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1.
By using the purified rat liver protein for reference in electrophoresis and peptide mapping experiments, I have identified the beta subunit of mitochondrial F1-ATPase and its cytoplasmic precursor in two-dimensional gel patterns of proteins from S49 mouse lymphoma cells. The beta subunit precursor is a substrate for cAMP-dependent phosphorylation during its synthesis. Normally, both nonphosphorylated and phosphorylated forms of beta subunit precursor are processed rapidly to the smaller, more acidic forms of mature beta subunit. When processing is inhibited with valinomycin, both nonphosphorylated and phosphorylated forms of beta subunit precursor are stabilized. Nonphosphorylated beta subunit is one of the most stable of cellular proteins, but the phosphorylated form is eliminated within minutes of processing. This suggests that phosphorylated beta subunit is recognized as aberrant and excluded from assembly into the ATPase complex. These results argue that cAMP-dependent phosphorylation of the beta subunit precursor is a physiological mistake that is remedied after mitochondrial import and processing.  相似文献   

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3.
《BBA》1987,890(1):55-65
The binding of five monoclonal antibodies to mitochondrial F1-ATPase has been studied. Competition experiments between monoclonal antibodies demonstrate that these antibodies recognize four different antigenic sites and provide information on the proximity of these sites. The accessibility of the epitopes has been compared for F1 integrated in the mitochondrial membrane, for purified β-subunit and for purified F1 maintained in its active form by the presence of nucleotides or inactivated either by dilution in the absence of ATP or by urea treatment. The three anti-β monoclonal antibodies bound more easily to the β-subunit than to active F1, and recognized equally active F1 and F1 integrated in membrane, indicating that their antigenic sites are partly buried similarly in purified or membrane-bound F1 and better exposed in the isolated β-subunit. In addition, unfolding F1 by urea strongly increased the binding of one anti-β monoclonal antibody (14 D5) indicating that this domain is at least partly shielded inside the β-subunit. One anti-α monoclonal antibody (20 D6) bound poorly to F1 integrated in the membrane, while the other (7 B3) had a higher affinity for F1 integrated in the membrane than for soluble F1. Therefore, 20 D6 recognizes an epitope of the α-subunit buried inside F1 integrated in the membrane, while 7 B3 binds to a domain of the α-subunit well exposed at the surface of the inner face of the mitochondrial membrane.  相似文献   

4.
The binding of calmodulin to the mitochondrial F1 · F0-ATPase has been studied. [125I]Iodoazidocalmodulin binds to the ε-subunit and to the endogeneous ATPase inhibitor peptide in a Ca2+-dependent reaction. The effect of the mitochondrial ATPase inhibitor peptide on the purified Ca2+-ATPase of erythrocytes has also been analyzed. The inhibitor peptide stimulates the ATPase when pre-incubated with the enzyme. The activation of the Ca2+-ATPase by calmodulin is not influenced by the inhibitor peptide, indicating that the two mechanisms of activation are different. These in vitro effects of the two regulatory proteins may reflect a common origin of the two ATPases considered and/or of the regulatory proteins.  相似文献   

5.
The usefulness of two monoclonal antibodies, ϵ-1 and ϵ-4, which recognize the ϵ subunit of Escherichia coli F1-ATPase, for removing that subunit from ATPase was assessed. The ϵ subunit is a tightly bound, but dissociable, inhibitor of the ATPase. ϵ-1 binds ϵ with 10-fold higher affinity than ϵ-4. ϵ-1 recognizes a site on ϵ which is hidden by the quaternary structure of ATPase, while ϵ-4 can recognize ϵ when it is part of ATPase. Each antibody was purified and coupled to Sepharose to generate affinity columns. Solutions of ATPase in a buffer which was designed to reduce the affinity of ϵ for the enzyme were pumped through the columns and the degree of ϵ depletion was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by Western blotting. Neither column retained ATPase significantly. At low ATPase concentrations and low flow rates, the ϵ-1 column was more efficient than the ϵ-4 column, removing in excess of 95% of the ϵ in a single passage compared with 93% removal by the ϵ-4 column. At higher protein concentrations or flow rates, however, the performance of the ϵ-1 column was substantially poorer, while that of the ϵ-4 column was much less affected. Very little ϵ emerged from the ϵ-4 column before most of the measured ϵ-binding capacity was filled. A second passage through the ϵ-4 column reduced residual ϵ to less than 2% of that which was originally present. Pure, active ϵ was eluted from either column by 1 m NH4OH, pH 11. The relatively poor performance of ϵ-1 is discussed in terms of the low availability of the epitope and the tendency of the ϵ-depleted complex to compete with ϵ-1 for residual ϵ subunit. From consideration of these factors it appears likely that antibodies which recognize exposed epitopes will generally be more effective than antibodies which recognize cryptic epitopes in removing spontaneously dissociable subunits from protein complexes.  相似文献   

