Functional characterization of the biogenic amine transporters on human macrophages.

Monocyte-derived macrophages (MDMs) are key players in tissue homeostasis and diseases regulated by a variety of signaling molecules. Recent literature has highlighted the ability for biogenic amines to regulate macrophage functions, but the mechanisms governing biogenic amine signaling in and around immune cells remain nebulous. In the CNS, biogenic amine transporters are regarded as the master regulators of neurotransmitter signaling. While we and others have shown that macrophages express these transporters, relatively little is known of their function in these cells. To address these knowledge gaps, we investigated the function of norepinephrine transporter (NET) and dopamine transporter (DAT) on human MDMs. We found that both NET and DAT are present and can uptake substrate from the extracellular space at baseline. Not only was DAT expressed in cultured MDMs, but it was also detected in a subset of intestinal macrophages in situ. Surprisingly, we discovered a NET-independent, DAT-mediated immunomodulatory mechanism in response to LPS. LPS induced reverse transport of dopamine through DAT, engaging an autocrine/paracrine signaling loop that regulated the macrophage response. Removing this signaling loop enhanced the proinflammatory response to LPS. Our data introduce a potential role for DAT in the regulation of innate immunity.

None of the rotor residues of F1-ATPase are essential for torque generation.

F1-ATPase is a powerful rotary molecular motor that can rotate an object several hundred times as large as the motor itself against the viscous friction of water. Forced reverse rotation has been shown to lead to ATP synthesis, implying that the mechanical work against the motor's high torque can be converted into the chemical energy of ATP. The minimal composition of the motor protein is α3ß3γ subunits, where the central rotor subunit γ turns inside a stator cylinder made of alternately arranged α3ß3 subunits using the energy derived from ATP hydrolysis. The rotor consists of an axle, a coiled coil of the amino- and carboxyl-terminal α-helices of γ, which deeply penetrates the stator cylinder, and a globular protrusion that juts out from the stator. Previous work has shown that, for a thermophilic F1, significant portions of the axle can be truncated and the motor still rotates a submicron sized bead duplex, indicating generation of up to half the wild-type (WT) torque. Here, we inquire if any specific interactions between the stator and the rest of the rotor are needed for the generation of a sizable torque. We truncated the protruding portion of the rotor and replaced part of the remaining axle residues such that every residue of the rotor has been deleted or replaced in this or previous truncation mutants. This protrusionless construct showed an unloaded rotary speed about a quarter of the WT, and generated one-third to one-half of the WT torque. No residue-specific interactions are needed for this much performance. F1 is so designed that the basic rotor-stator interactions for torque generation and control of catalysis rely solely upon the shape and size of the rotor at very low resolution. Additional tailored interactions augment the torque to allow ATP synthesis under physiological conditions.

Controlled rotation of the F1-ATPase reveals differential and continuous binding changes for ATP synthesis.

F(1)-ATPase is an ATP-driven rotary molecular motor that synthesizes ATP when rotated in reverse. To elucidate the mechanism of ATP synthesis, we imaged binding and release of fluorescently labelled ADP and ATP while rotating the motor in either direction by magnets. Here we report the binding and release rates for each of the three catalytic sites for 360° of the rotary angle. We show that the rates do not significantly depend on the rotary direction, indicating ATP synthesis by direct reversal of the hydrolysis-driven rotation. ADP and ATP are discriminated in angle-dependent binding, but not in release. Phosphate blocks ATP binding at angles where ADP binding is essential for ATP synthesis. In synthesis rotation, the affinity for ADP increases by >10(4), followed by a shift to high ATP affinity, and finally the affinity for ATP decreases by >10(4). All these angular changes are gradual, implicating tight coupling between the rotor angle and site affinities.

Torque generation in F1-ATPase devoid of the entire amino-terminal helix of the rotor that fills half of the stator orifice.

