Topological and functional study of subunit h of the F1Fo ATP synthase complex in yeast Saccharomyces cerevisiae

Subunit h, a 92-residue-long, hydrophilic, acidic protein, is a component of the yeast mitochondrial F1Fo ATP synthase. This subunit, homologous to the mammalian factor F6, is essential for the correct assembly and/or functioning of this enzyme since yeast cells lacking it are not able to grow on nonfermentable carbon sources. Chemical cross-links between subunit h and subunit 4 have previously been shown, suggesting that subunit h is a component of the peripheral stalk of the F1Fo ATP synthase. The construction of cysteine-containing subunit h mutants and the use of bismaleimide reagents provided insights into its environment. Cross-links were obtained between subunit h and subunits alpha, f, d, and 4. These results and secondary structure predictions allowed us to build a structural model and to propose that this subunit occupies a central place in the peripheral stalk between the F1 sector and the membrane. In addition, subunit h was found to have a stoichiometry of one in the F1Fo ATP synthase complex and to be in close proximity to another subunit h belonging to another F1Fo ATP synthase in the inner mitochondrial membrane. Finally, functional characterization of mitochondria from mutants expressing different C-terminal shortened subunit h suggested that its C-terminal part is not essential for the assembly of a functional F1Fo ATP synthase.     

ATP synthase complex from bovine heart mitochondria. Passive H+ conduction through mitochondrial coupling factor 6-depleted F0 complexes

Submitochondrial particles prepared by treatment of mitochondria with ammonia and silicotungstic acid were found to be deficient in coupling factor 6 according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting and had reduced ATP-Pi exchange activity. Requirement of coupling factor 6 for passive proton conductance through mitochondrial F0 was investigated by assaying the ability of depleted particles to sustain NADH-induced proton fluxes as measured by the quenching of 9-amino-6-chloro-2-methoxyacridine fluorescence. The depleted particles themselves showed negligible quenching, but the quenching increased markedly after treating the particles with oligomycin. The data show for the first time that coupling factor 6-depleted complexes have an active proton channel that can be blocked by oligomycin. Therefore, coupling factor 6 is not essential for inhibitor-sensitive proton conductance through mitochondrial F0.     

ATP synthase complex from beef heart mitochondria. Separation of protein bands in the region of 28-31 kDa

ATPase/ATP synthase preparations originally contain protein bands in the 28-31-kDa region. The present study demonstrates separation of the band at 29 kDa (adenine nucleotide translocator) from a band at approximately 31 kDa. In cholate/ammonium sulfate-extracted ATP synthases removal of the 31-kDa band results in decrease of ATP-Pi exchange and oligomycin sensitivity of the ATPase activity. It is suggested that the protein band at 31 kDa is heterogeneous, containing diverse activities, the identities of which are yet to be determined.     

Solution structure of subunit F(6) from the peripheral stalk region of ATP synthase from bovine heart mitochondria

The ATP synthase enzyme structure includes two stalk assemblies, the central stalk and the peripheral stalk. Catalysis involves rotation of the central stalk assembly together with the membrane-embedded ring of c-subunits driven by the trans-membrane proton-motive force, while the alpha and beta-subunits of F(1) are prevented from co-rotating by their attachment to the peripheral stalk. In the absence of structures of either the intact peripheral stalk or larger complexes containing it, we are studying its individual components and their interactions to build up an overall picture of its structure. Here, we describe an NMR structural characterisation of F(6), which is a 76-residue protein located in the peripheral stalk of the bovine ATP synthase and is essential for coupling between the proton-motive force and catalysis. Isolated F(6) has a highly flexible structure comprising two helices packed together through a loose hydrophobic core and connected by an unstructured linker. Analysis of chemical shifts, (15)N relaxation and RDC measurements confirm that the F(6) structure is flexible on a wide range of timescales ranging from nanoseconds to seconds. The relationship between this structure for isolated F(6) and its role in the intact peripheral stalk is discussed.     

