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)

Since the proposal of the chemiosmotic theory there has been a continuing debate about how protons that have been pumped across membranes reach another membrane protein that utilizes the established pH gradient. Evidence has been gathered in favour of a 'delocalized' theory, in which the pumped protons equilibrate with the aqueous bulk phase before being consumed, and a 'localized' one, in which protons move exclusively along the membrane surface. We report here that after proton release by an integral membrane protein, long-range proton transfer along the membrane surface is faster than proton exchange with the bulk water phase. The rate of lateral proton diffusion can be calculated by considering the buffer capacity of the membrane surface. Our results suggest that protons can efficiently diffuse along the membrane surface between a source and a sink (for example H(+)-ATP synthase) without dissipation losses into the aqueous bulk.
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PMID:Proton migration along the membrane surface and retarded surface to bulk transfer. 804 44

We have previously isolated the yeast nuclear gene OXA1 and showed that Oxa1p is required for the formation of the cytochrome c oxidase and ATP synthase complexes. We have expressed Oxa1p in E. coli and shown that it is toxic and rapidly degraded. Nevertheless, a truncated protein was successfully expressed and antibodies have been raised against this truncated protein. These antibodies recognise a protein in mitochondrially enriched fractions. In vitro mitochondrial import experiments demonstrate that the import of Oxa1p is accompanied by the cleavage of a long pre-sequence. Osmotic swelling and alkaline carbonate extraction show that Oxa1p is an integral membrane protein located in the inner membrane of mitochondria. The relationships between the sub-mitochondrial location and the function of Oxa1p are discussed.
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PMID:Oxa1p, which is required for cytochrome c oxidase and ATP synthase complex formation, is embedded in the mitochondrial inner membrane. 910 37

The Saccharomyces cerevisiae gene BTN1, encodes a 408 amino acid putative integral membrane protein, which is 39% identical and 59% similar to the human Cln3p, whose mutant forms are responsible for Batten's disease and for a diminished degradation of mitochondrial ATPase synthase subunit c. Disruption experiments established that Btn1p is not essential for viability, mitochondrial function, or degradation of mitochondrial ATP synthase in yeast.
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PMID:BTN1, a yeast gene corresponding to the human gene responsible for Batten's disease, is not essential for viability, mitochondrial function, or degradation of mitochondrial ATP synthase. 921 33

In order to ascertain whether there is one site for the import of precursor proteins into chloroplasts or whether different precursor proteins are imported via different import machineries, chloroplasts were incubated with large quantities of the precursor of the 33 kDa subunit of the oxygen-evolving complex (pOE33) or the precursor of the light-harvesting chlorophyll a/b-binding protein (pLHCP) and tested for their ability to import a wide range of other chloroplast precursor proteins. Both pOE33 and pLHCP competed for import into chloroplasts with precursors of the stromally-targeted small subunit of Rubisco (pSSu), ferredoxin NADP(+) reductase (pFNR) and porphobilinogen deaminase; the thylakoid membrane proteins LHCP and the Rieske iron-sulphur protein (pRieske protein); ferrochelatase and the gamma subunit of the ATP synthase (which are both associated with the thylakoid membrane); the thylakoid lumenal protein plastocyanin and the phosphate translocator, an integral membrane protein of the inner envelope. The concentrations of pOE33 or pLHCP required to cause half-maximal inhibition of import ranged between 0.2 and 4.9 microM. These results indicate that all of these proteins are imported into the chloroplast by a common import machinery. Incubation of chloroplasts with pOE33 inhibited the formation of early import intermediates of pSSu, pFNR and pRieske protein.
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PMID:Chloroplast precursor proteins compete to form early import intermediates in isolated pea chloroplasts. 1118 12

Subunit a of the Escherichia coli ATP synthase, a 30 kDa integral membrane protein, was purified to homogeneity by a novel procedure incorporating selective extraction into a monophasic mixture of chloroform, methanol and water, followed by Ni-NTA chromatography in the mixed solvent. Pure subunit a was reconstituted with subunits b and c and phospholipids to form a functional proton-translocating unit. Nuclear magnetic resonance (NMR) spectra of the pure subunit a in the mixed solvent show good chemical shift dispersion and demonstrate the potential of the solvent mixture for NMR studies of the large membrane proteins that are currently intractable in aqueous detergent solutions.
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PMID:Subunit A of the E. coli ATP synthase: reconstitution and high resolution NMR with protein purified in a mixed polarity solvent. 1470 21

The structure of the 30 KDa subunit a of the membrane component (F(0)) of E. coli ATP synthase is investigated in a mixture of chloroform, methanol and water, a solvent previously used for solving the structure of another integral membrane protein, subunit c. Near complete backbone chemical shift assignments were made from a set of TROSY experiments including HNCO, HNCA, HN(CA)CB, HN(CO)CACB and 4D HNCOCA and HNCACO. Secondary structure of subunit a was predicted from the backbone chemical shifts using TALOS program. The protein was found to consist of multiple elongated alpha-helical segments. This finding is generally consistent with previous predictions of multiple transmembrane alpha-helices in this polytopic protein.
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PMID:Backbone 1H, 15N and 13C assignments for the subunit a of the E. coli ATP synthase. 1521 58

