Revertant of the yeast Schizosaccharomyces pombe with modified alpha subunits of mitochondrial ATPase-ATPsynthase: impaired nucleotide interactions with soluble and membrane-bound enzyme

A partial revertant from a mutant with modified alpha subunits of mitochondrial ATPase-ATPsynthase has been obtained for the first time from the yeast Schizosaccharomyces pombe. The purified F1 contains a lower amount of endogenous nucleotides as compared to the wild-strain enzyme. In contrast to the wild-type, the F1 ATPase activity from the revertant does not exhibit bicarbonate-sensitive negative cooperativity. The revertant Michaelis constant for Mg-ATP is very similar to that of normal F1 in the presence of bicarbonate while the Vm is slightly lower. The revertant enzyme is much less sensitive to inhibitions by ADP and by azide. It is proposed that the lack of negative cooperativity of revertant F1 ATPase activity is due to lower affinity for ADP, the release of which is no longer the rate-limiting step.     

Purification from a yeast mutant of mitochondrial F1 with modified beta-subunit. High affinity for nucleotides and high negative cooperativity of ATPase activity

Mitochondrial F1 containing genetically modified beta-subunit was purified for the first time from a mutant of the yeast Schizosaccharomyces pombe. Precipitation by poly(ethylene glycol) allowed us to obtain a very stable and pure enzyme from either mutant or wild-type strain. In the presence of EDTA, purified F1 retained high amounts of endogenous nucleotides: 4.6 mol/mol and 3.7 mol/mol for mutant and wild-type F1, respectively. The additional nucleotide in mutant F1 was ATP; it was lost in the presence of Mg2+, which led to a total of 3.4 mol of nucleotides/mol whereas wild-type F1 retained all its nucleotides. Mutant F1 bound more exogenous ADP than wild-type F1 and the same total nucleotide amount was reached with both enzymes. Kinetics of ATPase activity revealed a much higher negative cooperativity for mutant than for wild-type F1. Bicarbonate abolished this negative cooperativity, but higher concentrations were required for mutant F1. The mutant enzyme was more sensitive than the wild-type one to azide inhibition and ADP competitive inhibition; this indicated stronger interactions between nucleotide and F1 in the mutant enzyme. The latter also showed increased sensitivity to N,N'-dicyclohexylcarbodiimide irreversible inhibition.     

8-Azido-adenosine 5'-triphosphate as a photoaffinity label for bacterial F1 ATPase.

1. 8-Azido-adenosine 5'-triphosphate (n83ATP) is a suitable photoaffinity label for F1 ATPase from Micrococcus luteus. The nucleotide is a substrate in the presence of bivalent cations and inhibits the enzyme irreversibly upon irradiation with ultraviolet light above 300 nm. 2. More than 80% of the label is covalently bound to the beta subunits in the presence of bivalent cations. Labeling and inactivation is decreased by protection with ADP, ATP or adenyl-5'-yl imidodiphosphate. To a much smaller degree the alpha subunits also become labeled. 3. n83AMP does not specifically bind to the beta subunits upon irradiation. Like n83ATP and n83ADP, it also labels the alpha subunits to a small extent. 4. The F1 ATPase is inactivated after a single beta subunit per F1 complex has become labeled. A cooperativity of the beta subunits carrying nucleotide binding sites is suggested.     

Photoaffinity labeling of the tight ADP binding site of the chloroplast coupling factor one (CF1): the effect on the CF1-ATPase activity

Chloroplast thylakoid membranes contain tightly bound ADP which is intimately involved in the mechanism of photophosphorylation. The photoaffinity analog 2-azido-ADP binds tightly to spinach thylakoid membrane-bound coupling factor one (CF1) and, in a manner similar to ADP, inhibits the light-triggered ATPase activity (Czarnecki, J.J., Abbott, M.S. and Selman, B.R. (1983) Eur. J. Biochem. 136, 19-24). Ultraviolet irradiation of thylakoid membranes containing noncovalently, tightly bound 2-azido[beta-32P]ADP results in the inactivation of both the methanol-stimulated MgATPase activity of the membrane-bound CF1 and the octylglucoside-dependent MgATPase activity of the solubilized enzyme. There is a linear correlation between the loss of enzyme activity and the covalent incorporation of the photoaffinity analog. Full inactivation of catalytic activity is estimated to occur upon incorporation of 1.07 mol analog and 0.65 mol analog per mol enzyme for the methanol- and octylglucoside-stimulated activities, respectively. Since 2-azido-ADP modifies only the beta subunit of the CF1 and since there are probably three beta subunits per CF1, these results indicate strong cooperativity among beta subunits and between the site of tightly bound nucleotides and the catalytic sites.     