F(1)-ATPase is an ATP-driven rotary molecular motor in which the central γ-subunit rotates inside a cylinder made of α(3)ß(3) subunits. The amino and carboxyl termini of the γ rotor form a coiled coil of α-helices that penetrates the stator cylinder to serve as an axle. Crystal structures indicate that the axle is supported by the stator at two positions, at the orifice and by the hydrophobic sleeve surrounding the axle tip. The sleeve contacts are almost exclusively to the longer carboxyl-terminal helix, whereas nearly half the orifice contacts are to the amino-terminal helix. Here, we truncated the amino-terminal helix stepwise up to 50 residues, removing one half of the axle all the way up and far beyond the orifice. The half-sliced axle still rotated with an unloaded speed a quarter of the wild-type speed, with torque nearly half the wild-type torque. The truncations were made in a construct where the rotor tip was connected to a ß-subunit via a short peptide linker. Linking alone did not change the rotational characteristics significantly. These and previous results show that nearly half the normal torque is generated if rotor-stator interactions either at the orifice or at the sleeve are preserved, suggesting that the make of the motor is quite robust.

Resolving stepping rotation in Thermus thermophilus H(+)-ATPase/synthase with an essentially drag-free probe.

Vacuole-type ATPases (V(o)V1) and F(o)F1 ATP synthases couple ATP hydrolysis/synthesis in the soluble V(1) or F1 portion with proton (or Na(+)) flow in the membrane-embedded V(o) or F(o) portion through rotation of one common shaft. Here we show at submillisecond resolutions the ATP-driven rotation of isolated V1 and the whole V(o)V1 from Thermus thermophilus, by attaching a 40-nm gold bead for which viscous drag is almost negligible. V1 made 120° steps, commensurate with the presence of three catalytic sites. Dwells between the steps involved at least two events other than ATP binding, one likely to be ATP hydrolysis. V(o)V1 exhibited 12 dwell positions per revolution, consistent with the 12-fold symmetry of the V(o) rotor in T. thermophilus. Unlike F1 that undergoes 80°-40° substepping, chemo-mechanical checkpoints in isolated V1 are all at the ATP-waiting position, and V(o) adds further bumps through stator-rotor interactions outside and remote from V1.

Chemo-mechanical coupling in F(1)-ATPase revealed by catalytic site occupancy during catalysis.

F(1)-ATPase is a rotary molecular motor in which the central gamma subunit rotates inside a cylinder made of alpha(3)beta(3) subunits. To clarify how ATP hydrolysis in three catalytic sites cooperate to drive rotation, we measured the site occupancy, the number of catalytic sites occupied by a nucleotide, while assessing the hydrolysis activity under identical conditions. The results show hitherto unsettled timings of ADP and phosphate releases: starting with ATP binding to a catalytic site at an ATP-waiting gamma angle defined as 0 degrees , phosphate is released at approximately 200 degrees , and ADP is released during quick rotation between 240 degrees and 320 degrees that is initiated by binding of a third ATP. The site occupancy remains two except for a brief moment after the ATP binding, but the third vacant site can bind a medium nucleotide weakly.

Stimulation of F(1)-ATPase activity by sodium dodecyl sulfate.

F(1)-ATPase is a rotary molecular motor in which the gamma subunit rotates inside the cylinder made of alpha(3)beta(3) subunits. We have studied the effects of sodium dodecyl sulfate (SDS) on the rotational and ATP hydrolysis activities of F(1)-ATPase. Bulk hydrolysis activity at various SDS concentrations was examined at 2mM ATP. Maximal stimulation was obtained at 0.003% (w/v) SDS, the initial (least inhibited) activity being about 1.4 times and the steady-state activity 3-4 times the values in the absence of SDS. Rotation rates observed with a 40-nm gold bead or a 0.29-mum bead duplex as well as the torque were unaffected by the presence of 0.003% SDS. The fraction of beads that rotated, in contrast, tended to increase in the presence of SDS. SDS seems to bring inactive F(1) molecules into an active form but it does not alter or enhance the function of already active F(1) molecules significantly.

Effect of epsilon subunit on the rotation of thermophilic Bacillus F1-ATPase.