6.
Although Saccharomyces cerevisiae can form petite mutants with deletions in mitochondrial DNA (mtDNA) (ρ?) and can survive complete loss of the organellar genome (ρo), the genetic factor(s) that permit(s) survival of ρ? and ρo mutants remain(s) unknown. In this report we show that a function associated with the F1-ATPase, which is distinct from its role in energy transduction, is required for the petite-positive phenotype of S. cerevisiae. Inactivation of either the α or β subunit, but not the γ, δ, or ? subunit of F1, renders cells petite-negative. The F1 complex, or a subcomplex composed of the α and β subunits only, is essential for survival of ρo cells and those impaired in electron transport. The activity of F1 that suppresses ρo lethality is independent of the membrane Fo complex, but is associated with an intrinsic ATPase activity. A further demonstration of the ability of F1 subunits to suppress ρo lethality has been achieved by simultaneous expression of S. cerevisiae F1α and γ subunit genes in Kluyveromyces lactis– which allows this petite-negative yeast to survive the loss of its mtDNA. Consequently, ATP1 and ATP2, in addition to the previously identified AAC2, YME1 and PEL1/PGS1 genes, are required for establishment of ρ? or ρo mutations in S. cerevisiae.  相似文献   

7.
《FEBS letters》1986,203(2):144-148
The α- and β-subunits of sweet potato mitochondrial F1ATPase were purified from the F1 complex by gel filtration and ion-exchange high-performance liquid chromatography. Isoelectric focussing and N-terminal amino acid sequencing indicated that the purified β-subunit contains at least two polypeptides similar to each other. The N-terminal 18 amino acid sequence of the β-subunit showed homology to the amino acid sequence of the tobacco mitochondrial F1ATPase β-subunit precursor deduced from the nucleotide sequence [(1985) EMBO J. 4, 2159-2165] between residues 56 and 73, suggesting that the N-terminal 55 amino acids of the tobacco precursor constitute the presequence required for mitochondrial targetting.  相似文献   

8.
The crystal structure of mitochondrial F1-ATPase indicatesthat the and subunits fold into a structure defined by threedomains: the top -barrel domain, the middle nucleotide-binding domain,and the C-terminal -helix bundle domain (Abraham et al.1994); Bianchet et al., 1998). The -barrel domains of the and subunits form a crown structure at the top ofF1, which was suggested to stabilize it (Abraham et al.1994). In this study. the role of the -barrel domain in the and subunits of the yeast Saccharomyces cerevisiae F1,with regard to its folding and assembly, was investigated. The -barreldomains of yeast F1 and subunits were expressedindividually and together in Escherichia coli. When expressedseperately, the -barrel domain of the subunit formed a largeaggregate structure, while the domain of the subunit waspredominately a monomer or dimer. However, coexpression of the -barreldomain of subunit domain. Furthermore, the two domains copurified incomplexes with the major portion of the complex found in a small molecularweight form. These results indicate that the -barrel domain of the and subunits interact specifically with each other and thatthese interactions prevent the aggregation of the -barrel domain of the subunit. These results mimic in vivo results and suggest thatthe interactions of the -barrel domains may be critical during thefolding and assembly of F1.  相似文献   

9.
Rat liver peroxisomes contain in their matrix the alpha-subunit of the mitochondrial F1-ATPase complex. The identification of this protein in liver peroxisomes has been achieved by immunoelectron microscopy and subcellular fractionation. No beta-subunit of the mitochondrial F1-ATPase complex was detected in the peroxisomal fractions obtained in sucrose gradients or in Nycodenz pelletted peroxisomes. The consensus peroxisomal targeting sequence (Ala-Lys-Leu) is found at the carboxy terminus of the mature alpha-subunit from bovine heart and rat liver mitochondria. Due to the dual subcellular localization of the alpha-subunit and to the structural homologies that exist between this protein and molecular chaperones [(1990) Biol. Chem. 265, 7713-7716] it is suggested that the protein should perform another functional role(s) in both organelles, plus to its characteristic involvement in the regulation of mitochondrial ATPase activity.  相似文献   