Accumulation of newly synthesized F1 in vivo in arabidopsis mitochondria provides evidence for modular assembly of the plant F1Fo ATP synthase

F(1) subcomplex in mitochondrial samples is often considered to be a breakage product of the F(1)F(O) ATP synthase during sample handling and electrophoresis. We have used a progressive (15)N incorporation strategy to investigate the plant F(1)F(O) ATP synthase assembly model and the apparently free F(1) in plant mitochondria which is found in both the inner membrane and matrix. We show that subunits within F(1) in the inner membrane and matrix had a relatively higher (15)N incorporation rate than corresponding subunits in intact membrane F(1)F(O). This demonstrates that free F(1) was a newer pool with a faster turnover rate consistent with it being an assembly intermediate in vivo. Import of [(35)S]Met-labeled F(1) subunit precursors into Arabidopsis mitochondria showed the rapid accumulation of F(1) assembly intermediates. The different (15)N incorporation rate in matrix F(1), inner membrane F(1) and intact F(1)F(O) demonstrates these three represent different protein populations and are likely step by step intermediates during the assembly process of plant mitochondrial ATP synthase. The potential biological implications of in vivo accumulation of enzymatically active F(1) in mitochondria are discussed.     

The native F0F1-inhibitor protein complex from beef heart mitochondria and its reconstitution in liposomes

A functional F₀F₁ ATP synthase that contains the endogenous inhibitor protein (F₀F₁I) was isolated by the use of two combined techniques [Adolfsen, R., McClung, J.A., and Moudrianakis, E. N. (1975).Biochemistry 14, 1727–1735; Dreyfus, G., Celis, H., and Ramirez, J. (1984).Anal. Biochem. 142, 215–220]. The preparation is composed of 18 subunits as judged by SDS-PAGE. A steady-state kinetic analysis of the latent ATP synthase complex at various concentrations of ATP showed aV _max of 1.28mol min^–1 mg^–1, whereas theV _max of the complex without the inhibitor was 8.3mol min^–1 mg^–1. In contrast, theK _m for Mg-ATP of F₀F₁ I was 148M, comparable to theK _m value of 142M of the F₀F₁ complex devoid of IF₁. The hydrolytic activity of the F₀F₁I increased severalfold by incubation at 60C at pH 6.8, reaching a maximal ATPase activity of 9.5mol min^–1 mg^–1; at pH 9.0 a rapid increase in the specific activity of hydrolysis was followed by a sharp drop in activity. The latent ATP synthase was reconstituted into liposomes by means of a column filtration method. The proteoliposomes showed ATP-Pi exchange activity which responded to phosphate concentration and was sensitive to energy transfer inhibitors like oligomycin and the uncouplerp-trifluoromethoxyphenylhydrazone.     

Isolation of the ADP/ATP translocator from beef heart mitochondria as the bongkrekate-protein complex

1. The isolation of the ADP/ATP translocator from beef heart mitochondria as the bongkrekateprotein complex is described, using hydroxyapatite chromatography and gel filtration in Triton X-100 solution. 2. The inhibitor is bound to the protein prior to solubilization with detergent for protection against denaturation. Only the intact bongkrekate-protein passes easily through the hydroxyapatite column. Bongkrekate shileds the protein in contrast to carboxyatractylate only partially against proteinases present in the crude extract. 3. The isolated bongkrekate protein shows the same molecular weights in dodecylsulfate and Triton X-100, the same amino acid composition and the same isoelectric point as the earlier isolated carboxyatractylate-protein complex. It differs by its higher sensitivity against trypsin and thermolysin. 4. The identity of both proteins is demonstrated by interconversion of the bongkrekate-protein into the carboxyatractylate-protein. The process requires the catalysis by ADP or ATP, the natural substrates of the protein. 5. The formation of the extractable [3H]bongkrekate-protein complex in mitochondria requires the presence of ADP or ATP. 6. These data, the immunological studies presented earlier, and the differences in the reactivity of -SH groups of the isolated bongkrekate and carboxyatractylate complexes (to be published) indicate that both proteins represent different conformational states of the translocator protein (m-state and c-state).     

Fo portion of Escherichia coli ATP synthase. Further resolution of trypsin-generated fragments from subunit b

F1-stripped everted membrane vesicles of the ATP synthase-overproducing Escherichia coli strain KY 7485 were treated with trypsin for different lengths of time. Subsequently, the Fo complex was isolated and analyzed by sodium dodecyl sulfate-gel electrophoresis, as well as immunoblotting using antibodies raised against subunit b. By these techniques 3 degradation products with apparent molecular masses of about 16 kDa could be detected in accordance with previous findings (Perlin, D.S., and Senior, A.E. (1985) Arch. Biochem. Biophys. 236, 603-611). Labeling of isolated trypsin-treated Fo fractions with the thiol-specific reagent N-(7-dimethylamino-4-methylcoumarinyl)-maleimide, which has been demonstrated recently to specifically modify subunit b (Schneider, E., and Altendorf, K. (1985) Eur. J. Biochem. 153, 105-109) revealed that the 16-kDa digestion products were degraded into two stable fragments of 12 and 8.3 kDa. These polypeptides do not react with the anti-b antibodies. Treatment of purified liposome-integrated Fo with trypsin resulted in a similar cleavage pattern. In both cases protease digestion inhibited F1 binding while proton-translocating activity remained unaffected. However, liposomes reconstituted with Fo isolated from trypsin-treated membranes were impaired in both binding of F1 and proton translocation. These activities could be restored when reconstitution was carried out in the presence of native subunit b. From this we conclude that the C-terminal region of subunit b is necessary for proper reconstitution of Fo into liposomes.     

