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Query: EC:3.6.3.14 (ATP synthase)
 7,042 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Secretory vesicles that accumulate in the temperature-sensitive sec6-4 strain of yeast have been shown to contain a vanadate-sensitive ATPase, presumably en route to the plasma membrane (Walworth, N. C., and Novick, P. J. (1987) J. Cell Biol. 105, 163-174). We have now established this enzyme to be a fully functional form of the PMA1 [H+]ATPase, identical in its catalytic properties to that found in the plasma membrane. In addition, the secretory vesicles are sealed tightly enough to permit the measurement of ATP-dependent proton pumping with fluorescent probes. We have gone on to exploit the vesicles as an expression system for site-directed mutants of the ATPase. For this purpose, a sec6-4 strain has been constructed in which the chromosomal PMA1 gene is under control of the GAL1 promoter; the mutant pma1 allele to be studied is introduced on a centromeric plasmid under the control of a novel heat shock promoter. In galactose medium at 23 degrees C, the wild-type ATPase is produced and supports normal vegetative growth. When the cells are switched to glucose medium at 37 degrees C, however, the wild-type gene turns off, the mutant gene turns on, and secretory vesicles accumulate. The vesicles contain a substantial amount of newly synthesized, plasmid-encoded ATPase (5-10% of total vesicle protein), but only traces of residual wild-type PMA1 ATPase and no detectable mitochondrial ATPase, vacuolar ATPase, or acid or alkaline phosphatase. To test the expression strategy, we have made use of pma1-105 (Ser368----Phe), a vanadate-resistant mutant previously characterized by standard methods (Perlin, D. S., Harris, S. L., Seto-Young, D., and Haber, J. E. (1989) J. Biol. Chem. 264, 21857-21864). In secretory vesicles, as expected, the plasmid-borne pma1-105 allele gives rise to a mutant enzyme with a reduced rate of ATP hydrolysis and a 100-fold increase in Ki for vanadate. Proton pumping is similarly resistant to vanadate. Thus, the vesicles appear well suited for the production and characterization of mutant forms of the PMA1 [H+]ATPase. They should also aid the study of other yeast membrane proteins that are essential for growth as well as heterologous proteins whose appearance in the plasma membrane may be toxic to the cell.
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PMID:Expression of the yeast plasma membrane [H+]ATPase in secretory vesicles. A new strategy for directed mutagenesis. 182 8

Using site-directed mutagenesis, Tyr-307, Tyr-341, or Tyr-364, supposedly located at the adenine nucleotide binding site(s) of the beta subunits of F1-ATPase from the thermophilic bacterium PS3, was replaced with Phe or Cys. The alpha 3 beta 3 complexes reconstituted from the alpha subunits and individual mutant beta subunits hydrolyzed ATP. Thus, neither the hydroxyl groups nor the aromatic rings in these positions are required for ATPase activity of F1-ATPase.
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PMID:Aromatic rings of tyrosine residues at adenine nucleotide binding sites of the beta subunits of F1-ATPase are not necessary for ATPase activity. 213 33

The ATP synthases of eubacteria and eukaryotes possess a conserved tyrosine (beta 331) that is labeled by ATP analogs and is believed to be at the catalytic site. In this report, this tyrosine was replaced by Phe, Ser, Cys, Gly, and Ala in an attempt to determine its role in catalysis. Each of the beta 331 mutant strains assembled an ATP synthase. Membranes from the beta 331-Ser, -Cys, -Ala, or -Gly strains showed strongly attenuated ATP hydrolysis and ATP-driven proton-pumping activities. The beta 331-Phe membranes showed nearly normal ATPase and functional proton pumping. A new purification procedure yielding highly active unc+ F1 (ATPase rates greater than 1000 s-1) allowed rapid isolation of soluble F1-ATPases. Kinetic analyses of purified enzymes confirmed that the structural and functional properties of beta 331-Tyr can be substituted by Phe but not effectively by Ser, Cys, Ala, or Gly. Since all of the beta 331 mutant enzymes catalyzed measurable ATP hydrolysis, it is clear that beta 331-Tyr is not directly involved in the bond making-breaking steps of catalysis. The ability of the beta 331-Phe enzyme to rapidly bind and hydrolyze ATP, and the results with other beta 331 mutant enzymes, suggests that a residue with an aromatic character is required at this position.
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PMID:Site-directed mutagenesis of the conserved beta subunit tyrosine 331 of Escherichia coli ATP synthase yields catalytically active enzymes. 214 32

