Properties of a novel ATPase enzyme in chromaffin granules

Membranes were isolated from mitochondria and chromaffin granules of bovine adrenal medullae. The cross-contamination between the two membranes was examined by comparing the radioactive bands on autoradiograms of gels after phosphorylation of the membranes with [-³²P]-ATP and decoration with [¹²⁵I]concanavalin A and [¹²⁵I]protein A with antibody that was raised against chromaffin-granule membranes. It was found that the membranes cross-contaminated each other by less than 10%. The technique of immunodecoration with antibodies against subunits of proton-ATPases from yeast mitochondria, spinach chloroplasts, andE. coli membranes was used for quantitative estimation of proton-ATPase complexes in chromaffin granules and mitochondrial membranes. It was found that chromaffin-granule membranes contain less than 10% of the amount of proton-ATPase complex in mitochondrial membranes. The specific ATPase activity of chromaffin-granule membranes was on the order of 30 to 50% of the mitochondrial membranes. The ATPase activity of the chromaffin-granule membranes was more sensitive to 4-acetamido-4-isothiocyano-2,2-disulfonic acid stilbene and 4-chloro-7-nitrobenzofurazan. It was much less sensitive than the mitochondrial membranes to antibody against subunit of proton-ATPase fromE. coli membranes. After solubilization of chromaffin-granule membranes by octyglucoside and cholate and subsequent centrifugation on sucrose gradient, two different ATPase enzymes were separated. The heavier enzyme was identical to the mitochondrial-ATPase complex, while the lighter enzyme was identified as a novel ATPase, which might be responsible for the special properties of the ATPase activity of chromaffin-granule membranes.Abbreviations DCCD dicyclohoxylcarbodiimide - NBD-Cl 4-chloro-7-nitrobenzofurazan - SITS 4-acetamido-4-isothiocyano-2,2-disulfonic acid stilbene - SDS sodium dodecyl sulfate - MES 2-(N-morpholino)ethane sulfonic acid - FITC fluorescein isothiocyanate     

Subunit composition of the H+-ATPase complex from anaerobic bacterium Lactobacillus casei

The H+-ATPase complex has been isolated from the membranes of the anaerobic bacterium Lactobacillus casei by two independent methods. 1. The crossed-immunoelectrophoresis of the 14C-labelled ATPase complex against antibodies to a highly purified soluble ATPase has been used. The subunit composition of the complex has been established by autoradiography. The soluble part of L. casei ATPase, in contrast to coupling factor F1-ATPases of aerobic bacteria, chloroplasts and mitochondria which include two kinds of large subunit (alpha and beta), consists of one kind of large subunit with a molecular mass of 43 kDa. Moreover, a minor polypeptide of 25 kDa has been found in the soluble ATPase. Factor F0 of L. casei ATPase complex consists of a 16-kDa subunit and two subunits with molecular masses less than 14 kDa. 2. A dicyclohexylcarbodiimide-sensitive ATPase complex has been isolated from L. casei membranes by treating them with a mixture of octyl glucoside and sodium cholate. The complex, purified by centrifugation on a sucrose density gradient, contains the main subunits with molecular masses of 43 kDa, 25 kDa and 16 kDa and a dicyclohexylcarbodiimide-binding subunit with a molecular mass less than 14 kDa.     

The biogenesis of rat liver mitochondrial ATPase. Subunit composition of the normal ATPase complex and of the deficient complex formed when mitochondrial protein synthesis is blocked.

1. An ATPase complex containing 12 subunits was isoalted from rat liver mitochondria. 2. In vivo inhibition of mitochondrial protein synthesis by the chloramphenicol analogue thiamphenicol leads to the formation of an oligomycin-insensitive membrane-bound ATPase complex in mitochondria of regenerating rat liver. 3. This oligomycin-insensitive, membrane-bound ATPase was isolated by the same procedure as the ATPase complex from regenerating livers of untreated animals. 4. SDS-polyacrylamide gel electrophoresis of in vivo labelled ATPase complexes from control and from thiamphenicol-treated rats reveals that three subunits out of the 12 are not synthesized or assembled when the mitochondrial translation activity is blocked. 5. From the subunits synthesized and assembled when mitochondrial pror (Fo) of the ATPase complex (subunit 5). 6. The oligomycin sensitivity-conferring protein seems absent in the ATPase complex formed in the presence of thiamphenicol.     

