Binding properties of an intrinsic ATPase inhibitor and occurrence in yeast mitochondria of a protein factor which stabilizes and facilitates the binding of the inhibitor to F1F0-ATPase

The content of an intrinsic ATPase inhibitor in mitochondria was determined by a radioimmunoassay procedure which showed the molar ratio of the inhibitor to ATPase to be 1:1. The ratio in submitochondrial particles, where half of the enzyme was activated, was the same as that of mitochondria, indicating that the inhibitor protein has affinity for the mitochondrial membrane as well as for F1-ATPase. The inhibitor protein could be removed from the mitochondrial membrane by incubation with 0.5 M Na2SO4 and concomitantly the enzyme was fully activated. The enzyme fully activated by the salt treatment was inactivated again by the externally added ATPase inhibitor in the presence of ATP and Mg2+. The enzyme-inhibitor complex (inactive) on the mitochondrial membrane was more stable than the solubilized enzyme-inhibitor complex but gradually dissociated in the absence of ATP and Mg2+. However, in mitochondria, the enzyme activity was inhibited even in the absence of the cofactors. A protein factor stabilizing the enzyme-inhibitor complex on the mitochondrial membrane was isolated from yeast mitochondria. This factor stabilized the inhibitor complex of membrane-bound ATPase while having no effect on that of purified F1-ATPase. It also efficiently facilitated the binding of the inhibitor to membrane-bound ATPase to form the complex, which reversibly dissociated at slightly alkaline pH.     

Activation of ATP hydrolysis by an uncoupler in mutant mitochondria lacking an intrinsic ATPase inhibitor in yeast

An intrinsic ATPase inhibitor and 9-kDa protein are regulatory factors of mitochondrial ATP synthase in Saccharomyces cerevisiae. A gene encoding the ATPase inhibitor was isolated from a yeast genomic library with synthetic oligonucleotides as hybridization probes and was sequenced. The deduced amino acid sequence showed that the precursor protein contains an amino-terminal presequence of 22 amino acid residues. Mutant strains that did not contain the inhibitor and/or the 9-kDa protein were constructed by transformation of cells with their in vitro disrupted genes. The disruption of the chromosomal copy in recombinant cells was verified by Southern blot analysis, and the absence of the proteins in the mutant cells was confirmed by Western blot analysis. All the mutants could grow on a nonfermentable carbon source and the oxidative phosphorylation activities of their isolated mitochondria were the same as that of normal mitochondria. However, an uncoupler, carbonylcyanide-m-chlorophenylhydrazone, induced marked ATP hydrolysis in the inhibitor-deficient mitochondria, but not in normal mitochondria. These observations suggest that the ATPase inhibitor inhibits ATP hydrolysis by F1F0-ATPase only when the membrane potential is lost.     

Binding of ADP to beef-heart mitochondrial ATPase (F1)

1. ADP binding to beef-heart mitochondrial ATPase (F1), in the absence of Mg2+, has been determined by separating the free ligand by ultrafiltration and determining it in the filtrate by a specially modified isotachophoretic procedure. 2. Since during the binding experiments the 'tightly' bound ADP (but not the ATP) dissociates, it is necessary to take this into account in calculating the binding parameters. 3. The binding data show that only one tight binding site (Kd about 0.5 microM) for ADP is present. 4. It is not possible to calculate from the binding data alone the number of or the dissociation constants for the weak binding sites. It can be concluded, however, that the latter is not less than about 50 microM.     

Caenorhabditis elegans MAI-1 protein, which is similar to mitochondrial ATPase inhibitor (IF1), can inhibit yeast F0F1-ATPase but cannot be transported to yeast mitochondria

In Caenorhabditis elegans, two proteins that are similar to mitochondrial ATPase inhibitor protein (IF₁) have been found and named MAI-1 and MAI-2. In this study, we overexpressed and purified both the proteins and examined their properties. Circular dichroism spectra indicated that both the MAI-1 and MAI-2 predominantly consisted of β- and random structure, and in contrast to mammalian IF₁, α-helixes were barely detected. Both MAI-1 and MAI-2 could inhibit yeast F₀F₁-ATPase, but the inhibition by MAI-1 was pH-independent. MAI-2-GFP fusion protein was transported to yeast mitochondria, but MAI-1-GFP was not. These results indicate that the MAI-2 is _{C. elegans} IF₁. MAI-1 seems to be a cytosolic protein and may regulate cytosolic ATPase(s).     

