Dicyclohexylcarbodiimide-binding Protein Research Articles

The Possible Role of Nonbilayer Structures in Regulating ATP Synthase Activity in Mitochondrial Membranes.

The effects of temperature and of the membrane-active protein CTII on the formation of nonbilayer structures in mitochondrial membranes were studied by 31P-NMR. Increasing the temperature of isolated mitochondrial fractions correlated with an increase in ATP synthase activity and the formation of nonbilayer packed phospholipids with immobilized molecular mobility. Computer modeling was employed for analyzing the interaction of mitochondrial membrane phospholipids with the molecular surface of CTII, which behaves like a dicyclohexylcarbodiimide-binding protein (DCCD-BP) of the F0 group in a lipid phase. Overall, our studies suggest that proton permeability toroidal pores formed in mitochondrial membranes consist of immobilized nonbilayer-packed phospholipids formed via interactions with DCCD-BP. Our studies support the existence of a proton transport along a concentration gradient mediated via transit toroidal permeability pores which induce conformational changes necessary for mediating the catalytic activity of ATP synthase in the subunits of the F0-F1 complex.

Mitochondrial DNA sequence analysis of the cytochrome oxidase subunit I and II genes, the ATPase9 gene, the NADH dehydrogenase ND4L and ND5 gene complex, and the glutaminyl, methionyl and arginyl tRNA genes from Trichophyton rubrum.

In this paper, we present the nucleotide sequence of a 5248 bp-long region of the mitochondrial (mt) genome of the dermatophyte Trichophyton rubrum. This region which represents about 1/4 of the total mt genome of this species reveals a compact organization of genes including: the glutaminyl tRNA, the methionyl tRNA, the cytochrome oxidase subunit I gene, the arginyl tRNA, the mitochondrial version of the ATPase subunit 9 gene, the cytochrome oxidase subunit II gene and a part of the NADH dehydrogenase ND4L and ND5 gene "complex". The main features of the part of mt DNA sequenced is the non-interrupted COXI gene and the presence in the mitochondrial version of the ATPase 9 gene of a small group IA intron. The extensive amino-acid sequence similarity with the equivalent gene in Aspergillus nidulans and Neuropora crassa indicates that this gene codes for a dicyclohexylcarbodiimide binding protein. The conserved arrangement of this portion of the mt genome and the presence of tRNAs between the protein-coding genes are compatible with a large polycistronic transcript processed by the excision of tRNAs, or similar secondary structures, as proposed for other fungal or mammalian mt DNAS.

Transcript levels for nuclear-encoded mammalian mitochondrial respiratory-chain components are regulated by thyroid hormone in an uncoordinated fashion.

Thyroid hormone is one of the few known physiological regulators of mammalian mitochondrial biogenesis. Although it exerts a global effect on biogenesis, it does so by regulating the expression of a limited number of unidentified mitochondrial proteins. We have investigated these hormone-regulated proteins in rat liver. Hormone injection induced a 30-fold increase in the levels of cytochrome-c1 mRNA after 3 d. In addition, the mRNA for the growth-activated adenine-nucleotide translocator, ANT2, was increased 13-fold and that for the ATPase N,N'-dicyclohexylcarbodiimide-binding protein increased 4-5-fold. Mitochondrial transcripts of cytochrome-oxidase subunit I also increased. No changes were found in the mRNA levels for the F1-ATPase beta-subunit or cytochrome oxidase IV. A single low dose of triiodothyronine induces rapid increases in cytochrome-c1 and ANT2 mRNA species which parallel changes in the activity of the hormone-responsive malic enzyme, but are earlier than other mitochondrial biogenetic events. These data strengthen the view that thyroid hormone regulates synthesis of specific components within each respiratory-chain complex and that these products apparently play key roles in inner-membrane biogenesis and assembly. The significance of ANT2 induction is also discussed with respect to the rapid respiratory response induced by thyroid hormone.

Plant mitochondrial F0F1 ATP synthase. Identification of the individual subunits and properties of the purified spinach leaf mitochondrial ATP synthase.