The rate of import and assembly of F1-ATPase in Saccharomyces cerevisiae

Subunit specific antiserum can be employed to study the course of ATPase assembly in mitochondria isolated from bakers' yeast. Comparing rates of subunit import with rates of enzyme assembly indicated that no substantial pool of unassembled subunits exists for the three largest ATPase peptides (alpha, beta, and gamma). Blocking import of specific ATPase subunits, however, did reveal a possible accumulation of unassembled alpha and gamma subunits in isolated mitochondria. The kinetic experiments also revealed a lag in the import of beta subunit relative to the uptake of alpha and gamma precursors. Experiments conducted in yeast cells confirmed that beta subunit is assembled soon after it is imported, but did not indicate a delay in import relative to the other subunits of F1.     

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.     

Aurovertin binds to the beta subunit of yeast mitochondrial ATPase.

Isolated beta subunit of ATPase (F1) from yeast mitochondria does not catalyze an ATPase reaction but still binds the specific F1 inhibitor aurovertin. Binding was measured by enhancement of aurovertin fluorescence; it was as tight as that to F1-ATPase. No binding was observed with F1 or with isolated beta subunit from a single-gene nuclear yeast mutant whose F1-ATPase was resistant to aurovertin.     

Immunological studies on beef-heart ubiquinol--cytochrome c reductase (complex III)

Antibodies against isolated beef-heart ubiquinol--cytochrome c reductase (complex III) have been characterized. Antibodies to complex III react strongly with isolated beef heart complex III and intact beef heart mitochondria, as shown by immunodiffusion and rocket electrophoresis experiments. The complex III content of intact mitochondria can be quantitated with rocket electrophoresis using isolated complex III as a standard. Antibodies to complex III also react with beef liver mitochondria and with both heart and liver mitochondria from rats. The latter are very weak antigens compared to beef heart material. Antibodies to complex III do not react with respiratory chain complexes I and IV, or F1-ATPase from beef heart mitochondria, but gives a slight, but variable, reaction with complex II and the membrane fraction isolated from complex V (oligomycin-sensitive ATPase). Antigenic sites are located on at least five of the seven peptides of complex III. These peptides are presumably lacking in respiratory chain complexes which do not react with antibodies to complex III, and are assumed to be uniquely located in complex III. Antiserum against complex III inhibitis duroquinol--cytochrome c reductase activity in isolated complex III and in complex III incorporated into phospholipid vesicles. Oxidation of NADH and succinate is not affected in submitochondrial particles treated with 6-times more antibody than required for complete inhibition of enzyme activity in free complex III or in complex III-phospholipid vesicles.     

ATP10, a yeast nuclear gene required for the assembly of the mitochondrial F1-F0 complex

A yeast nuclear gene (ATP10) is reported whose product is essential for the assembly of a functional mitochondrial ATPase complex. Mutations in ATP10 induce a loss of rutamycin sensitivity in the mitochondrial ATPase but do not affect respiratory enzymes. This phenotype has been correlated with a defect in the F0 sector of the ATPase. The wild type ATP10 gene has been cloned by transformation of an atp 10 mutant with a yeast genomic library. The gene codes for a protein of Mr = 30,293. The primary structure of the ATP10 product is not related to any known subunit of the yeast or mammalian mitochondrial ATPase complexes. To further clarify the role of this new protein in the assembly of the ATPase, an antibody was prepared against a hybrid protein expressed from a trpE/ATP 10 fusion gene. The antibody recognizes a 30-kDa protein present in wild type mitochondria. The protein is associated with the mitochondrial membrane but does not co-fractionate either with F1 or with the rutamycin-sensitive F1-F0 complex. These data suggest that the ATP10 product is not a subunit of the ATPase complex but rather is required for the assembly of the F0 sector of the complex.     

A mitochondrial presequence can transport a chloroplast-encoded protein into yeast mitochondria

Ribulose-1,5-bisphosphate carboxylase/oxygenase of chloroplasts contains eight large and eight small subunits. The small subunit is encoded by nuclear DNA, synthesized in the cytoplasm, and imported into chloroplasts. The large subunit is encoded by chloroplast DNA and synthesized within chloroplasts. We show in this communication that the large subunit of Chlamydomonas chloroplasts could be efficiently imported into isolated yeast mitochondria if it was attached to the presequence of a protein transported into the yeast mitochondrial matrix. Thus, synthesis of the large subunit within chloroplasts does not reflect the inability of this subunit to cross membranes. The same mitochondrial presequence could also transport the nuclear-encoded small subunit into yeast mitochondria. However, when the two types of subunits were coimported into mitochondria, they did not assemble with each other inside the heterologous organelle.     