F(1)-ATPase is an ATP-driven motor in which gammaepsilon rotates in the alpha(3)beta(3)-cylinder. It is attenuated by MgADP inhibition and by the epsilon subunit in an inhibitory form. The non-inhibitory form of epsilon subunit of thermophilic Bacillus PS3 F(1)-ATPase is stabilized by ATP-binding with micromolar K(d) at 25 degrees C. Here, we show that at [ATP]>2 microM, epsilon does not affect rotation of PS3 F(1)-ATPase but, at 200 nM ATP, epsilon prolongs the pause of rotation caused by MgADP inhibition while the frequency of the pause is unchanged. It appears that epsilon undergoes reversible transition to the inhibitory form at [ATP] below K(d).

Neither helix in the coiled coil region of the axle of F1-ATPase plays a significant role in torque production.

F(1)-ATPase is an ATP-driven rotary molecular motor in which the central gamma-subunit rotates inside the cylinder made of alpha(3)beta(3) subunits. The amino and carboxy termini of the gamma-subunit form the axle, an alpha-helical coiled coil that deeply penetrates the stator cylinder. We previously truncated the axle step by step, starting with the longer carboxy terminus and then cutting both termini at the same levels, resulting in a slower yet considerably powerful rotation. Here we examine the role of each helix by truncating only the carboxy terminus by 25-40 amino-acid residues. Longer truncation impaired the stability of the motor complex severely: 40 deletions failed to yield rotating the complex. Up to 36 deletions, however, the mutants produced an apparent torque at nearly half of the wild-type torque, independent of truncation length. Time-averaged rotary speeds were low because of load-dependent stumbling at 120 degrees intervals, even with saturating ATP. Comparison with our previous work indicates that half the normal torque is produced at the orifice of the stator. The very tip of the carboxy terminus adds the other half, whereas neither helix in the middle of the axle contributes much to torque generation and the rapid progress of catalysis. None of the residues of the entire axle played a specific decisive role in rotation.

Temperature dependence of the rotation and hydrolysis activities of F1-ATPase.

F(1)-ATPase, a water-soluble portion of the enzyme ATP synthase, is a rotary molecular motor driven by ATP hydrolysis. To learn how the kinetics of rotation are regulated, we have investigated the rotational characteristics of a thermophilic F(1)-ATPase over the temperature range 4-50 degrees C by attaching a polystyrene bead (or bead duplex) to the rotor subunit and observing its rotation under a microscope. The apparent rate of ATP binding estimated at low ATP concentrations increased from 1.2 x 10(6) M(-1) s(-1) at 4 degrees C to 4.3 x 10(7) M(-1) s(-1) at 40 degrees C, whereas the torque estimated at 2 mM ATP remained around 40 pN.nm over 4-50 degrees C. The rotation was stepwise at 4 degrees C, even at the saturating ATP concentration of 2 mM, indicating the presence of a hitherto unresolved rate-limiting reaction that occurs at ATP-waiting angles. We also measured the ATP hydrolysis activity in bulk solution at 4-65 degrees C. F(1)-ATPase tends to be inactivated by binding ADP tightly. Both the inactivation and reactivation rates were found to rise sharply with temperature, and above 30 degrees C, equilibrium between the active and inactive forms was reached within 2 s, the majority being inactive. Rapid inactivation at high temperatures is consistent with the physiological role of this enzyme, ATP synthesis, in the thermophile.

Axle-less F1-ATPase rotates in the correct direction.

F1-adenosine triphosphatase (ATPase) is an ATP-driven rotary molecular motor in which the central gamma subunit rotates inside a cylinder made of three alpha and three beta subunits alternately arranged. The rotor shaft, an antiparallel alpha-helical coiled coil of the amino and carboxyl termini of the gamma subunit, deeply penetrates the central cavity of the stator cylinder. We truncated the shaft step by step until the remaining rotor head would be outside the cavity and simply sat on the concave entrance of the stator orifice. All truncation mutants rotated in the correct direction, implying torque generation, although the average rotary speeds were low and short mutants exhibited moments of irregular motion. Neither a fixed pivot nor a rigid axle was needed for rotation of F1-ATPase.