Bacterial conjugation is an example of macromolecular trafficking between cells, based on the translocation of single-stranded DNA across membranes through a type IV secretion system. TrwBDeltaN70 is the soluble domain of TrwB, an essential integral membrane protein that couples the relaxosome (a nucleoprotein complex) to the DNA transport apparatus in plasmid R388 conjugation. TrwBDeltaN70 crystallographic structure revealed a hexamer with six equivalent subunits and a central channel. In this work, we characterize a DNA-dependent ATPase activity for TrwBDeltaN70. The protein displays positive cooperativity for ATP hydrolysis, with at least three catalytic sites involved. The activity is sensitive to pH and salt concentration, being more active at low pH values. The effective oligonucleotide size required for activation of the ATPase function is between 40 and 45 nucleotides, and the same length is required for the formation of high-molecular-weight TrwBDeltaN70-DNA complexes, as observed by gel filtration chromatography. A mutation in a tryptophan residue (W216A), placed in the central pore formed by the hexameric structure, resulted in a protein that did not hydrolyze ATP. In addition, it exerted a dominant negative effect, both on R388 conjugation frequency and ATP hydrolysis, underscoring the multimeric state of the protein. ATP hydrolysis was not coupled to a DNA unwinding activity under the tested conditions, which included forked DNA substrates. These results, together with TrwB structural similarity to F1-ATPase, lead us to propose a mechanism for TrwB as a DNA-translocating motor.
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PMID:TrwB, the coupling protein involved in DNA transport during bacterial conjugation, is a DNA-dependent ATPase. 1591 15

The c-subunit of ATP synthase (AtpH) is an 8 kD integral membrane protein with two transmembrane domains; we set out to demonstrate it amenable to top-down electrospray-ionization Fourier-transform mass spectrometry (FT-MS) using both collision activated and electron capture dissociation (CAD/ECD). Thermal activation concomitant with electron delivery was necessary for efficient ECD (activated-ion ECD; aiECD), yielding complementary information and greater sequence coverage in the transmembrane domains in comparison with CAD.
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PMID:Increased coverage in the transmembrane domain with activated-ion electron capture dissociation for top-down Fourier-transform mass spectrometry of integral membrane proteins. 1744 48

Conjugative systems contain an essential integral membrane protein involved in DNA transport called the Type IV coupling protein (T4CP). The T4CP of conjugative plasmid R388 is TrwB, a DNA-dependent ATPase. Biochemical and structural data suggest that TrwB uses energy released from ATP hydrolysis to pump DNA through its central channel by a mechanism similar to that used by F1-ATPase or ring helicases. For DNA transport, TrwB couples the relaxosome (a DNA-protein complex) to the secretion channel. In this work we show that TrwA, a tetrameric oriT DNA-binding protein and a component of the R388 relaxosome, stimulates TrwBDeltaN70 ATPase activity, revealing a specific interaction between the two proteins. This interaction occurs via the TrwA C-terminal domain. A 68-kDa complex between TrwBDeltaN70 and TrwA C-terminal domain was observed by gel filtration chromatography, consistent with a 1:1 stoichiometry. Additionally, electron microscopy revealed the formation of oligomeric TrwB complexes in the presence, but not in the absence, of TrwA protein. TrwBDeltaN70 ATPase activity in the presence of TrwA was further enhanced by DNA. Interestingly, maximal ATPase rates were achieved with TrwA and different types of dsDNA substrates. This is consistent with a role of TrwA in facilitating the interaction between TrwB and DNA. Our findings provide a new insight into the mechanism by which TrwB recruits the relaxosome for DNA transport. The process resembles the mechanism used by other DNA-dependent molecular motors, such as the RuvA/RuvB system, to be targeted to the DNA followed by hexamer assembly.
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PMID:The ATPase activity of the DNA transporter TrwB is modulated by protein TrwA: implications for a common assembly mechanism of DNA translocating motors. 1759 13

YidC, a 60-kDa integral membrane protein, plays an important role in membrane protein insertion in bacteria. YidC can function together with the SecYEG machinery or operate independently as a membrane protein insertase. In this paper, we describe two new yidC mutants that lead to a cold-sensitive phenotype in bacterial cell growth. Both alleles impart a cold-sensitive phenotype and result from point mutations localized to the third transmembrane (TM3) segment of YidC, indicating that this region is crucial for YidC function. We found that the yidC(C423R) mutant confers a weak phenotype on membrane protein insertion while a yidC(P431L) mutant leads to a stronger phenotype. In both cases, the affected substrates include the Pf3 coat protein and ATP synthase F(1)F(o) subunit c (F(o)C), while CyoA (the quinol binding subunit of the cytochrome bo3 quinol oxidase complex) and wild-type procoat are slightly affected or not affected in either cold-sensitive mutant. To determine if the different substrates require various levels of YidC activity for membrane insertion, we performed studies where YidC was depleted using an arabinose-dependent expression system. We found that -3M-PC-Lep (a construct with three negatively charged residues inserted into the middle of the procoat-Lep [PC-Lep] protein) and Pf3 P2 (a construct with the Lep P2 domain added at the C terminus of Pf3 coat) required the highest amount of YidC and that CyoA-N-P2 (a construct with the amino-terminal part of CyoA fused to the Lep P2 soluble domain) and PC-Lep required the least, while F(o)C required moderate YidC levels. Although the cold-sensitive mutations can preferentially affect one substrate over another, our results indicate that different substrates require different levels of YidC activity for membrane insertion. Finally, we obtained several intragenic suppressors that overcame the cold sensitivity of the C423R mutation. One pair of mutations suggests an interaction between TM2 and TM3 of YidC. The studies reveal the critical regions of the YidC protein and provide insight into the substrate profile of the YidC insertase.
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PMID:Isolation of cold-sensitive yidC mutants provides insights into the substrate profile of the YidC insertase and the importance of transmembrane 3 in YidC function. 1793 92


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