Some properties of isolated mitochondrial ATPase from salmon heart

A soluble Mg-dependent ATPase, similar to the mitochondrial ATPase from beef heart, has been isolated from heart mitochondria of salmon (Salmo salar). The salmon heart ATPase has 5 subunits with molecular weights similar to the beef heart enzyme, but the Stoke's radius of the intact salmon enzyme is larger. The salmon heart ATPase is less temperature labile than the beef heart enzyme. The salmon heart ATPase is strongly inhibited by ADP, and the inhibition is highly temperature dependent. The ITPase activity is also inhibited by IDP (Ki = 180 micron). 2,4-Dinitrophenol in small concentrations stimulates the ITPase activity as well as the ATPase activity of the "washed" salmon heart enzyme. However, in an enzyme preparation which had been freed of most of the bound nucleotides by dialysis in the presence of glycerol (Roveri et al., 1980) the ITPase activity is not stimulated by 2,4-dinitrophenol.     

Use of trypsin to monitor conformational changes of mitochondrial adenosinetriphosphatase induced by nucleotides and phosphate

Upon incubation with trypsin, the adenosine-5'-triphosphatase (ATPase) activity of the nucleotide-depleted F1 is first rapidly and slightly activated and then slowly inactivated. The first phase is simultaneous with the conversion of the alpha subunit into an alpha' fragment which migrates between alpha and beta on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The second phase is related to the proteolysis of the three main subunits, alpha', beta, and gamma. Preincubation of the enzyme with low concentrations of adenosine 5'-diphosphate (ADP) or adenosine 5'-triphosphate (ATP) does not modify the slight increase of activity but efficiently prevents the inactivation induced by trypsin. The alpha leads to alpha' conversion is not affected whereas the further proteolysis of alpha', beta, and gamma does not occur. On the contrary, even high concentrations of GDP only slightly lower the trypsin-induced inactivation. The presence of endogenous tightly bound nucleotides also partially lowers the sensitivity to trypsin since F1 is less rapidly inactivated and proteolyzed than the nucleotide-depleted F1. Phosphate, at high concentrations, both slows down the first phase of activation and simultaneous alpha leads to alpha' conversion and prevents the second phase of inactivation and proteolysis of the main subunits. Pretreatment of the nucleotide-depleted F1 with trypsin under conditions where the ATPase activity is largely inhibited only slightly modifies, however, the hysteretic behavior of the enzyme: the ADP binding and the concomitant hysteretic inhibition of the residual activity are not markedly diminished. The purified ATPase-ATP synthase complex binds very few ADP's and is not hysteretically inhibited. Its ATPase activity is rapidly activated but not further inhibited by trypsin. Preincubation of the complex with ADP does not modify the effects of trypsin.     

Site-directed mutagenesis of stable adenosine triphosphate synthase

Evidence was obtained that four ionizable residues in the alpha and beta subunits of thermophilic ATP synthase (TF0F1), corresponding to Lys-21 and Asp-119 in the MgATP binding segments of adenylate kinase, are essential for the normal catalytic activity. TF0F1 was used because it is the only ATP synthase whose alpha-, beta- and gamma-subunits can be reassembled into an active complex in the absence of both ATP and Mg. Lys-164 and Asp-252 of its beta-subunit were modified to isoleucine and asparagine, respectively, by site-directed mutagenesis using a multifunctional plasmid, and these genes were over-expressed in Escherichia coli. The resulting beta I164 and beta N252 subunits were both noncatalytic after re-assembly into the alpha beta gamma-complex, even though both subunits bound significant amounts of ADP. When Lys-175 and Asp-261 of the alpha-subunit were similarly replaced by isoleucine and asparagine, respectively, the resulting alpha I175 subunit reassembled weakly into an oligomer, while the alpha N261 subunit showed an increased dissociation constant for ADP and was reconstituted into an alpha beta gamma-complex that showed no inter-subunit cooperativity.     

