Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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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)

In order to better understand why higher eukaryotic membrane proteins, in contrast to soluble proteins, are not readily expressed in Escherichia coli, the gene encoding the liver mitochondrial phosphate transporter (H+/Pi symporter) (Ferreira, G. C., Pratt, R. D., and Pedersen, P. L. (1989) J. Biol. Chem. 264, 15628-15633), was subcloned into a plasmid (pFOG402) containing the alkaline phosphatase promoter and leader sequence. Although this system is highly efficient in overexpressing soluble mitochondrial proteins in E. coli, e.g. alpha and beta subunits of the liver ATP synthase, it fails to express the H+/Pi transporter. Expression is not obtained by truncation of the transporter gene from either the 3' or 5' end, by fusing the mature transporter gene to genes encoding either the alpha or beta ATP synthase subunits, or by using different expression plasmids. Significantly, the H+/Pi transporter is overexpressed in E. coli provided its cDNA is first truncated at the 3' end (carboxyl-terminal end) and fused to a cDNA fragment derived from the ATP synthase alpha subunit gene. In fact, progressive deletions from the 3' end of the transporter cDNA produce a ladder of increasingly overexpressed fusion proteins which account from the largest to the smallest for approximately 2.5-14% of the total bacterial cell protein. The minimal truncation necessary from the 3' end is 192 base pairs corresponding to 64 COOH-terminal amino acids. This corresponds to 20% of the transporter and involves removal of one of the six predicted membrane-spanning segments. In a variety of additional experiments designed to define the molecular basis for E. coli's inability to express the complete liver H+/Pi transporter, problems related to cell toxicity and transcription were ruled out. However, in vitro transcription-translation assays revealed that the complete transporter is readily expressed when eukaryotic, but not prokaryotic, ribosomes are present. Significantly, the fused transporter gene (i.e. Pi transporter cDNA truncated at the 3' end + ATP synthase alpha subunit cDNA) is expressed when prokaryotic ribosomes are present. These results support the view that the difficulty in expressing higher eukaryotic membrane proteins in bacteria may be related in some cases to a problem at the level of translation.
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PMID:Overexpression of higher eukaryotic membrane proteins in bacteria. Novel insights obtained with the liver mitochondrial proton/phosphate symporter. 153 83

A soluble form of the b subunit of the F0 sector of the F1F0-ATPase of Escherichia coli has been produced, purified, and characterized. In this form of the protein, designated bsol, residues 25-146 (the carboxyl terminus) of b have been fused to an amino-terminal octapeptide extension derived from the vector pUC8. The inferred subunit molecular weight of bsol is 15,459. bsol protein was expressed in E. coli as a soluble cytoplasmic protein and was readily purified to homogeneity by conventional methods. The molecular weight of bsol, determined by sedimentation equilibrium, was 31,200, indicating that the protein is dimeric. Chemical cross-linking studies supported this conclusion. However, bsol sedimented with a coefficient of just 1.8 S and behaved on size exclusion chromatography with an apparent molecular weight of 80,000-85,000. These results indicate that the protein exists in solution as a highly elongated dimer. The circular dichroism spectrum indicated that bsol is highly alpha-helical. Binding of bsol to F1-ATPase was directly demonstrated by size exclusion chromatography. bsol also inhibited the binding of F1-ATPase to F1-depleted membrane vesicles, as measured by reconstitution of energy-dependent quinacrine fluorescence quenching. This result implies that bsol and F0 compete for binding to the same site on F1. The apparently normal interaction of bsol with F1-ATPase strongly suggests that the recombinant protein assumes the correct structure. No substantial effects of bsol on the ATPase activity of purified F1 were observed.
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PMID:The polar domain of the b subunit of Escherichia coli F1F0-ATPase forms an elongated dimer that interacts with the F1 sector. 153 97