Simultaneous bindings of ATPase inhibitor and 9K protein to F1F0-ATPase in the presence of 15K protein in yeast mitochondria

Yeast mitochondrial ATP synthase has three regulatory proteins, ATPase inhibitor, 9K protein, and 15K protein. The 9K protein binds directly to purified F1-ATPase, as does the ATPase inhibitor, but the 15K protein does not [Hashimoto, T. et al. (1987) J. Biochem. 102, 685-692]. In the present study, we found that 15K protein bound to purified F1F0-ATPase, forming an equimolar complex with the enzyme. The apparent dissociation constant was calculated to be 1.4 x 10(-5) M. The ATPase inhibitor and 9K protein also bound to F1F0-ATPase in the presence of ATP and Mg2+, and the dissociation constants of their bindings were about 3 X 10(-6) M. They bound to the enzyme competitively in the absence of 15K protein, but in its presence, they bound in equimolar amounts to the enzyme. The ATP-hydrolyzing activity of the enzyme-ligand complex was greatly influenced by the order of bindings of ATPase inhibitor and 9K protein: when the ATPase inhibitor was bound first, the activity of the enzyme was inhibited completely and was not restored by 9K protein, but when 9K protein was added first, the activity was inhibited only partially even after equimolar binding of the ATPase inhibitor to the enzyme. These observations strongly suggest that the 15K protein binds to the F0 part and functions to hold the ATPase inhibitor or 9K protein on the F1 subunit.     

Binding of the inhibitor protein IF(1) to bovine F(1)-ATPase

In the structure of bovine F₁-ATPase inhibited with residues 1-60 of the bovine inhibitor protein IF₁, the α-helical inhibitor interacts with five of the nine subunits of F₁-ATPase. In order to understand the contributions of individual amino acid residues to this complex binding mode, N-terminal deletions and point mutations have been introduced, and the binding properties of each mutant inhibitor protein have been examined. The N-terminal region of IF₁ destabilizes the interaction of the inhibitor with F₁-ATPase and may assist in removing the inhibitor from its binding site when F₁F_o-ATPase is making ATP. Binding energy is provided by hydrophobic interactions between residues in the long α-helix of IF₁ and the C-terminal domains of the β_DP-subunit and β_TP-subunit and a salt bridge between residue E30 in the inhibitor and residue R408 in the C-terminal domain of the β_DP-subunit. Several conserved charged amino acids in the long α-helix of IF₁ are also required for establishing inhibitory activity, but in the final inhibited state, they are not in contact with F₁-ATPase and occupy aqueous cavities in F₁-ATPase. They probably participate in the pathway from the initial interaction of the inhibitor and the enzyme to the final inhibited complex observed in the structure, in which two molecules of ATP are hydrolysed and the rotor of the enzyme turns through two 120° steps. These findings contribute to the fundamental understanding of how the inhibitor functions and to the design of new inhibitors for the systematic analysis of the catalytic cycle of the enzyme.     

Binding of intrinsic ATPase inhibitor to mitochondrial ATPase--stoichiometry of binding of nucleotides, inhibitor, and enzyme

F1-ATPase inhibitor was purified from yeast, Saccharomyces cerevisiae. The purified inhibitor blocked ATPase activity in the presence of ATP and Mg2+ by forming a latent equimolar enzyme-inhibitor complex with ATP and ADP newly bound to loose sites on the enzyme. A small portion of externally added ATP was hydrolyzed before the latent complex was formed but the hydrolysis was not directly related to the complex formation. Newly bound ATP tended to be converted to ADP when the ATP concentration of the medium was low. ATP tightly bound to the enzyme was not directly involved in formation of the complex. The complex was fairly stable in the presence of excess inhibitor and ATP but at a high concentration of the enzyme (10(-5) M), the inhibition was not complete, although only about 0.03% of the original activity remained unblocked.     

Binding properties of 9K protein to F1-ATPase: a counterpart ligand to the ATPase inhibitor

A regulatory subunit of yeast mitochondrial ATP synthase, 9K protein, formed an equimolar complex with F1-ATPase in the presence of ATP and Mg2+, indicating that the binding of the protein to the enzyme took place in a similar manner to that of ATPase inhibitor. The ATP-hydrolyzing activity of F1-ATPase decreased 40% on binding of the 9K protein, and the remaining activity was resistant to external ATPase inhibitor. The apparent dissociation constant of the F1-ATPase-9K complex was determined by gel permeation chromatography to be 3.7 X 10(-6) M, which was in the same order of magnitude as that of enzyme-ATPase inhibitor complex (4.2 x 10(-6) M). When added simultaneously the binding of the inhibitor and 9K protein to F1-ATPase were competitive and the sum of their bindings did not exceed 1 mol per mol of enzyme. However, the binding of each protein ligand to F1-ATPase took more than 1 min for completion, and when one of these two proteins was added 10 min after the other, it did not replace the other. These observations strongly suggest that membrane-bound F1-ATPase always binds to either the 9K protein or ATPase inhibitor in intact mitochondria and that the complexes with the two ligands are active and inactive counterparts, respectively.     