Spinach leaf mitochondrial F0F1 ATPase has been purified and is shown to consist of twelve polypeptides. Five of the polypeptides constitute the F1 part of the enzyme. The remaining polypeptides, with molecular masses of 28 kDa, 23 kDa, 18.5 kDa, 15 kDa, 10.5 kDa, 9.5 kDa and 8.5 kDa, belong to the F0 part of the enzyme. This is the first report concerning identification of the subunits of the plant mitochondrial F0. The identification of the components is achieved on the basis of the N-terminal amino acid sequence analysis and Western blot technique using monospecific antibodies against proteins characterized in other sources. The 28-kDa protein crossreacts with antibodies against the subunit of bovine heart ATPase with N-terminal Pro-Val-Pro- which corresponds to subunit F0b of Escherichia coli F0F1. Sequence analysis of the N-terminal 32 amino acids of the 23-kDa protein reveals that this protein is similar to mammalian oligomycin-sensitivity-conferring protein and corresponds to the F1 delta subunit of the chloroplast and E. coli ATPases. The 18.5-kDa protein crossreacts with antibodies against subunit 6 of the beef heart F0 and its N-terminal sequence of 14 amino acids shows a high degree of sequence similarity to the conserved regions at N-terminus of the ATPase subunits 6 from different sources. ATPase subunit 6 corresponds to subunit F0a of the E. coli enzyme. The 15-kDa protein and the 10.5-kDa protein crossreact with antibodies against F6 and the endogenous ATPase inhibitor protein of beef heart F0F1-ATPase, respectively. The 9.5-kDa protein is an N,N'-dicyclohexylcarbodiimide-binding protein corresponding to subunit F0c of the E. coli enzyme. The 8.5-kDa protein is of unknown identity. The isolated spinach mitochondrial F0F1 ATPase catalyzes oligomycin-sensitive ATPase activity of 3.5 mumol.mg-1.min-1. The enzyme catalyzes also hydrolysis of GTP (7.5 mumol.mg-1.min-1) and ITP (4.4 mumol.mg-1.min-1). Hydrolysis of ATP was stimulated fivefold in the presence of amphiphilic detergents, however the hydrolysis of other nucleotides could not be stimulated by these agents. These results show that the plant mitochondrial F0F1 ATPase complex differs in composition from the other mitochondrial, chloroplast and bacterial ATPases. The enzyme is, however, more closely related to the yeast mitochondrial ATPase and to the animal mitochondrial ATPase than to the chloroplast enzyme. The plant mitochondrial enzyme, however, exhibits catalytic properties which are characteristic for the chloroplast enzyme.

Dicyclohexylcarbodiimide-binding protein is a subunit of the [formula omitted] ATPase complex

Membrane ATPase of Methanosarcina barkeri was inhibited by N, N′-dicyclohexylcarbodiimide (DCCD), whereas the extrinsic αβ complex of the same enzyme was not. Consistent with this finding, a 6,000 dalton (6 kDa) membrane protein was preferentially labeled with radioactive DCCD. The DCCD-sensitive ATPase was solubilized from the membranes with octylglucoside and purified in the presence of this detergent. The purified ATPase contained the α and β subunits and also at least four additional proteins (40, 27, 23 and 6 kDa). The 6 kDa protein in the purified enzyme reacted with DCCD, indicating that it is a subunit of an integral part of the M. barkeri ATPase complex.

The Paracoccus denitrificans cytochrome aa3 has a third subunit.

The presence of a third polypeptide subunit in Paracoccus cytochrome c oxidase is demonstrated. This protein (apparent molecular mass 23 kDa) binds dicyclohexylcarbodiimide in membranes of aerobically grown bacteria and in the purified enzyme. The N-terminal amino-acid sequence of this dicyclohexylcarbodiimide-binding protein is identical to the deduced sequence of the COIII gene product [Raitio et al. (1987) EMBO J. 6, 2825-2833]. We conclude that the aa3-type oxidase in Paracoccus is composed of at least three subunits, which correspond to the three mitochondrially coded polypeptides in the eukaryotic enzyme.

Proton translocation by the H+-ATPase of mitochondria. Effect of modification by monofunctional reagents of thiol residues in F0 polypeptides.