10.
We investigated the effect of L and D enantiomers of a 25-residue peptide derived from the N-terminal region of the presequence of Nicotiana plumbaginifolia F1 subunit of the ATP synthase, pF1(1, 25), on import into spinach leaf mitochondria. Three in vitro synthesized precursor proteins using different import pathways were used. Import of the precursor proteins of F1 subunit of the ATP synthase, pre-F1, and the alternative oxidase, pre-AOX, required addition of external ATP, whereas the chimeric precursor containing the N-terminal 84 amino acids of the cytochrome b 2 precursor protein linked to dihydrofolate reductase, pre-b 2(1, 84)-DHFR was not dependent on ATP. Import of pre-F1, and pre-AOX was inhibited already at 1 M and 3 M concentration of the L and D enantiomers, whereas inhibition of import of pre-b 2(1, 84)-DHFR, occurred at concentrations >10 M of both enantiomers. Binding efficiency of the precursor proteins was not affected by addition of the L and D enantiomers. There was no correlation between inhibition of import of pre-F1 and pre-AOX and dissipation of membrane potential measured as a decrease of Rhodamine 123 fluorescence quenching. The inhibitory effect of the L and D presequence enantiomers on import of pre-F1 and pre-AOX was concluded to occur within the outer membrane translocase machinery beyond the initial precursor receptor interaction. Furthermore, the fact that the D enantiomer had the same effect as the natural peptide showed that interaction of the presequence with the import machinery was not dependent on chiral properties of the presequence.  相似文献   

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In observations of single molecule behavior under V(max) conditions with minimal load, the F(1) sector of the ATP synthase (F-ATPase) rotates through continuous cycles of catalytic dwells (~0.2 ms) and 120° rotation steps (~0.6 ms). We previously established that the rate-limiting transition step occurs during the catalytic dwell at the initiation of the 120° rotation. Here, we use the phytopolyphenol, piceatannol, which binds to a pocket formed by contributions from α and β stator subunits and the carboxyl-terminal region of the rotor γ subunit. Piceatannol did not interfere with the movement through the 120° rotation step, but caused increased duration of the catalytic dwell. The duration time of the intrinsic inhibited state of F(1) also became significantly longer with piceatannol. All of the beads rotated at a lower rate in the presence of saturating piceatannol, indicating that the inhibitor stays bound throughout the rotational catalytic cycle. The Arrhenius plot of the temperature dependence of the reciprocal of the duration of the catalytic dwell (catalytic rate) indicated significantly increased activation energy of the rate-limiting step to trigger the 120° rotation. The activation energy was further increased by combination of piceatannol and substitution of γ subunit Met(23) with Lys, indicating that the inhibitor and the β/γ interface mutation affect the same transition step, even though they perturb physically separated rotor-stator interactions.  相似文献   

14.
Rotation of the γ subunit of the F1-ATPase plays an essential role in energy transduction by F1-ATPase. Hydrolysis of an ATP molecule induces a 120° step rotation that consists of an 80° substep and 40° substep. ATP binding together with ADP release causes the first 80° step rotation. Thus, nucleotide binding is very important for rotation and energy transduction by F1-ATPase. In this study, we introduced a βY341W mutation as an optical probe for nucleotide binding to catalytic sites, and a βE190Q mutation that suppresses the hydrolysis of nucleoside triphosphate (NTP). Using a mutant monomeric βY341W subunit and a mutant α3β3γ subcomplex containing the βY341W mutation with or without an additional βE190Q mutation, we examined the binding of various NTPs (i.e., ATP, GTP, and ITP) and nucleoside diphosphates (NDPs, i.e., ADP, GDP, and IDP). The affinity (1/Kd) of the nucleotides for the isolated β subunit and third catalytic site in the subcomplex was in the order ATP/ADP > GTP/GDP > ITP/IDP. We performed van’t Hoff analyses to obtain the thermodynamic parameters of nucleotide binding. For the isolated β subunit, NDPs and NTPs with the same base moiety exhibited similar ΔH0 and ΔG0 values at 25°C. The binding of nucleotides with different bases to the isolated β subunit resulted in different entropy changes. Interestingly, NDP binding to the α3β(Y341W)3γ subcomplex had similar Kd and ΔG0 values as binding to the isolated β(Y341W) subunit, but the contributions of the enthalpy term and the entropy term were very different. We discuss these results in terms of the change in the tightness of the subunit packing, which reduces the excluded volume between subunits and increases water entropy.  相似文献   