Rhodamine 6G inhibits the matrix-catalyzed processing of precursors of rat-liver mitochondrial proteins

Several inner membrane proteins from rat liver mitochondria have been translated for the first time in rabbit reticulocyte lysates. These include the Rieske iron-sulfur protein, cytochrome c1 and core protein I of the cytochrome bc1 complex, the alpha and beta subunits of F1 ATPase, and subunit IV of cytochrome oxidase. All were translated from free polysomes as larger-molecular-mass precursors, and were processed to their mature forms by isolated liver mitochondria or by the isolated mitochondrial matrix fraction. In vitro processing, catalyzed by the isolated matrix fraction, is inhibited by rhodamine 6G. The latter is a fluorescent probe, which accumulates specifically in mitochondria of whole cells and which is used extensively to visualize mitochondrial morphology. The concentration of rhodamine 6G required for inhibition in vitro is similar to that of o-phenanthroline. Rhodamine 6G inhibits matrix-catalyzed processing of all precursors tested, indicating that the mechanism of inhibition is common for a variety of functionally unrelated precursors. The novel action of rhodamine 6G reported here can form the basis for its inhibition of precursor processing in intact hepatoma cells [Kolarov, J. & Nelson, B.D. (1984) Eur. J. Biochem. 144, 387-392].     

Changes in activity and F1 content of mitochondrial H+-ATPase in regenerating rat liver

Submitochondrial particles prepared from rat liver during hepatic regeneration exhibit a depressed ATPase activity which is correlated with a decrease in F1 subunit content as shown by SDS-PAGE. Use of an antibody directed against the F1 portion of the H+-ATPase complex demonstrated that there is a definite decrease in the amount of beta-subunit of F1 in both submitochondrial particles and mitochondria from rat liver 24 h after partial hepatectomy.     

The deactivation process of the proton-ATPase in isolated chloroplasts and whole leaves

Using rapid micromethods for chloroplast isolation and ATPase solubilization from preilluminated leaves, the deactivation of the proton-ATPase after different treatments was compared. The rate of decay of the ‘in vivo’ light-activated membrane-bound Mg²⁺-ATPase was highly dependent on temperature. However, the soluble Ca²⁺-ATPase, extracted from the temperature-inactivated membrane-bound ATPase, was active. Coupling factor 1 with a manifest and stable Ca²⁺-ATPase activity was also solubilized from chloroplasts activated by light in whole leaves and deactivated after chloroplast isolation with gramicidin D. Deactivation of the proton-ATPase in isolated chloroplasts was only associated with the dissipation of the proton gradient. Reaction of the accessible sulfhydryl groups of the membrane-bound proton-ATPase with iodoacetamide prevent inactivation of the enzyme by oxidants. However, the iodoacetamide treatment had not effect on the temperature-dependent decay. The rate of deactivation of the proton-ATPase in whole leaves was similar for both membrane-bound and soluble ATPases. Thus, the oxidation process may play an important role in physiological conditions.     

Acyl carrier protein is present in the mitochondria of plants and eucaryotic micro-organisms

Proteins antigenically similar to the acyl carrier protein (ACP) found in the mitochondria of Neurospora crassa were detected by immunoblotting and radioimmunoassay techniques in mitochondria isolated from yeast, potatoes, and pea leaves. These mitochondrial proteins were similar to Neurospora ACP both in their electrophoretic mobility and in their unusual decrease in mobility upon reduction. Authentic ACP(s) show this type of change upon conversion of the acylated to the unacylated form. Purified ACP from both spinach chloroplasts and Escherichia coli cells cross-reacted with antibodies raised against Neurospora ACP. Purified ACP from Neurospora cross-reacted with antibodies raised against spinach chloroplast ACP and E. coli ACP. Mitochondria isolated from beef heart and rat brain were tested extensively and exhibited no cross-reaction with any of the three anti-ACP preparations. The discovery of ACP in the mitochondria of other organisms raises questions concerning the possible relationship between ACP and beta-oxidation in mitochondria, the involvement of ACP in de novo biosynthesis of some of the acyl chains in mitochondria and the subcellular locations of fatty acid biosynthesis in plants and eucaryotic micro-organisms.     