RNA editing, a process that results in the production of RNA molecules having a nucleotide sequence different from that of the initial DNA template, has been demonstrated in several organisms using different biochemical pathways. Very recently RNA editing was described in plant mitochondria following the discovery that the sequence of certain wheat and Oenothera cDNAs is different from the nucleotide sequence of the corresponding genes. The main conversion observed was C to U, leading to amino acid changes in the deduced protein sequence when these modifications occurred in an open reading frame. In this communication we show the first attempt to isolate and sequence a protein encoded by a plant mitochondrial gene. Subunit 9 of the wheat mitochondrial ATP synthase complex was purified to apparent homogeneity and the sequence of the first 32 amino acid residues was determined. We have observed that at position 7 leucine was obtained by protein sequencing, instead of the serine predicted from the previously determined genomic sequence. Also we found phenylalanine at position 28 instead of a leucine residue. Both amino acid conversions, UCA (serine) to UUA (leucine) and CUC (leucine) to UUC (phenylalanine), imply a C to U change. Thus our results seem to confirm, at the protein level, the RNA editing process in plant mitochondria.
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PMID:Direct protein sequencing of wheat mitochondrial ATP synthase subunit 9 confirms RNA editing in plants. 219 74

We have determined the nucleotide sequences of three mutant rho genes encoding hyperfunctional rho proteins (rho S) together with their parent allele, rho-ts702. These mutant rho factors contain the following amino acid changes as deduced from their sequences: (1) the thermo-labile mutant, rho-ts702, has Thr304 substituting for Ala; (2) rho S-77 and rho S-81, which are selectively altered in the primary polynucleotide binding site, share an identical mutation, Leu3----Phe; (3) rho S-82, which is altered in both the primary and secondary polynucleotide binding sites, carries three amino acid substitutions together, Leu3----Phe, Asp156----Asn and Thr323----Ile. Dissection and functional characterization of each mutation in rho S-82 have revealed that Ile323 alone is responsible for alterations in both the secondary RNA interaction and the terminator selectivity observed with the original mutant, rho S-82. Taken together, these results not only confirm our proposal in the accompanying paper that the primary and secondary RNA binding sites differently contribute in determining the overall efficiency and site-specificity of termination, respectively, but also support the possibility that these binding sites exist as structurally distinct domains in rho protein. In contrast, Asn156 was shown to cause decreased termination efficiency, though it had no influence on RNA interactions. Thus, this amino acid residue appears to be associated with still another rate-determining step of termination, for instance, interactions between rho and RNA polymerase. On the basis of Chou-Fasman secondary structure predictions as well as amino acid sequence comparison with F1-ATPase, we discuss how the proposed domains are structurally and functionally related to the putative ATPase reactive center of rho protein.
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PMID:Mutant rho factors with increased transcription termination activities. II. Identification and functional dissection of amino acid changes. 247 57

[32P]Azidonitrophenyl phosphate [( 32P]ANPP) is a photoactivatable analogue of Pi. It competes efficiently with Pi for binding to the F1 sector of beef heart mitochondrial ATPase and photolabels the Pi binding site located in the beta subunit of F1 [Lauquin, G. J. M., Pougeois, R., & Vignais, P. V. (1980) Biochemistry 19, 4620-4626]. By cleavage of the photolabeled beta subunit of F1 with cyanogen bromide, trypsin, and chymotrypsin, bound [32P]ANPP was localized in a fragment spanning Thr 299-Phe 326. By Edman degradation of the radiolabeled tryptic peptide spanning Ile 296-Arg 337, [32P]ANPP was found to be attached covalently by its photoreactive group to Ile 304, Gln 308, and Tyr 311. These results are discussed in terms of a model in which the phosphate group of [32P]ANPP interacts with a glycine-rich sequence of the beta subunit, spanning Gly 156-Lys 162, which is spatially close to the photolabeled Ile 304-Tyr 311 segment of the same subunit.
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PMID:Photolabeling of the phosphate binding site of mitochondrial F1-ATPase by [32P]azidonitrophenyl phosphate. Identification of the photolabeled amino acid residues. 252 9