This paper deals with a truncated derivative of subunit 8 of yeast mitochondrial ATP synthase in which a conserved positively charged residue (Lys47) has been removed by site-directed mutagenesis together with the C-terminal residue (Leu48). This derivative has been expressed as a chimaeric precursor N9L/Y8-1(K47-STP) carrying an N-terminal cleavable leader sequence (N9L), fused by a short bridging sequence to the truncated subunit-8 passenger protein. Allotopic expression of N9L/Y8-1(K47-STP) in vivo in an aap1 mit- host yeast strain lacking endogenous subunit 8 leads to partial restoration of bioenergetic function in the transformant strain denoted T475. Import and assembly studies were carried out in vitro using target mitochondria from strain YGL-1 partially depleted in subunit 8; such controlled depletion has been previously shown to be required for the efficient assembly (monitored immunochemically) of full-length subunit 8 imported in vitro as the precursor N9L/Y8-1. It was found that N9L/Y8-1(K47-STP) synthesized in vitro was imported successfully into YGL-1 mitochondria, but no significant assembly of the truncated subunit 8 was observed in these or any other mitochondria tested. The bioenergetic defects in T475 mitochondria are ascribed to the impaired assembly of the subunit-8 variant in vivo, resulting from the truncation at Lys47. In consequence, T475 mitochondria behave as though partially depleted of subunit 8. This conclusion was supported by the ability of isolated T475 mitochondria to provide a vehicle for the efficient import and assembly of subunit 8 processed from full-length N9L/Y8-1. Two related aspects of import and assembly have been addressed as part of the analysis of truncated subunit 8. First, mitochondria from strain T2-1, an aap1 mit- mutant genetically reconstituted by allotopic expression of N9L/Y8-1, were also found to be effective in the in vitro assembly of subunit 8 derived from imported N9L/Y8-1. This suggests an intramitochondrial shortage of subunit 8 delivered by allotopic expression of N9L/Y8-1 in vivo, which may underlie the incomplete restoration of energy coupling in T2-1 mitochondria compared to those of wild-type yeast. Second, on allotopic expression of N9L/Y8-2 (containing subunit 8 directly fused to N9L) in the aap1 mit- host, a rescued transformant strain T10-1 was generated which displays bioenergetic defects superficially similar to those of T475. Processed subunit 8 clearly assembled into the ATP synthase of isolated YGL-1 mitochondria, in spite of the relatively weak import of N9L/Y8-2 in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The C-terminal positively charged region of subunit 8 of yeast mitochondrial ATP synthase is required for efficient assembly of this subunit into the membrane F0 sector. 182 79

We report the first lateral diffusion measurements of redox components in normal-sized, matrix-containing, intact mitoplasts (inner membrane-matrix particles). The diffusion measurements were obtained by submicron beam fluorescence recovery after photobleaching measurements of individual, intact, rat liver mitoplasts bathed in different osmolarity media to control the matrix density and the extent of inner membrane folding. The data reveal that neither the extent of mitochondrial matrix density nor the complexity of the inner membrane folding have a significant effect on the mobility of inner membrane redox components. Diffusion coefficients for Complex I (NADH:ubiquinone oxidoreductase), Complex III (ubiquinol: cytochrome c oxidoreductase), Complex IV (cytochrome oxidase), ubiquinone, and phospholipid were found to be effectively invariant with the matrix density and/or membrane folding and essentially the same as values we reported previously for spherical, fused, ultralarge, matrix-free, inner membranes. Diffusion of proton-transporting Complex V (ATP synthase) appeared to be 2-3-fold slower at the greatest matrix density and degree of membrane folding. Consistent with a diffusion-coupled mechanism of electron transport, comparison of electron transport frequencies (productive collisions) with the theoretical, diffusion-controlled, collision frequencies (maximum collisions possible) revealed that there were consistently more calculated than productive collisions for all redox partners. Theoretical analyses of parameters for submicron fluorescence recovery after photobleaching measurements in intact mitoplasts support the finding of highly mobile redox components diffusing at the same rates as determined in conventional fluorescence recovery after photobleaching measurements in fused, ultralarge inner membranes. These findings support the Random Collision Model of Mitochondrial Electron Transport at the level of the intact mitoplast and suggest a similar conclusion for the intact mitochondrion.
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PMID:Lateral diffusion of redox components in the mitochondrial inner membrane is unaffected by inner membrane folding and matrix density. 200 33

A set of chimaeric precursors which contain the same leader sequences but different passenger proteins has been analyzed for the site of protease cleavage following import into yeast mitochondria. Each precursor comprises the leader of Neurospora crassa subunit 9 of mitochondrial ATP synthase fused to subunit 8 or 9 of the corresponding yeast enzyme. Precursors containing the first five residues of mature N. crassa subunit 9 interposed between the leader and the yeast passenger protein were cleaved at the natural site of the N. crassa subunit 9 precursor. Direct fusions without interposed sequences were cleaved at novel sites. Cleavage occurred between the 3rd and 4th residues of yeast subunit 8, but for yeast subunit 9, cleavage occurred within the leader, 8 residues upstream of the passenger protein.
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PMID:Aberrant mitochondrial processing of chimaeric import precursors containing subunits 8 and 9 of yeast mitochondrial ATP synthase. 215 Oct 20

Sequence analysis of the mitochondrial ATPase 6 gene from chicken revealed that its 3' region is virtually identical with a chicken muscle-specific 7 S RNA which was reported to induce the expression of tissue-specific functions in blastoderm explants. Using chicken and quail cell lines depleted of mitochondrial DNA, we demonstrate that the 7 S RNA is encoded by the mitochondrial genome and not by nuclear (repetitive) DNA as suggested previously. Moreover, no 7 S RNA-homologous transcript of the expected length (about 400 bases) is detected, either in these cell lines or in heart and liver tissues. The only RNA species hybridizing with a 7 S RNA-specific probe is an abundant, 900 base long transcript of mitochondrial origin that we identify as the ATPase 8-ATPase 6 fused messenger. We suggest that the characterized muscle-specific 7 S RNA cDNA is derived from an unrelated contaminant in the blastoderm-inducing fraction.
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PMID:Putative chicken "muscle-specific 7 S RNA" is related to the mitochondrial ATPase 6 gene. 247 59