Characterization of epitopes of the yeast mitochondrial H+-ATPase complex recognized by monoclonal antibodies

Nine monoclonal antibodies which react with the beta subunit of the yeast mitochondrial H+-ATPase and three which react with a 25 kDa subunit of the enzyme complex (P25) have been characterized. Competitive binding studies indicated the presence of at least four antigenic regions on the beta subunit of the enzyme complex. One antigenic region of the beta subunit is recognized by two monoclonal antibodies RH 57.1 and RH 45.5 which inhibit the ATPase activity to different degrees. Antibody RH 48.6 appears to bind to a second region on the beta subunit and has no effect on the ATPase activity. A third region of the beta subunit is recognized by antibodies RH 51.4 and RH 72.1. RH 51.4 has no effect on the ATPase activity, whereas RH 72.1 stimulates ATPase activity. Antibody RH 32.4 which has no effect on the ATPase activity appears to bind to the fourth epitope of the beta subunit. All three monoclonal anti-P25 antibodies, RH 66.3, RH 41.2 and RH 37.0, apparently bind to the same antigenic region on this subunit. Two of the monoclonal anti-beta antibodies RH 48.6 and RH 51.4 were found to be very effective in immunoprecipitating the whole H+-ATPase complex in a solid phase system. However, the other monoclonal antibodies (and also a polyclonal antiserum) appear to induce the dissociation of one or more of the H+-ATPase subunits by their binding to the epitopes on the beta or the P25 subunits.     

The association of the autoantigens of primary biliary cirrhosis with the mitochondrial H+-ATPase--a reassessment

The claimed association between the M2 autoantigens of primary biliary cirrhosis (PBC) and the mitochondrial H+-ATPase has been re-examined in view of the recent reports that PBC autoantibodies react specifically with the lipoate acetyl transferases of 2-oxo acid dehydrogenases. Study of F0F1-ATPase purified from human and yeast mitochondria, and the comparison between immunoprecipitates obtained with antibodies against the H+-ATPase beta subunit and anti-M2 antibodies of PBC, established that the M2 antigens are not associated with the H+-ATPase complex. The M2 antigens did copurify with a crude bovine heart F1-ATPase preparation, but not with F1-ATPase from yeast, human heart or human liver.     

Immunological comparison of the pyruvate dehydrogenase complexes from pea mitochondria and chloroplasts

The pyruvate dehydrogenase complex (PDC) in pea (Pisum sativum L., cv. Little Marvel) was studied immunologically using antibodies to specific subunits of mammalian PDC. Pea mitochondria and chloroplasts were both found to contain PDC, but distinct differences were noted in the subunit relative molecular mass (M_r) values of the individual enzymes in the mitochondrial and chloroplast PDC complexes. In particular, the mitochondrial E3 enzyme (dihydrolipoamide dehydrogenase; EC 1.8.1.4) has a high subunit M_r value of 67 000, while the chloroplast E3 enzyme has a subunit M_r value of 52 000, similar in size to the prokaryotic, yeast ad mammalian E3 enzymes. In addition, component X (not previously noted in plant PDC) was also found to be present in two distinct forms in pea mitochondrial and chloroplast complexes. As in the case of E3, mitochondrial component X has a higher subunit M_r value (67 000) than component X from chloroplasts (48 000), which is similar in size to its mammalian counterpart. The subunit M_r value of E2 (dihydrolipoamide acetyltransferase; EC 2.3.1.12) in both mitochondria and chloroplasts (50 000) is lower than that of mammalian E2 (74 000) but similar to that of yeast E2 (58 000), and is consistent with the presence of only a single lipoyl domain. Neither mitochondria nor chloroplasts showed any appreciable cross-reactivity with antiserum to mammalian E1 (pyruvate dehydrogenase; EC 1.2.4.1). However, mitochondria cross-reacted strongly with antiserum to yeast E1, giving a single band (M_r 41 000) which is thought to be E1_a. Chloroplasts showed no cross-reactivity with yeast E1, indicating that the mitochondrial E1_a subunit and its chloroplast equivalent are antigenically distinct polypeptides.Abbreviations E1 pyruvate dehydrogenase - E2 dihydrolipoamide acetyltransferase - E3 dihydrolipoamide dehydrogenase - M_r relative molecular mass - PDC pyruvate dehydrogenase multienzyme complex - SDS sodium dodecyl sulphate The financial support of the Agricultural and Food Research Council is gratefully acknowledged. We thank Steve Hill (Department of Botany, University of Edinburgh, UK) for advice on mitochondrial isolation, and James Neagle (Department of Biochemistry, University of Glasgow) and Ailsa Carmichael for helpful discussion.     

