bacillus PS3 has altered ATPase activity after cross-linking alpha and beta subunits at noncatalytic site interfaces

In crystal structures of bovine MF(1), the side chains of alpha F(357) and beta R(372) are near the adenines of nucleotides bound to noncatalytic sites. To determine if during catalysis these side chains must pass through the different arrangements in which they are present in crystal structures, the catalytic properties of the (alpha F(357)C)(3)(beta R(372)C)(3)gamma subcomplex of the TF(1)-ATPase were characterized before and after cross-linking the introduced cysteines with CuCl(2). The unmodified mutant enzyme hydrolyzes MgATP at 50% the rate exhibited by wild type. Detailed comparison of the catalytic properties of the double mutant enzyme before and after cross-linking with those of the wild-type subcomplex revealed the following. Before cross-linking, the (alpha F(357)C)(3)(beta R(372)C)(3)gamma subcomplex has less tendency than wild type to release inhibitory MgADP entrapped in a catalytic site during turnover when MgATP binds to noncatalytic sites. Following cross-linking, ATPase activity is reduced 5-fold, and inhibitory MgADP entrapped in a catalytic site during turnover does not release under conditions wherein binding of ATP to noncatalytic sites of the wild-type enzyme promotes release of MgADP from the affected catalytic site. When assayed in the presence of lauryldimethylamine oxide, which prevents turnover-dependent entrapment of inhibitory MgADP in a catalytic site, ATPase activity of the cross-linked form is 47% that of the unmodified mutant enzyme. These results suggest that, during catalysis, the side chains of alpha F(357) and beta R(372) do not pass through the extremely different relative positions in which they exist at the three noncatalytic site interfaces in crystal structures.     

Human F1-ATPase: molecular cloning of cDNA for the beta subunit

F1-ATPase is the major enzyme for ATP synthesis, and its beta subunit is the catalytic site. To date, no full-length cDNA for the eukaryotic F1 gene has been reported. Human F1 was studied because of its importance in medicine and cell biology. Here we report molecular cloning of a full-length cDNA for the human F1 beta subunit and purification of the human F1 beta subunit. The HeLa cell cDNA library constructed in an expression vector gamma gt11 was screened with antiserum against the yeast F1 beta subunit. One of the positive phage DNAs containing the human F1 beta gene and its flanking regions (1.8 kilobase pairs) was sequenced by the dideoxy chain termination method. The open reading frame started from a putative signal presequence, which was rich in both serine and arginine. There was a homologous segment in the signal presequence of human ornithine transcarbamoylase and that of F1 beta. The precursor of F1 beta was expressed in E. coli harboring a plasmid which had been constructed with T5 promotor and the F1 beta cDNA. Both the precursor and mature form of F1 beta were detected in HeLa cells in a pulse-chase experiment. The amino acid sequence of 480 residues (51,568.3 daltons) following the presequence was highly homologous with that of mature beef heart F1 beta (97.5%) and E. coli F1 beta (71.7%), but the codon usage in the human gene was very different from those of reported genes coding for F1 beta of other species.     

Cross-linking of two beta subunits in the closed conformation in F1-ATPase

In the crystal structure of mitochondrial F1-ATPase, two beta subunits with a bound Mg-nucleotide are in "closed" conformations, whereas the third beta subunit without bound nucleotide is in an "open" conformation. In this "CCO" (beta-closed beta-closed beta-open) conformational state, Ile-390s of the two closed beta subunits, even though they are separated by an intervening alpha subunit, have a direct contact. We replaced the equivalent Ile of the alpha3beta3gamma subcomplex of thermophilic F1-ATPase with Cys and observed the formation of the beta-beta cross-link through a disulfide bond. The analysis of conditions required for the cross-link formation indicates that: (i) F1-ATPase takes the CCO conformation when two catalytic sites are filled with Mg-nucleotide, (ii) intermediate(s) with the CCO conformation are generated during catalytic cycle, (iii) the Mg-ADP inhibited form is in the CCO conformation, and (iv) F1-ATPase dwells in conformational state(s) other than CCO when only one (or none) of catalytic sites is filled by Mg-nucleotide or when catalytic sites are filled by Mg2+-free nucleotide. The alpha3beta3gamma subcomplex containing the beta-beta cross-link retained the activity of uni-site catalysis but lost that of multiple catalytic turnover, suggesting that open-closed transition of beta subunits is required for the rotation of gamma subunit but not for hydrolysis of a single ATP.     