Sequencing of an open reading frame associated with cytoplasmic male sterility (CMS) in Petunia has revealed a gene fusion (the Pcf gene) containing the 5'-flanking and amino-terminal transmembrane segment of the ATP synthase proteolipid gene (atp9), parts of the cytochrome oxidase subunit II (coxII) coding region, and the carboxyl terminus and 3'-flanking region of an unidentified reading frame (urfS). The coxII region has several small deletions and tandem repeats that remove all of the segments coding for the residues involved in copper binding, but may possibly maintain the cytochrome c binding site. Normal atp9 and coxII genes and their transcripts are also present in the sterile cytoplasm. S1 nuclease protection studies identify fused gene transcripts only in CMS lines, with an increase in transcript amount in anthers relative to leaves.
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PMID:A fused mitochondrial gene associated with cytoplasmic male sterility is developmentally regulated. 288 95

A mitochondrial gene from Saccharomyces cerevisiae encoding a hydrophobic membrane protein, subunit 8 of the F0/F1-type mitochondrial ATPase complex, has been functionally replaced by an artificial nuclear gene specifying an imported version of this protein. The experiments reported here utilized a multicopy expression vector (pLF1) that replicates in the nucleus of yeast cells and that carries an inserted DNA segment, specifying a precursor protein (N9/Y8) consisting of subunit 8 fused to an N-terminal cleavable transit peptide (the leader sequence from Neurospora crassa ATPase subunit 9). The successful incorporation of the imported subunit 8 into functional ATPase complexes after transformation with pLF1 expressing N9/Y8 was indicated by the efficient genetic complementation of respiratory growth defects of aap1 mit- mutants, which lack endogenous subunit 8. The reconstitution of ATPase function was confirmed by biochemical assays of ATPase performance in mitochondria and by immunochemical analyses that demonstrated the assembly of the cytoplasmically synthesized subunit 8 into the ATPase complex. Reconstitution of ATPase function required the cytoplasmically synthesized subunit to have a transit peptide. The strategy for importation and reconstitution developed for subunit 8 leads to a systematic approach to the directed manipulation of mitochondrially encoded membrane-associated proteins that has general implications for exploring membrane biogenesis mechanistically and evolutionarily.
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PMID:Assembly of functional proton-translocating ATPase complex in yeast mitochondria with cytoplasmically synthesized subunit 8, a polypeptide normally encoded within the organelle. 289 70

We have investigated the energy requirement of mitochondrial protein import with a simplified system containing only isolated yeast mitochondria, energy sources and a purified precursor protein. This precursor was a fusion protein composed of 22 residues of the cytochrome oxidase subunit IV pre-sequence fused to mouse dihydrofolate reductase. Import of this protein required not only an energized inner membrane, but also ATP. ATP could be replaced by GTP, but not by CTP, TTP or non-hydrolyzable ATP analogs. Added ATP did not increase the membrane potential of respiring mitochondria; it supported import even if the proton-translocating mitochondrial ATPase and the entry of ATP into the matrix were blocked. We conclude that ATP exerts its effect on mitochondrial protein import outside the inner membrane.
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PMID:Both ATP and an energized inner membrane are required to import a purified precursor protein into mitochondria. 303 90

Most mitochondrial proteins are encoded by nuclear genes and are synthesized as precursors containing a presequence at the N terminus. In yeast and in mammalian cells, the function of the presequence in mitochondrial targeting has been revealed by chimaeric gene studies. Fusion of a mitochondrial presequence to a foreign protein coding sequence enables the protein to be imported into mitochondria in vitro as well as in vivo. Whether plant mitochondrial presequences function in the same way has been unknown. We have previously isolated and characterized a nuclear gene (atp2-1) from Nicotiana plumbaginifolia that encodes the beta-subunit of the mitochondrial ATP synthase. We have constructed a chimaeric gene comprising a putative atp2-1 presequence fused to the bacterial chloramphenicol acetyltransferase (CAT) coding sequence and introduced it into the tobacco genome. We report here that a segment of 90 amino acids of the N terminus of the beta-subunit precursor is sufficient for the specific targeting of the CAT protein to mitochondria in transgenic plants. Our results demonstrate a high specificity for organelle targeting in plant cells.
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PMID:Targeting of bacterial chloramphenicol acetyltransferase to mitochondria in transgenic plants. 347 28


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