A mutation conferring aurovertin resistance on Escherichia coli F1-ATPase was identified as R398----H in the F1 beta-subunit. Beta-subunit from the mutant does not bind aurovertin; therefore our results suggest the region of sequence around residue beta-398 is involved in aurovertin binding. Since nucleotide and aurovertin binding to isolated beta-subunit are not mutually exclusive, the data further suggest that the beta-subunit catalytic nucleotide-binding domain does not include residue 398. The mutation prevented aurovertin inhibition of ATPase at pH 6 and 8.5, implying charge on the arginine side-chain is not a major determinant of aurovertin binding or that the pK of R398 is shifted due to a peculiar environment. The equivalent residue is usually arginine in F1 beta-subunits of different species; notably in the aurovertin-insensitive thermophilic bacterium PS3 F1-ATPase, this residue is phenylalanine.
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PMID:Identification of a mutation in Escherichia coli F1-ATPase beta-subunit conferring resistance to aurovertin. 252 66

Three missense mutants in subunit a of the Escherichia coli F1F0-ATPase were isolated and characterized after hydroxylamine mutagenesis of a plasmid carrying the uncB (subunit a) gene. The mutations resulted in Asp119----His, Ser152----Phe, or Gly197----Arg substitutions in subunit a. Function was not completely abolished by any of the mutations. The F0 membrane sector was assembled in all three cases as judged by restoration of dicyclohexylcarbodiimide sensitivity to the F1F0-ATPase. The H+ translocation capacity of F0 was reduced in all three mutants. ATP-driven H+-translocation was also reduced, with the response in the Gly197----Arg mutant being almost nil and that in the Asp119----His and Ser152----Phe mutants less severely affected. The substituted residues are predicted to lie in the second, third, and fourth transmembrane helices suggested in most models for subunit a. The Gly197----Arg mutation lies in a very conserved region of the protein and the substitution may disrupt a structure that is critical to function. The Asp119----His and Ser152----Phe mutations also lie in areas with sequence conservation. A further analysis of randomly generated mutants may provide more information on regions of the protein that are crucial to function. Heterodiploid transformants, carrying plasmids with either the wild-type uncB gene or mutant uncB genes in an uncB (Trp231----stop) background, were characterized biochemically. The truncated subunit a was not detected in membranes of the background strain by Western blotting, and the uncB+ plasmid complemented strain showed normal biochemistry. The uncB mutant genes were shown to cause equivalent defects in either the heterodiploid background configuration, or after incorporation into an otherwise wild-type unc operon. The subunit a (Trp231----stop) background strain was shown to bind F1-ATPase nearly normally despite lacking subunit a in its membrane.
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PMID:Mutations in three of the putative transmembrane helices of subunit a of the Escherichia coli F1F0-ATPase disrupt ATP-driven proton translocation. 252 29

Site directed mutagenesis has been performed on the gene coding for the beta-subunit of the yeast mitochondrial F1-ATPase. Two different regions were studied. First, the corresponding yeast amino acid, Tyr-344, which was affinity labeled in the bovine enzyme was changed to Phe-344 and Ala-344. The Phe-344 enzyme was completely active and less sensitive to the affinity reagent, 4-chloro-7-nitrobenzofurazan. In contrast, the in vivo level of the Ala-344 enzyme was greatly diminished and apparently inactive. The second region studied is in the glycine rich region homologous in nucleotide binding proteins. Five different replacements were made and all mutations but one completely eliminated the biological activity and reduced the in vivo level of the mutant peptides. These results support the importance of these amino acids in the function of the ATPase.
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PMID:Site directed mutagenesis of the beta-subunit of the yeast mitochondrial ATPase. 252 56

A series of mitochondrially inherited mutants of yeast has been analysed, which were previously identified as showing resistance to the antibiotics venturicidin or ossamycin and whose mutations showed tight linkage to oligomycin-resistance alleles affecting subunit 9 of the mitochondrial ATP synthase. DNA sequence analysis of the oli1 gene of these mutants has been used to define the nature of amino acid substitution in the subunit 9 protein. In the case of the two venturicidin-resistant mutants, mutations affect amino acids on the N-terminal stem of the protein, namely Gly25----Ser (venR ossS oliR) and Ala 27----Gly (venR ossS oliS). The mutations found in the two ossamycin-resistant mutants affect amino acids on the C-terminal stem of the protein; namely Leu53----Phe (vanS ossR oliR) and Leu57----Phe (venS ossR oliS). These results allow us to further develop a fine structure map of domains within the subunit 9 protein involved in antibiotic interaction.
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PMID:Amino acid substitutions in mitochondrial ATP synthase subunit 9 of Saccharomyces cerevisiae leading to venturicidin or ossamycin resistance. 266 Dec 66


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