Ultrafast purification and reconstitution of His-tagged cysteine-less Escherichia coli F1Fo ATP synthase

His-tagged cysteine-less F1Fo ATP synthase from Escherichia coli was purified using Ni-NTA affinity chromatography. During the purification procedure the loss of total ATPase activity did not exceed 50%, and the extent of purification was about 80-fold. The purified enzyme was essentially free of other proteins, was highly active in ATP hydrolysis (75 units/mg at pH 8 and 37 degrees C), and was sensitive to N,N'-dicyclohexylcarbodiimide (70%). Incorporation of F1Fo into soybean liposomes yielded well-coupled and highly active proteoliposomes. The entire procedure, from the disruption of cells by French press to the preparation of proteoliposomes, took only about 8 h. Some improvements in procedures for the estimation of rates of both ATP hydrolysis and ATP-dependent 9-amino-6-chloro-2-methoxyacridine (ACMA) fluorescence quenching are described.     

Purification and characterization of the F1 portion of the ATP synthase (F1Fo) of Streptomyces lividans

The F1 complex of the ATP synthase of Streptomyces lividans was isolated and purified. The procedure involved the solubilization of F1 from membranes with buffer of low ionic strength in the presence of EDTA, ion-exchange chromatography and gel filtration. The purified F1 complex from S. lividans (SLF1) consists of five subunits alpha, beta, gamma, delta and epsilon with molecular masses of 58,000, 50,000, 36,000, 28,000 and 13,000, respectively and exhibits immunological cross-reactivity with the F1 portion purified from Escherichia coli (ECF1). The enzymatic properties of SLF1 were determined by the use of microtiter-plate-based assay and compared with data obtained for ECF1. ATPase activity of SLF1 (specific activity: 20-30 U/mg) was only observed in the presence of high concentrations of Ca2+ (10mM). Stimulation of the ATPase activity by Mg2+ was not detectable; quite to the contrary, Mg2+ inhibited the Ca(2+)-stimulated activity of SLF1. SLF1 was re-bound to F1-stripped membranes of S. lividans, but not to F1-stripped membrane vesicles of E. coli. In contrast, ECF1 could be cross-reconstituted with F1-stripped membranes of S. lividans; however, a structural but not a functional reconstitution of the hybrid F1Fo complex was observed.     

A functionally inactive, cold-stabilized form of the Escherichia coli F1Fo ATP synthase

An unusual effect of temperature on the ATPase activity of E. coli F1Fo ATP synthase has been investigated. The rate of ATP hydrolysis by the isolated enzyme, previously kept on ice, showed a lag phase when measured at 15 degrees C, but not at 37 degrees C. A pre-incubation of the enzyme at room temperature for 5 min completely eliminated the lag phase, and resulted in a higher steady-state rate. Similar results were obtained using the isolated enzyme after incorporation into liposomes. The initial rates of ATP-dependent proton translocation, as measured by 9-amino-6-chloro-2-methoxyacridine (ACMA) fluorescence quenching, at 15 degrees C also varied according to the pre-incubation temperature. The relationship between this temperature-dependent pattern of enzyme activity, termed thermohysteresis, and pre-incubation with other agents was examined. Pre-incubation of membrane vesicles with azide and Mg2+, without exogenous ADP, resulted in almost complete inhibition of the initial rate of ATPase when assayed at 10 degrees C, but had little effect at 37 degrees C. Rates of ATP synthesis following this pre-incubation were not affected at any temperature. Azide inhibition of ATP hydrolysis by the isolated enzyme was reduced when an ATP-regenerating system was used. A gradual reactivation of azide-blocked enzyme was slowed down by the presence of phosphate in the reaction medium. The well-known Mg2+ inhibition of ATP hydrolysis was shown to be greatly enhanced at 15 degrees C relative to at 37 degrees C. The results suggest that thermohysteresis is a consequence of an inactive form of the enzyme that is stabilized by the binding of inhibitory Mg-ADP.     

