Molecular architecture of the rotary motor in ATP synthase.
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Adenosine triphosphate (ATP) synthase contains a rotary motor involved in biological energy conversion. Its membrane-embedded F0 sector has a rotation generator fueled by the proton-motive force, which provides the energy required for the synthesis of ATP by the F1 domain. An electron density map obtained from crystals of a subcomplex of yeast mitochondrial ATP synthase shows a ring of 10 c subunits. Each c subunit forms an alpha-helical hairpin. The interhelical loops of six to seven of the c subunits are in close contact with the gamma and delta subunits of the central stalk. The extensive contact between the c ring and the stalk suggests that they may rotate as an ensemble during catalysis. (+info)
Mechanical rotation of the c subunit oligomer in ATP synthase (F0F1): direct observation.
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F0F1, found in mitochondria or bacterial membranes, synthesizes adenosine 5'-triphosphate (ATP) coupling with an electrochemical proton gradient and also reversibly hydrolyzes ATP to form the gradient. An actin filament connected to a c subunit oligomer of F0 was able to rotate by using the energy of ATP hydrolysis. The rotary torque produced by the c subunit oligomer reached about 40 piconewton-nanometers, which is similar to that generated by the gamma subunit in the F1 motor. These results suggest that the gamma and c subunits rotate together during ATP hydrolysis and synthesis. Thus, coupled rotation may be essential for energy coupling between proton transport through F0 and ATP hydrolysis or synthesis in F1. (+info)
Crucial role of the membrane potential for ATP synthesis by F(1)F(o) ATP synthases.
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ATP, the universal carrier of cell energy, is manufactured from ADP and phosphate by the enzyme ATP synthase using the free energy of an electrochemical gradient of protons (or Na(+)). The proton-motive force consists of two components, the transmembrane proton concentration gradient (delta pH) and the membrane potential. The two components were considered to be not only thermodynamically but also kinetically equivalent, since the chloroplast ATP synthase appeared to operate on delta pH only. Recent experiments demonstrate, however, that the chloroplast ATP synthase, like those of mitochondria and bacteria, requires a membrane potential for ATP synthesis. Hence, the membrane potential and proton gradient are not equivalent under normal operating conditions far from equilibrium. These conclusions are corroborated by the finding that only the membrane potential induces a rotary torque that drives the counter-rotation of the a and c subunits in the F(o) motor of Propionigenium modestum ATP synthase. (+info)
The cellular biology of proton-motive force generation by V-ATPases.
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The vacuolar H(+)-ATPase (V-ATPase) is one of the most fundamental enzymes in nature. It functions in almost every eukaryotic cell and energizes a wide variety of organelles and membranes. In contrast to F-ATPases, whose primary function in eukaryotic cells is to form ATP at the expense of the proton-motive force, V-ATPases function exclusively as ATP-dependent proton pumps. The proton-motive force generated by V-ATPases in organelles and across plasma membranes of eukaryotic cells is utilized as a driving force for numerous secondary transport processes. The enzyme is also vital for the proper functioning of endosomes and the Golgi apparatus. In contrast to yeast vacuoles, which maintain an internal pH of approximately 5. 5, it is believed that the vacuoles of lemon fruit may have a pH as low as 2. Similarly, some brown and red algae maintain an internal pH as low as 1 in their vacuoles. It was yeast genetics that allowed the identification of the special properties of individual subunits and the discovery of the factors that are involved in V-ATPase biogenesis and assembly. Null mutations in genes encoding V-ATPase subunits of Saccharomyces cerevisiae result in a phenotype that is unable to grow at high pH and is sensitive to high and low metal-ion concentrations. Treatment of these null mutants with ethyl methanesulphonate causes mutations that suppress the V-ATPase null phenotype, and these cells are able to grow at pH 7.5. The suppressor mutants were denoted as svf (Suppressor of V-ATPase Function). The svf mutations are recessive: crossing the svf mutants with their corresponding V-ATPase null mutants resulted in diploid strains that were not able to grow at pH 7.5. A novel gene family in which null mutations cause pleiotropic effects on metal-ion resistance or on the sensitivity and distribution of membrane proteins in different targets was discovered. We termed this gene family VTC (Vacuolar Transporter Chaperon) and discovered four genes in S. cerevisiae that belong to the family. Inactivation of one of them, VTC1, in the background of V-ATPase null mutations resulted in an svf phenotype that was able to grow at pH 7.5. Apparently, Vtc1p is one of a few membrane organizers that determine the relative amounts of different membrane proteins in the various cellular membranes. We utilize the numerous yeast mutants generated in our laboratory to identify the specific organelle whose acidification is vital. The interaction between V-ATPase and the secretory pathway is investigated. (+info)
A spectroscopic assay for the analysis of carbohydrate transport reactions.