The alpha subunit of a plant mitochondrial F1-ATPase is translated in mitochondria

The mitochondrial F1-ATPase from bean (Vicia faba L.) was solubilized by a chloroform treatment of mitochondrial membranes and purified by centrifugation on a glycerol gradient. The active fraction contained 5 subunits: alpha (Mr = 52,000), beta (Mr = 51,000), gamma (Mr = 34,000), delta (Mr = 23,800), and epsilon (Mr = 22,900). Purified coupled mitochondria were incubated in the presence of [ 35S ]methionine and malate to allow mitochondrial translation to occur. The largest labeled polypeptide (Mr = 52,000) was present in the chloroform extract, co-sedimented with the F1-ATPase on glycerol gradient and co-migrated with the alpha subunit upon two-dimensional electrophoresis. The results indicate that the alpha subunit of bean mitochondrial ATPase is translated on mitoribosomes, in contrast to the situation in other organisms.     

The structure of bovine F1-ATPase in complex with its regulatory protein IF1

In mitochondria, the hydrolytic activity of ATP synthase is prevented by an inhibitor protein, IF1. The active bovine protein (84 amino acids) is an alpha-helical dimer with monomers associated via an antiparallel alpha-helical coiled coil composed of residues 49-81. The N-terminal inhibitory sequences in the active dimer bind to two F1-ATPases in the presence of ATP. In the crystal structure of the F1-IF1 complex at 2.8 A resolution, residues 1-37 of IF1 bind in the alpha(DP)-beta(DP) interface of F1-ATPase, and also contact the central gamma subunit. The inhibitor opens the catalytic interface between the alpha(DP) and beta(DP) subunits relative to previous structures. The presence of ATP in the catalytic site of the beta(DP) subunit implies that the inhibited state represents a pre-hydrolysis step on the catalytic pathway of the enzyme.     

Nucleotide binding drives conformational changes in the isolated alpha and beta subunits of the F(1)-ATPase from Escherichia coli

The modeling of the rotatory mechanism performed by the F(1)-ATPase complex during ATP synthesis shows that the beta, but not the alpha subunit, undergoes large conformational changes that depend on the occupancy of the catalytic site. Here we determined by fluorescence spectroscopy the changes in tertiary structure and hydrophobic exposed area of the isolated alpha and beta subunits of the F(1)-ATPase complex from Escherichia coli upon adenine nucleotide binding. The results show that in the absence of intersubunit contacts, the two subunits exhibit markedly similar conformational movements.     

Functions of alpha3beta1 integrin

alpha3beta1 integrin is a laminin receptor with apparently diverse functions. In epithelial cells it acts as a receptor for the basement membrane, whereas in neuronal and possibly tumor cells it mediates migration. Interactions of alpha3beta1 integrin with tetraspanin proteins may provide clues to how it transduces signals that affect cell behavior.     

Nuclear genes coding the yeast mitochondrial adenosine triphosphatase complex. Isolation of ATP2 coding the F1-ATPase beta subunit

A yeast nuclear pet mutant of Saccharomyces cerevisiae lacking any detectable mitochondrial F1-ATPase activity was genetically complemented upon transformation with a pool of wild type genomic DNA fragments carried in the yeast Escherchia coli shuttle vector YEp 13. Plasmid-dependent complementation restored both growth of the pet mutant on a nonfermentable carbon source as well as functional mitochondrial ATPase activity. Characterization of the complementing plasmid by plasmid deletion analysis indicated that the complementing gene was contained on adjoining BamH1 fragments with a combined length of 3.05 kilobases. Gel analysis of the product of this DNA by in vitro translation in a rabbit reticulocyte lysate programmed with yeast mRNA hybrid selected by the plasmid revealed a product which could be immunoprecipitated by antisera against the beta subunit of the yeast mitochondrial ATPase complex. A comparison of the protein sequence derived from partial DNA sequence analysis indicated that the beta subunit of the yeast mitochondrial ATPase complex exhibits greater than 70% conservation of protein sequence when compared to the same subunit from the ATPase of E. coli, beef heart, and chloroplast. The gene coding the beta subunit (subunit 2) of yeast mitochondrial adenosine triphosphatase is designated ATP2. The utilization of cloned nuclear structural genes of mitochondrial proteins for the analysis of the post-translational targeting and import events in organelle assembly is discussed.     

CD3 delta deficiency arrests development of the alpha beta but not the gamma delta T cell lineage.

The CD3 complex found associated with the T cell receptor (TCR) is essential for signal transduction following TCR engagement. During T cell development, TCR-mediated signalling promotes the transition from one developmental stage to the next and controls whether a thymocyte undergoes positive or negative selection. The roles of particular CD3 components in these events remain unclear. Indeed, it is unknown whether they have specialized or overlapping roles. However, the multiplicity of CD3 components and their evolutionary conservation suggest that they serve distinct functions. Here the developmental requirement for the CD3 delta chain is analyzed by generating a mouse line specifically lacking this component (delta-/- mice). Strikingly, CD3 delta is shown to be differentially required during development. In particular, CD3 delta is not needed for steps in development mediated by pre-TCR or gamma delta TCR, but is required for further development of thymocytes expressing alpha beta TCR. Absence of CD3 delta specifically blocks the thymic selection processes that mediate the transition from the double-positive to single-positive stages of development.     