Studies on polypeptide composition, hydrolytic activity and proton conduction of mitochondrial FoF1 H+ ATPase in regenerating rat liver

A study of the FoF1 ATPase complex of mitochondria isolated from regenerating rat liver following partial (70%) hepatectomy is presented. As we have previously reported, ATPase activity in submitochondrial particles prepared from regenerating rat liver 24 h following partial hepatectomy was depressed by 75% with respect to controls (submitochondrial particles from sham-operated animals). Polyacrylamide gel electrophoresis and immunodecoration using an antibody raised against isolated bovine heart F1 sector of the FoF1 ATPase indicated a substantial decrease in F1 content in the mitochondrial membrane from regenerating rat liver. Proton conduction by the FoF1 ATPase complex was studied by following the anaerobic relaxation of the transmembrane proton gradient (delta mu H+) generated by succinate-driven respiration. In control rat-liver submitochondrial particles containing the FoF1 moiety of the ATPase complex, anaerobic relaxation of delta mu H+ showed biphasic kinetics, whilst the same process in particles derived from regenerating rat liver exhibited monophasic kinetics and was significantly more rapid. Oligomycin and N,N-dicyclohexyl carbodiimide [(cHxN)2C] inhibited proton conductance by the F1-Fo ATPase complex in submitochondrial particles from both control and regenerating rat liver. Binding of [14C](cHxN)2C and immunodecoration using an antibody raised against bovine heart oligomycin-sensitivity-conferring protein (OSCP) indicated no difference in the content of either the (cHxN)2C binding protein or OSCP between control and regenerating rat-liver mitochondrial membranes. The results reported show that the structural and functional integrity of the Fo-F1 ATPase of rat liver is severely perturbed during regeneration.     

Control processes in oxidative phosphorylation: kinetic constraints and stoichiometry

Control processes in oxidative phosphorylation have been studied in three experimental models. (1) In isolated yeast mitochondria, external ATP is a regulatory effector of cytochrome-c oxidase activity. In phosphorylating or uncoupling states, the relationships between respiratory rate and delta mu H+, and the respiratory rate and cytochrome-c oxidase reduction level are dependent on this kinetic regulation. (2) In rat liver mitochondria, the response of the respiratory rate to uncoupler addition is age-dependent: liver mitochondria isolated from young rats maintain a greater delta mu H+ than liver mitochondria isolated from adults, with the same respiratory rate obtained with the same concentration of uncoupler. This behaviour is linked to redox proton pump properties, i.e., to the degree of intrinsic uncoupling induced by uncoupler addition. (3) The effect of almitrine, a new kind of ATPase/ATPsynthase inhibitor, was studied in mammalian mitochondria. (i) Almitrine inhibits oligomycin-sensitive ATPase - it decreases the ATPase/O value without any change in delta mu H+; (ii) almitrine increased the mechanistic H+/ATP stoichiometry of ATPase/ATPsynthase; (iii) almitrine-induced changes in H+/ATPase stoichiometry depend on the flux magnitude through ATPase. These results are discussed in terms of the following interdependent parameters; flux value, force, pump efficiency and control coefficient.     

Subunit VII, the ubiquinone-binding protein, of the cytochrome b-c1 complex of yeast mitochondria is involved in electron transport at center o and faces the matrix side of the membrane

The functional role and topographical orientation in the inner membrane of subunit VII, the ubiquinone-binding protein, of the cytochrome b-c1 complex of yeast mitochondria has been investigated. The apparent molecular weight of this subunit on sodium dodecyl sulfate-urea gels was calculated to be 15,500, while its amino acid composition was similar to that of the Q-binding proteins present in the cytochrome b-c1 complexes isolated from both beef heart and yeast mitochondria. The specific antibody obtained against subunit VII inhibited 30-47% of the ubiquinol-cytochrome c reductase activity in the isolated cytochrome b-c1 complex and in submitochondrial particles but had no effect on cytochrome c reductase activity in mitoplasts, mitochondria from which the outer membrane has been removed. Furthermore, the antibody against subunit VII strongly inhibited (74%) the reduction of cytochrome b by succinate in the presence of antimycin, an inhibitor of center i, but had no effect on cytochrome b reduction in the presence of myxothiazol, an inhibitor of center o. These results suggest that subunit VII, the Q-binding protein, is involved in electron transport at center o of the cytochrome b-c1 complex of the respiratory chain and that subunit VII is localized facing the matrix side of the inner mitochondrial membrane.