Assembly of imported subunit 8 into the ATP synthase complex of isolated yeast mitochondria

This study concerns the assembly into a multisubunit enzyme complex of a small hydrophobic protein imported into isolated mitochondria. Subunit 8 of yeast mitochondrial ATPase (normally a mitochondrial gene product) was expressed in vitro as a chimaeric precursor N9L/Y8-1, which includes an N-terminal-cleavable transit peptide to direct its import into mitochondria. Assembly into the enzyme complex of the imported subunit 8 was monitored by immunoadsorption using an immobilized anti-F1-beta monoclonal antibody. Preliminary experiments showed that N9L/Y8-1 imported into normal rho+ mitochondria, with its complement of fully assembled ATPase, did not lead to an appreciable assembly of the exogenous subunit 8. With the expectation that mitochondria previously depleted of subunit 8 could allow such assembly in vitro, target mitochondria were prepared from genetically modified yeast cells in which synthesis of subunit 8 was specifically blocked. Initially, mitochondria were prepared from strain M31, a mit- mutant completely incapable of intramitochondrial biosynthesis of subunit 8. These mit- mitochondria however were unsuitable for assembly studies because they could not import protein in vitro. A controlled depletion strategy was then evolved. An artificial nuclear gene encoding N9L/Y8-1 was brought under the control of a inducible promoter GAL1. This regulated gene construct, in a low copy number yeast expression vector, was introduced into strain M31 to generate strain YGL-1. Galactose control of the expression of N9L/Y8-1 was demonstrated by the ability of strain YGL-1 to grow vigorously on galactose as a carbon source, and by the inability to utilize ethanol alone for prolonged periods of growth. The measurement of bioenergetic parameters in mitochondria from YGL-1 cells experimentally depleted of subunit 8, by transferring growing cells from galactose to ethanol, was consistent with the presence in mitochondria of a mosaic of ATPase, namely fully assembled functional ATPase complexes and partially assembled complexes with defective F0 sectors. These mitochondria demonstrated very efficient import of N9L/Y8-1 and readily incorporated the imported processed subunit 8 protein into ATPase. Comparison of the kinetics of import and assembly of subunit 8 showed that assembly was noticeably delayed with respect to import. These findings open the way to a new systematic analysis of the assembly of imported proteins into multisubunit mitochondrial enzyme complexes.     

ATP synthase complex from bovine heart mitochondria. Passive H+ conduction through F0 does not require oligomycin sensitivity-conferring protein

Oligomycin sensitivity-conferring protein (OSCP) is a water-soluble subunit of bovine heart mitochondrial H(+)-ATPase (F1-F0). In order to investigate the requirement of OSCP for passive proton conductance through mitochondrial F0, OSCP-depleted membrane preparations were obtained by extracting purified F1-F0 complexes with 4.0 M urea. The residual complexes, referred to as UF0, were found to be deficient with respect to OSCP, as well as alpha, beta, and gamma subunits of F1-ATPase, but had a full complement of coupling factor 6 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting techniques. These UF0 complexes had no intrinsic ATPase activity and were able to bind nearly the same amount of F1-ATPase in the presence of either OSCP or NH4+ ions alone, or a combination of the two. However, the preparations exhibited an absolute dependency on OSCP for conferral of oligomycin sensitivity to membrane-bound ATPase. The passive proton conductance in UF0 proteoliposomes was measured by time-resolved quenching of 9-amino-6-chloro-2-methoxyacridine or 9-aminoacridine fluorescence following a valinomycin-induced K(+)-diffusion potential. The data clearly establish that OSCP is not a necessary component of the F0 proton channel nor is its presence required for conductance blockage by the inhibitors oligomycin or dicyclohexylcarbodiimide. Furthermore, OSCP does not prevent or block passive H+ leakage. Comparisons of OSCP with the F1-F0 subunits from Escherichia coli and chloroplast lead us to suggest that mitochondrial OSCP is, both structurally and functionally, a hybrid between the beta and delta subunits of the prokaryotic systems.     