Covalent binding of 3'-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (BzATP) to the isolated alpha and beta subunits and the alpha 3 beta 3 core complex of TF1. Covalent binding of BzATP prevents association of alpha and beta subunits and induces dissociation of the alpha 3 beta 3 core complex.

Binding of the photoreactive ATP analog, 3'-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (BzATP), to the isolated alpha and beta subunits of TF1 and to the alpha 3 beta 3 "core" complex of the holoenzyme is described. About 1 mol of BzATP/mol of subunit was incorporated to isolated alpha and beta subunits. The incorporation of BzATP was prevented by ATP. Covalent binding of BzATP to the alpha subunit was in general somewhat lower than that observed with the beta subunit. No complex was formed upon mixing of either of the modified subunits with the complementary nontreated subunits. Covalent binding of 3 mol of BzATP/alpha 3 beta 3 complex completely inhibited ATPase activity and resulted in the dissociation of the complex. The labeled nucleotide analog was specifically incorporated into the beta subunit of the complex. The holoenzyme TF1, in contrast to the core complex, did not dissociate to the individual subunits upon covalent binding of BzATP. These results are discussed in relation to the location of the catalytic nucleotide binding site(s) and the conformation stability of the alpha 3 beta 3 core complex of TF1.     

Molecular dissection of the beta subunit of F1-ATPase into peptide fragments.

Partial digestion of the native beta subunit of F1-ATPase from the thermophilic Bacillus strain PS3 by three different proteases produced a limited number of peptide fragments. In most cases, the peptides remained associated, and the gross structure of the beta subunit was not destroyed. Furthermore, most peptides were able to reassociate into the form of the beta subunit after denaturating urea treatment. Therefore, the cleaved sites are most likely located in water-exposed loop regions in the tertiary structure of the protein. Almost all peptides were analyzed, and 17 cleaved sites were determined. From the analysis of the distribution of cleaved sites and deletions or insertions in the multiple amino acid sequence alignment of proteins homologous to the beta subunit, locations of five loops and four candidate loops in the beta subunit are suggested. There are two large loops in the central region of the beta subunit sequence, and dicyclohexylcarbodiimide-reactive Glu190 is located in one of them. Tyr341, involved in putative catalytic ATP binding, is also found in one of the loops. Then, taking cleaved sites as a reference, two kinds of expression plasmids, each of which carried genes of two complementary peptide fragments, 1-193 and 198-473 or 1-284 and 285-473, were constructed and expressed in Escherichia coli. For each plasmid, two peptides were coexpressed, associated into a stable beta subunit form in E. coli cells, and purified without dissociation. When these beta subunits were denatured by urea and applied to polyacrylamide gel without denaturant, a protein band with the same mobility as that of the beta subunit appeared, indicating that reassociation of peptide fragments into the form of the beta subunit occurred upon removal of urea. These beta subunits retained the ability to reconstitute the alpha 3 beta 3 gamma complexes even though the efficiency of reconstitution and the recovered ATPase activities were decreased. These complexes were stable at high or low temperature, and ATPase activities were sensitive to inhibition by N3-.     

Steady state kinetics of proton translocation catalyzed by thermophilic F0F1-ATPase reconstituted in planar bilayer membranes

The proton-translocating ATPase of the thermophilic bacterium PS3 was reconstituted into planar phospholipid bilayers by the previously reported method (Hirata, H., Ohno, K., Sone, N., Kagawa, Y., and Hamamoto, T. (1986) J. Biol. Chem. 261, 9839-9843), and the relationship between the electric current induced by ATP and the concentration of ATP was examined. The magnitude of the electric current generated upon addition of ATP followed simple Michaelis-Menten type kinetics, and the Michaelis constant was found to be 0.14 mM under our conditions. This value is close to the values reported for F1- or F0F1-ATPase in its steady state catalytic cycle, indicating that the proton translocation is coupled to the steady state ATPase reaction. The relationship between the Km value and the membrane potential was also examined under the voltage-clamped condition, and we found that there was no apparent dependence of the Km on membrane voltage. These results together with the previous data suggest that the voltage dependence residues in some step that defines the apparent Vmax rather than Km in the reaction cycle, and proton translocation is not directly coupled to this ATP binding step.