The role of dissimilar subunits of NAD-specific isocitrate dehydrogenase from pig heart. Evaluation using affinity labeling

NAD-specific isocitrate dehydrogenase from pig heart is composed of three dissimilar subunits present in the native enzyme as 2 alpha:1 beta: 1 gamma, with a tetramer being the smallest form of complete enzyme. The role of these subunits has been explored using affinity labeling. Specifically labeled subunits are separated and then recombined with unmodified subunits to form dimers. Recombination of beta or gamma subunits modified by the isocitrate analogues, 3-bromo-2-ketoglutarate and 3,4-didehydro-2-ketoglutarate, with unmodified alpha subunit led to the same activity in the dimer as when unmodified beta or gamma was combined with alpha. Contrastingly, modification of alpha with these isocitrate analogues led to loss in activity either alone or when recombined with beta or gamma. Hence, the isocitrate site on alpha is required for catalytic activity but the isocitrate sites on beta or gamma are not necessary for the activity of the functional dimer. Reaction of isolated subunits with 3-bromo-2-ketoglutarate shows that alpha and the alpha beta dimer are modified at about the same rate as holoenzyme, suggestive of similarity of the isocitrate site in native enzyme and in isolated active entities containing alpha subunit; in contrast, beta and gamma subunits react more slowly. Modification by the 2',3'-dialdehyde derivative of the allosteric effector, ADP, led to loss of activity in reconstituted dimers, independent of which subunit was modified. Reaction of isolated subunits with the dialdehyde derivative of ADP is slow compared to the initial reaction with native enzyme, indicating differences in the effects of ADP on intact enzyme and subunits. The ADP sites on all subunits may thus be important in intersubunit interactions, which in turn modulate catalytic activity.     

F1-ATPase with cysteine instead of serine at residue 373 of the alpha subunit.

Escherichia coli strain AN718 contains the alpha S373F mutation in F1F0-ATP synthase which blocks ATP synthesis (oxidative phosphorylation) and steady-state F1-ATPase activity. The revertant strain AN718SS2 containing the mutation alpha C373 was isolated and shown to confer a phenotype of higher growth yield than that of the wild type in liquid medium containing limiting glucose, succinate, or LB. Purified F1 from strain AN718SS2 was found to have 30% of wild-type steady-state ATPase activity and 60% of wild-type oxidative phosphorylation activity. Azide sensitivity of ATPase activity and ADP-induced enhancement of bound aurovertin fluorescence, both of which are lost in alpha S373F mutant F1, were regained in alpha C373 F1. N-Ethylmaleimide (NEM) inactivated alpha C373 F1 steady-state ATPase potently but had no effect on unisite ATPase. Complete inactivation of alpha C373 F1 steady-state ATPase corresponded to incorporation of one NEM per F1 (mol/mol), in just one of the three alpha subunits. NEM-inactivated enzyme showed azide-insensitive residual ATPase activity and loss of ADP-induced enhancement of bound aurovertin fluorescence. The data confirm the view that placement at residue alpha 373 of a bulky amino acid side-chain (phenylalanyl or NEM-derivatized cysteinyl) blocks positive catalytic cooperativity in F1. The fact that NEM inhibits steady-state ATPase when only one alpha subunit of three is reacted suggests a cyclical catalytic mechanism.     