A study is presented on the effect of chemical modification of thiol groups on proton conduction by the H+-ATPase complex in 'inside out' submitochondrial particles, before and after removal of the F1 moiety, and by F0 liposomes. The results obtained show that modification with monofunctional reagents [N-ethylmaleimide, 2,2'-dithiobispyridine, mersalyl and N-(7-dimethylamino-4-methyl-coumarinyl)-maleimide] of thiol residues in membrane integral proteins of F0 results in inhibition of proton conduction. Comparison of the inhibitory effects with the binding of [14C]N-ethylmaleimide to the various F0 polypeptides indicates that the inhibition of proton conduction by thiol reagents was correlated with modification of the 25-kDa, 11-kDa and 9-kDa (N,N'-dicyclohexylcarbodiimide-binding protein) proteins. Involvement of the last component is supported by the observation that modification by thiol reagents depressed the binding of N,N'-dicyclo[14C]hexylcarbodiimide to the 9-kDa protein.

Kinetics of inhibition and binding of dicyclohexylcarbodiimide to the 82,000-dalton mitochondrial K+/H+ antiporter.

Inhibition of K+/H+ antiport by N,N'-dicyclohexylcarbodiimide in Mg2+ depleted mitochondria follows first order kinetics, exhibiting a half-time of 13 min when mitochondria are incubated with 50 nmol/mg inhibitor at 0 degrees C. 14C radiolabeled N,N'-dicyclohexylcarbodiimide binds to the 82,000-dalton protein, and the second order rate constant for binding is found to be approximately the same as the second order rate constant for inhibition. These findings provide additional confirmation of the identification of this porter with the 82,000-dalton protein and permit us to estimate that rat liver mitochondria contain about 8 pmol/mg of K+/H+ antiporter with a turnover number of 700 s-1. The K+/H+ antiporter of rat liver mitochondria is protected from N,N'-dicyclohexylcarbodiimide inhibition and binding by quinine and by endogenous Mg2+. An 82,000-dalton, [14C]N,N'-dicyclohexylcarbodiimide-binding protein is also observed in rat liver submitochondrial particles, establishing this as an integral protein of the inner membrane. Submitochondrial particles, presumed to be inverted in membrane orientation, are protected from radiolabeling by external Mg2+, supporting the contention that the Mg2+ binding site is localized to the matrix side of the K+/H+ antiporter.

H+-ATPase of Escherichia coli. An uncE mutation impairing coupling between F1 and Fo but not Fo-mediated H+ translocation.

The uncE114 mutation from Escherichia coli strain KI1 (Nieuwenhuis, F. J. R. M., Kanner, B. I., Gutnick, D. L., Postma, P. W., and Van Dam, K. (1973) Biochim. Biophys. Acta 325, 62-71) was characterized after transfer to a new genetic background. A defective H+-ATPase complex is formed in strains carrying the mutation. Based upon the genetic complementation pattern of other unc mutants by a lambda uncE114 transducing phage, and complementation of uncE114 recipients by an uncE+ plasmid (pCP35), the mutation was concluded to lie in the uncE gene. The uncE gene codes for the omega subunit ("dicyclohexylcarbodiimide binding protein") of the H+-ATPase complex. The mutation was defined by sequencing the mutant gene. The G----C transversion found results in a substitution of Glu for Gln at position 42 of the omega subunit in the Fo sector of the H+-ATPase. The substitution did not significantly impair H+ translocation by Fo or affect inhibition of H+ translocation by dicyclohexylcarbodiimide. Wild-type F1 was bound by uncE114 Fo with near normal affinity, but the functional coupling between F1 and Fo was disrupted. The uncoupling was indicated by an H+-leaky membrane, even when saturating levels of wild-type F1 were bound. Disassociation of F1 from Fo under conditions of assay did partially contribute to the H+ leakiness, but the major contributor to the high H+ conductance was Fo with bound F1. The F1 bound to uncE114 membranes exhibited normal ATPase activity, but ATP hydrolysis was uncoupled from H+ translocation and was resistant to inhibition by dicyclohexylcarbodiimide. The F1 isolated from the uncE114 mutant was modified with partial loss of coupling function. However, this modification did not account for the uncoupled properties of the mutant Fo described above, since these properties were retained after reconstitution of mutant membrane (Fo) with wild-type F1.