Formation of the yeast F1F0-ATP synthase dimeric complex does not require the ATPase inhibitor protein,Inh1

The yeast F1F0-ATP synthase forms dimeric complexes in the mitochondrial inner membrane and in a manner that is supported by the F0-sector subunits, Su e and Su g. Furthermore, it has recently been demonstrated that the binding of the F1F0-ATPase natural inhibitor protein to purified bovine F1-sectors can promote their dimerization in solution (Cabezon, E., Arechaga, I., Jonathan P., Butler, G., and Walker J. E. (2000) J. Biol. Chem. 275, 28353-28355). It was unclear until now whether the binding of the inhibitor protein to the F1 domains contributes to the process of F1F0-ATP synthase dimerization in intact mitochondria. Here we have directly addressed the involvement of the yeast inhibitor protein, Inh1, and its known accessory proteins, Stf1 and Stf2, in the formation of the yeast F1F0-ATP synthase dimer. Using mitochondria isolated from null mutants deficient in Inh1, Stf1, and Stf2, we demonstrate that formation of the F(1)F(0)-ATP synthase dimers is not adversely affected by the absence of these proteins. Furthermore, we demonstrate that the F1F0-ATPase monomers present in su e null mutant mitochondria can be as effectively inhibited by Inh1, as its dimeric counterpart in wild-type mitochondria. We conclude that dimerization of the F1F0-ATP synthase complexes involves a physical interaction of the membrane-embedded F0 sectors from two monomeric complexes and in a manner that is independent of inhibitory activity of the Inh1 and accessory proteins.     

Existence of stoichiometric amounts of an intrinsic ATPase inhibitor and two stabilizing factors with mitochondrial ATP synthase in yeast

Two proteinaceous factors, 15K and 9K proteins, which acted together to stabilize the inactivated yeast F1F0-ATPase-inhibitor complex [Hashimoto, T., et al. (1984) J. Biochem. 95, 131-136] were hardly distinguishable from the sigma and epsilon subunits, respectively, of yeast F1-ATPase by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. However, they were clearly distinguishable from these subunits by analyses of the sequences at their amino terminals and by immunoblotting combined with SDS polyacrylamide gel electrophoresis. The two stabilizing factors and an ATPase inhibitor existed in mitochondria in equimolar ratios to F1-ATPase. These three protein factors were not present in purified F1-ATPase or in F1F0-ATPase preparations, but remained in the mitochondrial membranes after extraction of F1F0-ATPase with Triton X-100. These observations strongly suggest that the two stabilizing factors and the ATPase inhibitor form a regulatory substructure of mitochondrial ATP synthase, in addition to the F1 and F0 subunits.     

Cell-free synthesis of mitochondrial ATPase inhibitor precursor and its transport into yeast mitochondria

ATPase inhibitor protein, which blocks mitochondrial ATPase activity by forming an enzyme-inhibitor complex, was found to be synthesized as a larger precursor in a cell-free translation system directed by yeast mRNA. Other protein factors, which stabilize latent ATPase by binding to the enzyme-inhibitor complex, were also found to be formed as larger precursors. The precursor of ATPase inhibitor protein was transported into isolated yeast mitochondria and was cleaved to the mature peptide in the mitochondria. Impaired mitochondria lacking phosphorylation activity could not convert the precursor to the mature form. Neither antimycin A nor oligomycin alone exhibited a marked effect on the transport-processing of the precursor by intact mitochondria. However, when antimycin A was added with oligomycin, the transport-processing was markedly inhibited. The processing was also strongly inhibited by an uncoupler, carbonylcyanide p-trifluoro-methoxyphenyl hydrazone. The inhibition by the uncoupler was not relieved by ATP added externally. It is concluded that the transport-processing of precursor proteins requires intact mitochondria with a potential difference across the inner membrane.     

The epsilon subunit as an ATPase inhibitor of the F1-ATPase in Escherichia coli

The isolation of protein ATPase inhibitor was attempted directly from Escherichia coli membrane extracts to examine the possible presence of a Pullman-Monroy-type inhibitor [M. E. Pullman and G. C. Monroy (1963) J. Biol. Chem. 238, 3762-3769] distinct from the epsilon subunit of E. coli ATPase. Purification to homogeneity was achieved in a sequence of steps involving trichloracetic acid precipitation, DEAE-cellulose, Sephadex G75 chromatography, and a terminal isoelectric focusing step. An inhibitory protein was obtained and was identified by its physicochemical and inhibitory properties as the epsilon subunit of E. coli ATPase. The other inhibitory fraction observed in the purification procedure consisted of aggregated epsilon subunits.     

Solution structure of a C-terminal coiled-coil domain from bovine IF(1): the inhibitor protein of F(1) ATPase

Bovine IF(1) is a basic, 84 amino acid residue protein that inhibits the hydrolytic action of the F(1)F(0) ATP synthase in mitochondria under anaerobic conditions. Its oligomerization state is dependent on pH. At a pH value below 6.5 it forms an active dimer. At higher pH values, two dimers associate to form an inactive tetramer. Here, we present the solution structure of a C-terminal fragment of IF(1) (44-84) containing all five of the histidine residues present in the sequence. Most unusually, the molecule forms an anti-parallel coiled-coil in which three of the five histidine residues occupy key positions at the dimer interface.     

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.