Characterization of revertants of stmF mutants of Dictyostelium discoideum: evidence that stmF is the structural gene of the cGMP-specific phosphodiesterase

stmF mutants of Dictyostelium discoideum produce long, banded aggregation streams on growth plates and exhibit altered cGMP metabolism. To learn more about the role of cGMP in chemotaxis and the nature of the defect in these mutants, 15 nonstreaming (Stm+) revertants of two stmF mutants were isolated and characterized. Fourteen of the revertants continued to show the elevated cAMP-induced cGMP response and very low cGMP-specific phosphodiesterase (cGPD) activity characteristic of their stmF parents. Parasexual genetic analysis revealed that many of these Stm+ revertants carried phenotypic suppressors unlinked to stmF. One Stm+ revertant, strain HC344, exhibited a low, prolonged cGMP response and relatively high cGPD activity throughout development. To determine whether the elevated cGPD activity in this revertant resulted from increased enzyme production or enhanced enzyme activity, cGPDs were partially purified from the wild-type strain, the stmF parent and revertant HC344, and properties of the enzymes were compared. cGPDs from the stmF mutant and the revertant showed similar differences from the wild-type enzyme in kinetic properties, thermal stability, and sensitivity to certain inhibitors. These results suggest that stmF is the structural gene of the cGPD. In addition, the unusual cGMP response in revertant HC344 appeared to be due to increased production of an altered cGPD.     

Characterization of exchangeable and nonexchangeable bound adenine nucleotides in F1-ATPase from Escherichia coli

The total amount of bound exchangeable and nonexchangeable adenine nucleotides in Escherichia coli F1-ATPase (BF1) was determined; three exchangeable nucleotides were assessed by equilibrium dialysis in a [14C]ADP-supplemented medium. When BF1 was purified in a medium supplemented with ATP, a stoichiometry of nearly 6 mol of bound nucleotides/mol of enzyme was found; three of the bound nucleotides were ATP and the others ADP. When BF1 was filtered on Sephadex G-50 in a glycerol medium (Garrett, N.E., and Penefsky, H.S. (1975) J. Biol. Chem. 250, 6640-6647), bound ADP was rapidly released, in contrast to bound ATP which remained firmly attached to the enzyme. Upon incubation of BF1 with [14C]ADP, the bound ADP rather than the bound ATP was exchanged. Of the three [14C]ADPs which have bound to BF1 by exchange after equilibrium dialysis, one was readily lost by gel filtration on Sephadex G-50; the loss of bound [14C]ADP was markedly reduced by incubation of BF1 with aurovertin, a specific ligand of the beta subunit which is known to increase the affinity of the beta subunit for nucleotides (Issartel, J.-P., and Vignais, P. V. (1984) Biochemistry 23, 6591-6595). Upon photoirradiation of BF1 with [alpha-32P]2-azido-ADP, only the beta subunit was labeled; concomitantly, bound ADP was released, but the content in bound ATP remained stable. These results suggest that specific sites located on the three beta subunits bind nucleotides in a reversible manner. Consequently, the tightly bound ATP of native BF1 would be located on the alpha subunits.     

Isolation of subunits from Methanosarcina barkeri ATPase: nucleotide-binding site in the alpha subunit.

The alpha (62,000-dalton) and beta (49,000-dalton) subunits of Methanosarcina barkeri ATPase were purified to homogeneity. The subunits and ATPase complex were trypsinized in the presence of various nucleotides. ATP and ADP changed the trypsin sensitivity of the alpha subunit in the complex and isolated forms, suggesting the presence of a nucleotide-binding site in the alpha subunit.     

Ground state structure of F1-ATPase from bovine heart mitochondria at 1.9 A resolution

The structure of bovine F(1)-ATPase, crystallized in the presence of AMP-PNP and ADP, but in the absence of azide, has been determined at 1.9A resolution. This structure has been compared with the previously described structure of bovine F(1)-ATPase determined at 1.95A resolution with crystals grown under the same conditions but in the presence of azide. The two structures are extremely similar, but they differ in the nucleotides that are bound to the catalytic site in the beta(DP)-subunit. In the present structure, the nucleotide binding sites in the beta(DP)- and beta(TP)-subunits are both occupied by AMP-PNP, whereas in the earlier structure, the beta(TP) site was occupied by AMP-PNP and the beta(DP) site by ADP, where its binding is enhanced by a bound azide ion. Also, the conformation of the side chain of the catalytically important residue, alphaArg-373 differs in the beta(DP)- and beta(TP)-subunits. Thus, the structure with bound azide represents the ADP inhibited state of the enzyme, and the new structure represents a ground state intermediate in the active catalytic cycle of ATP hydrolysis.     