The 35 kDa DCCD-binding protein from pig heart mitochondria is the mitochondrial porin

The protein which can be labelled by low concentrations of dicyclohexylcarbodiimide in the M r region of 30000–35000 has been purified from pig heart mitochondria with a high yield and as a single band of apparent M r 35000 in dodecyl sulphate-containing gels. The protein is not identical with the phosphate carrier as suggested before, since the two proteins behave differently during isolation. Incorporation of the isolated 35 kDa dicyclohexylcarbodiimide-binding protein into lipid bilayer membranes causes an increase of the membrane conductance in definite steps, due to the formation of pores. The specific pore-forming activity increases during the purification procedure. The single pore conductance is about 4.0 nS, suggesting a diameter of 1.7 nm of the open pore. The pore conductance is dependent on the voltage across the membrane. Anion permeability of the pore is higher than cation permeability. These properties are similar to those described for isolated mitochondrial and bacterial porins. It is concluded that the 35 kDa dicyclohexylcarbodiimide-binding protein from pig heart mitochondria is identical with porin from outer mitochondrial membrane.

Nucleotide sequence of F 0 -ATPase proteolipid (subunit 9) gene of maize mitochondria

The F(0)-ATPase proteolipid, also referred to as subunit 9 or the dicyclohexylcarbodiimide-binding protein, is encoded by a mitochondrial gene in maize that we have designated atp 9. The clone containing atp 9 was selected for investigation from a mitochondrial DNA library because of its abundant transcript in total maize mitochondrial RNA preparations. Sequence analysis of the clone revealed an open reading frame that was readily identified by its nucleotide homology with the ATPase subunit 9 gene of yeast. As deduced from the nucleotide sequence, the maize ATPase subunit 9 protein contains 74 amino acids with a molecular weight of 7368. Substantial amino acid sequence homology is conserved among maize, yeast, bovine, and Neurospora mitochondrial ATPase subunit 9 proteins, regardless of whether the gene is nuclearly encoded (bovine and Neurospora) or mitochondrially encoded (yeast and maize). RNA transfer blot analysis indicated that the gene sequence is actively transcribed, producing an initial transcript that is large and extensively processed.

Processing peptidase of Neurospora mitochondria. Two-step cleavage of imported ATPase subunit 9.

Subunit 9 (dicyclohexylcarbodiimide binding protein, 'proteolipid') of the mitochondrial F1F0-ATPase is a nuclearly coded protein in Neurospora crassa. It is synthesized on free cytoplasmic ribosomes as a larger precursor with an NH2-terminal peptide extension. The peptide extension is cleaved off after transport of the protein into the mitochondria. A processing activity referred to as processing peptidase that cleaves the precursor to subunit 9 and other mitochondrial proteins is described and characterized using a cell-free system. Precursor synthesized in vitro was incubated with extracts of mitochondria. Processing peptidase required Mn2+ for its activity. Localization studies suggested that it is a soluble component of the mitochondrial matrix. The precursor was cleaved in two sequential steps via an intermediate-sized polypeptide. The intermediate form in the processing of subunit 9 was also seen in vivo and upon import of the precursor into isolated mitochondria in vitro. The two cleavage sites in the precursor molecule were determined. The data indicate that: the correct NH2-terminus of the mature protein was generated, the NH2-terminal amino acid of the intermediate-sized polypeptide is isoleucine in position -31. The cleavage sites show similarity of primary structure. It is concluded that processing peptidase removes the peptide extension from the precursor to subunit 9 (and probably other precursors) after translocation of these polypeptides (or the NH2-terminal part of these polypeptides) into the matrix space of mitochondria.

Comparison of the vacuolar membrane ATPase of Neurospora crassa with the mitochondrial and plasma membrane ATPases.