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A carbohydrate-transport assay was developed that does not require isotopically labelled substrates, but allows transport reactions to be followed spectrophotometrically. It makes use of a membrane system (hybrid membranes or proteoliposomes) bearing the transport system of interest, and a pyrroloquinoline quinone-dependent aldose dehydrogenase [soluble glucose dehydrogenase (sGDH)] and the electron acceptor 2,6-dichloroindophenol (Cl2Ind) enclosed in the vesicle lumen. After transport across the vesicular membrane, the sugar is oxidized by sGDH. The accompanying reduction of Cl2Ind results in a decrease in A600. The assay was developed and optimized for the lactose carrier (LacS) of Streptococcus thermophilus, and both solute/H+ symport and exchange types of transport could be measured with high sensitivity in crude membranes as well as in proteoliposomes. To observe exchange transport, the membranes were preloaded with a nonoxidizable substrate analogue and diluted in assay buffer containing an oxidizable sugar. Transport rates measured with this assay are comparable with those obtained with the conventional assay using isotopically labelled substrates. The method is particularly suited for determining transport reactions that are not coupled to any form of metabolic energy such as uniport reactions, or for characterizing mutant proteins with a defective energy-coupling mechanism or systems with high-affinity constants for sugars. (+info)
Regulation and reversibility of vacuolar H(+)-ATPase.
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Arabidopsis thaliana vacuolar H(+)-translocating pyrophosphatase (V-PPase) was expressed functionally in yeast vacuoles with endogenous vacuolar H(+)-ATPase (V-ATPase), and the regulation and reversibility of V-ATPase were studied using these vacuoles. Analysis of electrochemical proton gradient (DeltamuH) formation with ATP and pyrophosphate indicated that the proton transport by V-ATPase or V-PPase is not regulated strictly by the proton chemical gradient (DeltapH). On the other hand, vacuolar membranes may have a regulatory mechanism for maintaining a constant membrane potential (DeltaPsi). Chimeric vacuolar membranes showed ATP synthesis coupled with DeltamuH established by V-PPase. The ATP synthesis was sensitive to bafilomycin A(1) and exhibited two apparent K(m) values for ADP. These results indicate that V-ATPase is a reversible enzyme. The ATP synthesis was not observed in the presence of nigericin, which dissipates DeltapH but not DeltaPsi, suggesting that DeltapH is essential for ATP synthesis. (+info)
Light-induced stimulation of carbonic anhydrase activity in pea thylakoids.
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Stimulation of the bicarbonate dehydration reaction in thylakoid suspension under conditions of saturating light at pH 7.6-8.0 was discovered. This effect was inhibited by nigericin or the lipophilic carbonic anhydrase (CA) inhibitor ethoxyzolamide (EZ), but not by the hydrophilic CA inhibitor, acetazolamide. It was shown that the action of EZ is not caused by an uncoupling effect. It was concluded that thylakoid CA is the enzyme utilizing the light-generated proton gradient across the thylakoid membrane thus facilitating the production of CO(2) from HCO(3)(-) and that this enzyme is covered from the stroma side of thylakoids by a lipid barrier. (+info)
Protonmotive force regulates the membrane conductance of Streptococcus bovis in a non-ohmic fashion.
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Because the DCCD (dicyclohexylcarbodiimide)-sensitive, F-ATPase-mediated, futile ATP hydrolysis of non-growing Streptococcus bovis JB1 cells was not affected by sodium or potassium, ATP hydrolysis appeared to be dependent only upon the rate of proton flux across the cell membrane. However, available estimates of bacterial proton conductance were too low to account for the rate of ATP turnover observed in S. bovis. When de-energized cells were subjected to large pH gradients (2.75 units, or -170 mV), internal pH declined at a rate of 0.15 pH units s(-1). Based on an estimated cellular buffering capacity of 200 nmol H+ (mg protein)(-1) per pH unit, H+ flux across the cell membrane (at -170 mV) was 108 mmol (g protein)(-1) h(-1). When potassium-loaded cells were treated with valinomycin in low-potassium buffers, initial K+ efflux generated membrane potentials in close agreement with values predicted by the Nernst equation. These artificial membrane potentials drove H+ uptake, and H+ influx was counterbalanced by a further loss of cellular K+. Flame photometry indicated that the rate of K+ loss was 215 (+/-26) mmol K+ (g protein)(-1) h(-1) at -170 mV, but the potassium-sensitive fluorescent compound CD222 indicated that this rate was only 110 (+/-44) mmol K+ (g protein)(-1) h(-1). As pH gradients or membrane potentials were reduced, the rate of H+ flux declined in a non-ohmic fashion, and all rates were <25 mmol (g protein)(-1) h(-1) at a driving force of -80 mV. Previous estimates of bacterial proton flux were based on low and unphysiological protonmotive forces, and the assumption that H+ influx rate would be ohmic. Rates of H+ influx into S. bovis cells [as high as 9x10(-11) mol H+ (cm membrane)(-2) s(-1)] were similar to rates reported for respiring mitochondria, but were at least 20-fold greater than any rate previously reported in lactic acid bacteria. (+info)