Demonstration of two exchangeable non-catalytic and two cooperative catalytic sites in isolated bovine heart mitochondrial F1, using the photoaffinity labels [2-3H]8-azido-ATP and [2-3H]8-azido-ADP

The photoreactive nucleotides [2-3H]8-azido-ATP and [2-3H]8-azido-ADP could be used to label the nucleotide binding sites on isolated mitochondrial F1-ATPase to a maximum of 4 mol of nucleotide per mol F1, also when the F1 was depleted of tightly bound nucleotides. At a photolabel concentration of 300-1000 microM, label was found on both alpha and beta subunits in a typically 1:3 ratio, independent of the total amount bound. Under these conditions the covalent binding of two nucleotides is needed for full inactivation (Wagenvoord, R.J., Van der Kraan, I. and Kemp, A. (1977) Biochim. Biophys. Acta 460, 17-24). At lower concentrations of [2-3H]8-azido-ATP (20 microM), it was found that covalent binding of only 1 mol of nucleotide per mole F1 was required for complete inactivation to take place indicating catalytic site cooperativity in the mechanism of ATP hydrolysis. Under those conditions, radioactivity was only found on the beta subunits, which would indicate that the catalytic site is located on a beta subunit and that a second site is located on the alpha/beta interface. It is found that four out of the six nucleotide binding sites are exchangeable and can be labelled with 8-azido-AT(D)P, i.e., two catalytic sites and two non-catalytic sites.     

Recessive (mediator-) revertants from c-H-ras oncogene-transformed NIH 3T3 cells: tumorigenicity in nude mice and transient anchorage and serum independence of the recovered tumor cells in culture.

We have reported earlier the isolation of two recessive, serum- and anchorage-dependent revertants (R116 and R260) from a c-H-ras oncogene-transformed NIH 3T3 line. In both revertants, the oncogene was fully expressed and fusion of either revertant with (untransformed) NIH 3T3 cells, or of the two revertants with one another, resulted in transformed progeny. These, and other data, indicated that the transforming activity of the oncogene was impaired in the two revertants in consequence of defects in distinct genes needed to mediate this activity. We report here that neither revertant could be re-transformed by the K-ras or N-ras oncogene (though they could be re-transformed by several other oncogenes). The two revertants turned out to be tumorigenic in nude mice (though less so than the parental transformed cells). The tumor cells, as recovered, formed foci and had a transformed morphology and a greatly diminished serum and anchorage dependence. Growth of the cells in culture (for 20 passages) resulted in their regaining the characteristics (i.e., anchorage and serum dependence) of cultured R116 and R260 cells. Proliferation of the cells in nude mice was not accompanied by a change in the level of ras oncogene expression or in gene amplification, at least as manifested in the lack of appearance of double-minute chromosomes. The addition of the growth factors TGF alpha and beta to the medium of either revertant did not support anchorage-independent growth.     

Partial characterization of a soluble ATPase from pea cotyledon mitochondria.

A partially purified soluble ATPase (ATP phosphohydrolase, EC 3.6.1.3) from pea cotyledon mitochondria was characterized. Inhibition patterns with azide, NaF, and cold, and a stimulation by 2,4-dinitrophenol were typical of F1-ATPases from mammalian mitochondria. The enzyme hydrolysed GTP, ITP, and ATP, but not CTP, UTP, ADP, or IDP. ATPase and ITPase activities were strongly inhibited by ADP and to a lesser extent by IDP. Distinctive properties of the pea mitochondrial enzyme were activation by high concentrations of CaCl2 and stimulation by NaCl.     