The vacuolar membrane ATPase of Neurospora crassa closely resembles the mitochondrial ATPase in its substrate specificity, substrate affinity, and sensitivity to the inhibitor N,N'-dicyclohexylcarbodiimide. Three different mutants with altered mitochondrial ATPase activity, exhibited as 1) resistance to N,N'-dicyclohexylcarbodiimide, 2) enhanced sensitivity to N,N'-dicyclohexylcarbodiimide, and 3) very low specific activity, were found to be unaltered in the vacuolar membrane ATPase. The vacuolar membrane ATPase was similar to the mitochondrial ATPase and approximately 10-fold more sensitive than the plasma membrane ATPase in its sensitivity to the inhibitors 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate, and 5'-adenylylimidodiphosphate. By contrast, the vacuolar ATPase resembled the plasma membrane ATPase in its response to quercetin (both 10-fold more sensitive than the mitochondrial ATPase); it was unique in its sensitivity to KNO3. A N,N'-dicyclohexylcarbodiimide-binding protein, migrating between molecular weight markers of 14,400 and 21,500, was identified as a putative component of the vacuolar membrane ATPase. Taken together, these findings support the argument that the vacuolar membrane ATPase is a distinct enzyme, more like the mitochondrial F0F1 ATPase than the plasma membrane ATPase.

H+-ATPase of Escherichia coli uncB402 mutation leads to loss of chi subunit of subunit of F0 sector.

The uncB402 mutation in Escherichia coli results in formation of an H+-ATPase complex that is defective in energy-transducing capacity. The mutation, originally described by Butlin et al. (Butlin, J.D., Cox, G.B., and Gibson, F. (1973) Biochim. Biophys. Acta 292, 366-375), alters the F0 sector of the H+-ATPase complex. Here, we show that uncB402 is an amber-suppressible, chain-terminating mutation that results in loss of the chi subunit from F0. This was demonstrated in crude membrane fractions after overproduction of the ATPase complex by heat induction of a lambda transducing phage carrying the unc operon of uncB402. The lambda-uncB402 DNA was used as a template in an in vitro transcription-translation system. A synthesis product that may correspond to the truncated form of the chi subunit was observed. Despite the absence of chi, the F1-ATPase was still bound to the membrane, although more weakly than in wild type. The omega subunit of F0 ("dicyclohexylcarbodiimide-binding protein") shows normal reactivity with dicyclohexylcarbodiimide, indicating that at least this portion of F0 integrates properly in the membrane in the absence of the chi subunit. The F0 of uncB402 was not functional in H+ translocation activity. This was shown by direct H+ flux measurements with crude membrane vesicles that were treated with guanidine to disrupt the binding of F1 to F0. Secondly, a method was developed for isolation of F0 from F1-depleted membranes. The F0 from uncB402 was shown to have less than 5% the proton-translocase activity of wild type F0 when reconstituted into liposomes. Although the uncB402 mutant shows these defects, the question of whether the chi subunit plays a direct role in F1-binding or H+ translocation remains open, since the loss of chi may lead to subtle changes in the assembly of the other F0 subunits. Analysis of other mutants should permit a more definitive assignment of function.

Amino acid composition of a new mitochondrially translated proteolipid isolated from yeast mitochondria and from the OSATPase complex

A new mitochondrially translated 10000 Mr proteolipid was isolated from yeast mitochondria. This proteolipid was purified by phosphocellulose chromatography, followed by reverse phase HPLC. This proteolipid was also extracted from the oligomycin sensitive ATPase complex and purified by HPLC. Its amino acid composition is different from the Dicyclohexylcarbodiimide binding protein.

The dicyclohexylcarbodiimide-binding protein c of ATP synthase from Escherichia coli is not sufficient to express an efficient H+ conduction.

Bacteriophage Mu was inserted into the unc genes of Escherichia coli. The resulting mutation AS12 had a polar effect on the unc operon: membranes of the mutant AS12 contained the dicyclohexylcarbodiimide-binding protein c and the protein a as sole subunits of the ATP synthase. It was shown by peptide mapping and amino acid analysis of the fragments that protein c from mutant AS12 was identical with the wild-type protein c. The absence of subunit b in mutant AS12 drastically lowered the H+ conduction dependent on the membrane-integrated moiety (F0) of the ATP synthase. This suggests that both subunits b and c are necessary for an efficient expression of H+ conduction.

Photoaffinity labeling of a mitochondrial hydrophobic protein by an anisotropic inhibitor of energy transduction in oxidative phosphorylation.

The monoazide derivative of ethidium, the parent compound of which is an anisotropic inhibitor of energy transduction in oxidative phosphorylation, was synthesized and shown to be useful as a photoaffinity probe. Results showed that monoazide ethidium specifically binds to a hydrophobic protein of mitochondria (with an apparent molecular weight of about 6200 in the presence of 0.1% sodium dodecyl sulfate). The molar binding ratios of monoazide ethidium to protein were about 5 and 17 with protein in the nonenergized and energized states, respectively. This protein differed from the dicyclohexylcarbodiimide-binding protein. We refer to this new hydrophobic protein, anisotropic inhibitor-binding protein, in this paper.