Dicyclohexylcarbodiimide binds to cytochrome b and subunit VIII in soluble complex III from beef heart mitochondria

Incubation of soluble complex III isolated from either yeast or beef heart mitochondria with 25-100 nmol of [14C]dicyclohexylcarbodiimide (DCCD)/nmol of cytochrome b followed by centrifugation through 10% sucrose or precipitation with trichloroacetic acid did not result in any changes in the appearance of the subunits of either complex. The [14C]DCCD was bound to cytochrome b and phospholipids in the yeast complex and with similar kinetics to both cytochrome b and subunit VIII (Mr = 4000-8000) plus phospholipids of the beef complex. Subunit VIII of the beef complex was partially extracted with chloroform:methanol; however, no subunit of this mobility was present in the yeast complex. Incubation of the beef complex in phosphate buffer for short times resulted in a doubling of the [14C]DCCD bound to cytochrome b relative to that to subunit VIII. Preincubation of both complexes with venturicidin prior to treatment with DCCD resulted in a 50% decrease in the binding of [14C]DCCD to cytochrome b. Reisolation of the beef complex III by precipitation with (NH4)2SO4 after incubation with [14C]DCCD resulted in the formation of a new band with an apparent molecular weight of 39,000 even in the zero time control. The [14C]DCCD was bound to subunit VIII and the core proteins but not to cytochrome b at all times, suggesting that precipitation with (NH)2SO4 in the presence of DCCD causes cross-linking of the subunits of complex III.     

Second stalk of ATP synthase. Cross-linking of gamma subunit in F1 to truncated Fob subunit prevents ATP hydrolysis

ATP synthase consists of two portions, F(1) and F(o), connected by two stalks: a central rotor stalk containing gamma and epsilon subunits and a peripheral, second stalk formed by delta and two copies of F(o)b subunits. The second stalk is expected to keep the stator subunits from spinning along with the rotor. We isolated a TF(1)-b'(2) complex (alpha(3)beta(3)gammadeltaepsilonb'(2)) of a thermophilic Bacillus PS3, in which b' was a truncated cytoplasmic fragment of F(o)b subunit, and introduced a cysteine at its N terminus (bc'). Association of b'(2) or bc'(2) with TF(1) did not have significant effect on ATPase activity. A disulfide bond between the introduced cysteine of bc' and cysteine 109 of gamma subunit was readily formed, and this cross-link caused inactivation of ATPase. This implies that F(o)b subunit bound to stator subunits of F(1) with enough strength to resist rotation of gamma subunit and to prevent catalysis. Contrary to this apparent tight binding, some detergents such as lauryldodecylamine oxide tend to cause release of b'(2) from TF(1).     

F0 complex of the Escherichia coli ATP synthase. Not all monomers of the subunit c oligomer are involved in F1 interaction.

The antigenic determinants of mAbs against subunit c of the Escherichia coli ATP synthase were mapped by ELISA using overlapping synthetic heptapeptides. All epitopes recognized are located in the hydrophilic loop region and are as follows: 31-LGGKFLE-37, 35-FLEGAAR-41, 36-LEGAAR-41 and 36-LEGAARQ-42. Binding studies with membrane vesicles of different orientation revealed that all mAbs bind to everted membrane vesicles independent of the presence or absence of the F1 part. Although the hydrophilic region of subunit c and particularly the highly conserved residues A40, R41, Q42 and P43 are known to interact with subunits gamma and epsilon of the F1 part, the mAb molecules have no effect on the function of F0. Furthermore, it could be demonstrated that the F1 part and the mAb molecule(s) are bound simultaneously to the F0 complex suggesting that not all c subunits are involved in F1 interaction. From the results obtained, it can be concluded that this interaction is fixed, which means that subunits gamma and epsilon do not switch between the c subunits during catalysis and furthermore, a complete rotation of the subunit c oligomer modified with mAb(s) along the stator of the F1F0 complex, proposed to be composed of at least subunits b and delta, seems to be unlikely.     

Increased expression of the F(1)F(o) ATP synthase in response to iron in heart mitochondria

The objective of the present study was to identify mitochondrial components associated with the damage caused by iron to the rat heart. Decreased cell viability was assessed by increased presence of lactate dehydrogenase (LDH) in serum. To assess the functional integrity of mitochondria, Reactive Oxygen Species (ROS), the Respiratory Control Ratio (RCR), ATP and chelatable iron content were measured in the heart. Chelatable iron increased 15-fold in the mitochondria and ROS increased by 59%. Deterioration of mitochondrial function in the presence of iron was demonstrated by low RCR (46% decrease) and low ATP content (96% decrease). Using two dimensional gel electrophoresis (2DE), we identified alterations in 21 mitochondrial proteins triggered by iron overload. Significantly, expression of the alpha, beta, and d subunits of F(1)F(o) ATP synthase increased along with the loss of ATP. This suggests that the F(1)F(o) ATP synthase participates in iron metabolism.