Recent developments on structural and functional aspects of the F1 sector of H+-linked ATPases

This review concerns the catalytic sector of F1 factor of the H+-dependent ATPases in mitochondria (MF1), bacteria (BF1) and chloroplasts (CF1). The three types of F1 have many similarities with respect to the structural parameters, subunit composition and catalytic mechanism. An alpha 3 beta 3 gamma delta epsilon stoichiometry is now accepted for MF1 and BF1; the alpha 2 beta 2 gamma 2 delta 2 epsilon 2 stoichiometry for CF1 remains as matter of debate. The major subunits alpha, beta and gamma are equivalent in MF1, BF1 and CF1; this is not the case for the minor subunits delta and epsilon. The delta subunit of MF1 corresponds to the epsilon subunit of BF1 and CF1, whereas the mitochondrial subunit equivalent to the delta subunit of BF1 and CF1 is probably the oligomycin sensitivity conferring protein (OSCP). The alpha beta gamma assembly is endowed with ATPase activity, beta being considered as the catalytic subunit and gamma as a proton gate. On the other hand, the delta and epsilon subunits of BF1 and CF1 most probably act as links between the F1 and F0 sectors of the ATPase complex. The natural mitochondrial ATPase inhibitor, which is a separate protein loosely attached to MF1, could have its counterpart in the epsilon subunit of BF1 and CF1. The generally accepted view that the catalytic subunit in the different F1 species is beta comes from a number of approaches, including chemical modification, specific photolabeling and, in the case of BF1, use of mutants. The alpha subunit also plays a central role in catalysis, since structural alteration of alpha by chemical modification or mutation results in loss of activity of the whole molecule of F1. The notion that the proton motive force generated by respiration is required for conformational changes of the F1 sector of the H+-ATPase complex has gained acceptance. During the course of ATP synthesis, conversion of bound ADP and Pi into bound ATP probably requires little energy input; only the release of the F1-bound ATP would consume energy. ADP and Pi most likely bind at one catalytic site of F1, while ATP is released at another site. This mechanism, which underlines the alternating cooperativity of subunits in F1, is supported by kinetic data and also by the demonstration of partial site reactivity in inactivation experiments performed with selective chemical modifiers. One obvious advantage of the alternating site mechanism is that the released ATP cannot bind to its original site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification of F1-ATPase with impaired catalytic activity from partial revertants of Escherichia coli uncA mutant strains

It is shown that F1-ATPase preparations having impaired catalytic rates may be purified from partial revertants of uncA mutant strains of Escherichia coli. Recovery of catalytic activity in the partial revertant F1 was accompanied by recovery of alpha in equilibrium beta intersubunit conformational interaction, supporting the hypothesis that such interaction is required for normal catalysis in F1. The specific ATPase activities of the partial revertant F1 preparations were in the range 1-29% of normal, and some of the preparations showed unusual insensitivity to inhibitors. The properties of a new uncA mutant F1 preparation (uncA498) which has approximately half of normal catalytic rate are also briefly described.     

Evidence for coupling between membrane and cytoplasmic domains of the yeast plasma membrane H(+)-ATPase. An analysis of intragenic revertants of pma1-105.

A genetic approach was used to identify interacting portions of the plasma membrane H(+)-ATPase from Saccharomyces cerevisiae. The cellular sensitivity of the pma1-105 strain (S368F) to low external pH and to NH4+ was used to select intragenic revertants of two classes: phenotypically wild-type full revertants and partial revertants that were low pH-resistant but retained resistance to hygromycin B. All 10 full revertants had S368 restored. Among five partial revertants mapping to the original site within the phosphorylation domain, S368L and S368V were each found twice. One revertant contained an E367V substitution adjacent to the original S368F alteration. Four of 13 independently isolated second-site revertants mapped to one site, V289F, in the proposed phosphatase domain. Mutations within the proposed phosphatase and phosphorylation domains resulted in enzymes with increased vanadate sensitivity relative to the vanadate-insensitive S368F enzyme. These results suggest that sites S368, E367, and V289 contribute to a vanadate (Pi) binding domain or are able to interact with such a site within the catalytic domain. The remaining nine partial second-site revertants mapped to six sites within the putative transmembrane regions. Mutations within the transmembrane region had less of an effect on vanadate sensitivity. Most revertant enzymes showed small but significant increases in the rate of ATP hydrolysis relative to the S368F enzyme. Several enzymes no longer displayed the acid-sensitive pH-dependence seen in the S368F enzyme. These data provide novel evidence for an interaction between putative transmembrane helices 1-3 and 7 and the ATP hydrolytic portion of the enzyme.