Studies on the immunological properties of complex V (mitochondrial ATP synthetase complex)

Antibody raised in rabbits against Complex V (miochondrial ATP synthetase complex) purified from beef heart mitochondria cross-reacted with Complex V and submitochondrial particles from beef heart, beef adrenals, and rat liver as shown by double-diffusion and rocket immunoelectrophoresis analysis. Of the various isolated and purified components of Complex V, only the oligomycin sensitivity-conferring protein showed strong reactivity with the anti-Complex V antibody, soluble F 1-ATPase reacted very faintly, while F 6 and ATPase inhibitor protein showed no precipitin lines. Crossed immunoelectrophoresis indicated that antigenic determinants recognized by the antibody were present on OSCP and possibly on the dicyclohexylcarbodiimide-binding protein. The components of Complex V could be precipitated from beef heart submitochondrial particles dissolved in Triton X-100 and pretreated with control IgG. When the composition of the immunoprecipitate was compared to that of purified Complex V, all the constituent polypeptides of the latter were present in the immunoprecipitate, except for one polypeptide in the low-molecular-weight region. Incubation of Complex V or submitochondrial particles with the anti-Complex V antibody in the absence of Triton X-100 caused inhibition of ATP- P i exchange but not of ATPase activity. In the presence of Triton X-100, oligomycin sensitivity of Complex V was lost and the antibody was able to inhibit also the ATPase activity. The enzymic activity of soluble F 1-ATPase was unaffected by the antibody in the absence or presence of Triton X-100. These results suggest that the anti-Complex V antibody might be a useful tool for identifying and probing the role of Complex V components involved in energy transduction.

Nucleotide sequence of the gene coding for the δ subunit of proton-translocating ATPase of Escherichia coli

The DNA sequence of a part of the gene cluster for the proton-translocating ATPase of E. coli was determined. Two reading frames were found between the genes for dicyclohexylcarbodiimide-binding protein and α subunit. The following evidence indicates that one frame codes the δ subunit; i) The primary structure deduced from DNA sequence agreed with amino acid composition of the protein. ii) Five residues from the amino terminal were the same as those deduced from DNA sequence. The α helix content of this protein (estimated from the primary structure) was 60.2% of the total residues with the longest helical domain of 50 residues. The intercistronic sequence between the genes for δ and α had 15 base pairs, suggesting that the synthesis of the α is not regulated transcriptionally. The organization of the gene cluster was also shown on the physical map.

Structural gene coding for the dicyclohexylcarbodiimide-binding protein of the proton-translocating ATPase from escherichia coli: Locus of the gene in the F 1F 0 gene cluster

The proton-translocating ATPase (F,-F,) is an apparatus for the synthesis of ATP common to different energy-transducing organelles, such as mitochondria, chloroplasts and bacterial cytoplasmic membranes (reviews [l-4] ). The ATPase consists of two portions, F, and F,. F, is a peripheral membrane component with 5 different subunits (cw,P.rJ ,E), and it is bound to the integral membrane component, Fo, which functions as a proton pathway. Two or more subunits have been found in F, from different sources. The proteolipid subunits, dicyclohexylcarbodiimide (DCCD)-binding proteins, have been purified from several organisms [2,5,6] and their primary sequences have been determined [7]. Two types of E. coli mutant of this subunit, which both result in inability to bind DCCD, have been reported; the F, of mutant strain DG7/1 is defective in proton translocation [8,9], whereas that of the other type of mutant RF7 [lo] is normal. Thus DG7/1 cannot grow with succinate as the sole carbon source because of defective oxidative phosphorylation, whereas RF7 can grown normally. Both mutant strains have functional F,, although their membrane ATPase activities are not sensitive to DCCD because the mutant protein cannot bind this compound. DCCD binds to an aspartyl residue at position 61 of the polypeptide, and this residue is replaced by a glytine residue in the protein of DG7/1 [8,9]. It has not been shown directly whether these mutations are in