15.
The mgi1-4 and mgi2-1 mutants of the petite-negative yeast Kluyveromyces lactis have mutations in the β- and α-subunits of the mitochondrial F1-ATPase, respectively. The mutants are respiratory competent but can form petites with deletions in mitochondrial DNA. In this study a cryptic nuclear mutation (lipB-1) was identified which, in combination with the mgi alleles, displays a synergistic respiratory-deficient phenotype on glycerol medium. The gene defined by the mutation was cloned and shown to encode a polypeptide of 332 amino acids with an N-terminal sequence characteristic of a mitochondrial targeting signal. The deduced protein shares 27% sequence identity with the product of the Escherichia coli lipB gene, which encodes a lipoyl-protein ligase involved in the attachment of lipoyl groups to lipoate-dependent apoproteins. A K. lactis strain carrying a disrupted lipB allele is severely compromised for growth on glycerol medium. The growth defect cannot be rescued by addition of lipoic acid, but cell growth can be restored on medium containing ethanol plus succinate. In addition, it was observed that lipB mutants of K. lactis, unlike the wild-type, are unable to utilize glycine as sole nitrogen source, indicating that activity of the glycine decarboxylase complex (GDC) is also affected. Taken together, these findings suggest that LIPB is the main determinant of the lipoyl-protein ligase activity required for lipoylation of enzymes such as α-ketoacid dehydrogenases and GDC.  相似文献   

16.
In this study we report the first comparison of the mitochondrial protein import and processing events in two different tissues from the same organism. Both spinach leaf and root mitochondria were able to import and process the in vitro transcribed and translated Neurospora crassa F1 subunit of ATP synthase to the mature size product. Temperature optimum for protein import, 20 °C, was considerably lower than that found in other systems. In spinach leaf mitochondria, the processing peptidase has been shown to constitute an integral part of the bc1 complex of the respiratory chain. In accordance with these results, the majority of the processing activity in root mitochondria was also localized in the membrane. However, although the same amount of the processing peptidase was present per mg of membrane protein in both leaf and root mitochondria, as determined immunologically, the specific processing activity was several-fold higher in roots. Furthermore, in contrast to the processing enzyme in leaf, a portion of the processing activity could be disassociated from the root membrane with relatively weak salt treatment. The processing event in both the leaf and root membranes was always accompanied by a degradation of the F1 precursor. The degradation activity was found to be several-fold higher in roots than in leaves and was also partially dissociated from the membrane after salt treatment. Both the processing and degradation activities were inhibited by orthophenanthroline, a known metalloprotease inhibitor. These results show tissue-specific differencies of the processing event catalyzed by the bc1 complex and indicate the presence of two populations of the processing peptidase in root mitochondria.  相似文献   

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Energy coupling between the A1 ATPase of archaea type A1AO ATP synthase and its integral membrane sub-complex AO occurs via the stalk part, formed by the subunits C, D and F. To provide a molecular basis of the energy coupling, we performed NMR studies. Here, we report the assignment of the subunit F. Shovanlal Gayen and Subramanian Vivekanandan contributed equally to this work.  相似文献   

20.
《BBA》1987,892(1):30-41
The fluorescence properties of 3′-O(1-naphthoyl)adenosine di- and triphosphates (termed N-ADP and N-ATP, respectively) were investigated in detail. Of special importance for the use of these analogues as environmental probes is their high quantum yield (0.58 in water) and the polarity dependence of shape and wavelength position of the emission spectrum. Upon binding of N-ADP and N-ATP to mitochondrial F1-ATPase, the fluorescence intensity is markedly decreased, due to polarity changes and ‘ground-state’ quenching. Using this signal for equilibrium binding studies, three (at least a priori) equivalent nucleotide-binding sites were detected on the enzyme. The perspective intrinsic dissociation constants are as follows: N-ADP/Mg2+ 120 nM; N-ATP/Mg2+ 160 nM; N-ADP/EDTA 560 nM; N-ATP/EDTA 3500 nM. For bound ligand the environment was found to be rather unpolar; the rotational mobility of the fluorophore is restricted, its accessibility for iodide anions (as a quencher) is hindered. These facts show a location of the binding sites quite deeply embedded in the protein. The conformation of the binding domains is strongly dependent on the absence or presence of Mg2+, as can be seen from the relative efficiencies of the singlet-singlet energy transfer from tyrosine residues in the protein to bound naphthoyl moieties. Investigation of the binding kinetics revealed this process as biphasic (in presence of Mg2+). After the first fast step (kon > 1 · 106 M−1 · s−1), in which the analogue is bound to the enzyme, a slow local conformational rearrangement occurs.  相似文献   

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