Coupling of rotation and catalysis in F(1)-ATPase revealed by single-molecule imaging and manipulation.

F(1)-ATPase is a rotary molecular motor that proceeds in 120 degrees steps, each driven by ATP hydrolysis. How the chemical reactions that occur in three catalytic sites are coupled to mechanical rotation is the central question. Here, we show by high-speed imaging of rotation in single molecules of F(1) that phosphate release drives the last 40 degrees of the 120 degrees step, and that the 40 degrees rotation accompanies reduction of the affinity for phosphate. We also show, by single-molecule imaging of a fluorescent ATP analog Cy3-ATP while F(1) is forced to rotate slowly, that release of Cy3-ADP occurs at approximately 240 degrees after it is bound as Cy3-ATP at 0 degrees . This and other results suggest that the affinity for ADP also decreases with rotation, and thus ADP release contributes part of energy for rotation. Together with previous results, the coupling scheme is now basically complete.

The rotor tip inside a bearing of a thermophilic F1-ATPase is dispensable for torque generation.

F(1)-ATPase is an ATP-driven rotary molecular motor in which the central gamma-subunit rotates inside a stator cylinder made of alpha(3)beta(3) subunits. To elucidate the role of rotor-stator interactions in torque generation, we truncated the gamma-subunit at its carboxyl terminus, which forms an alpha helix that penetrates deeply into the stator cylinder. We used an alpha(3)beta(3)gamma subcomplex of F(1)-ATPase derived from thermophilic Bacillus PS3 and expressed it in Escherichia coli. We could obtain purified subcomplexes in which 14, 17, or 21 amino-acid residues were deleted. The rotary characteristics of the truncated mutants, monitored by attaching a duplex of 0.49-microm beads to the gamma-subunit, did not differ greatly from those of the wild-type over the ATP concentrations of 20 nM-2 mM, the most conspicuous effect being approximately 50% reduction in torque and approximately 70% reduction in the rate of ATP binding upon deletion of 21 residues. The ATP hydrolysis activity estimated in bulk samples was more seriously affected. The 21-deletion mutant, in particular, was >10-fold less active, but this is likely due to instability of this subcomplex. For torque generation, though not for rapid catalysis, most of the rotor-stator contacts on the deeper half of the penetrating portion of the gamma-subunit are dispensable.

One rotary mechanism for F1-ATPase over ATP concentrations from millimolar down to nanomolar.

F(1)-ATPase is a rotary molecular motor in which the central gamma-subunit rotates inside a cylinder made of alpha(3)beta(3)-subunits. The rotation is driven by ATP hydrolysis in three catalytic sites on the beta-subunits. How many of the three catalytic sites are filled with a nucleotide during the course of rotation is an important yet unsettled question. Here we inquire whether F(1) rotates at extremely low ATP concentrations where the site occupancy is expected to be low. We observed under an optical microscope rotation of individual F(1) molecules that carried a bead duplex on the gamma-subunit. Time-averaged rotation rate was proportional to the ATP concentration down to 200 pM, giving an apparent rate constant for ATP binding of 2 x 10(7) M(-1)s(-1). A similar rate constant characterized bulk ATP hydrolysis in solution, which obeyed a simple Michaelis-Menten scheme between 6 mM and 60 nM ATP. F(1) produced the same torque of approximately 40 pN.nm at 2 mM, 60 nM, and 2 nM ATP. These results point to one rotary mechanism governing the entire range of nanomolar to millimolar ATP, although a switchover between two mechanisms cannot be dismissed. Below 1 nM ATP, we observed less regular rotations, indicative of the appearance of another reaction scheme.

Rotation of F1-ATPase: how an ATP-driven molecular machine may work.

F1-ATPase is a rotary motor made of a single protein molecule. Its rotation is driven by free energy obtained by ATP hydrolysis. In vivo, another motor, Fo, presumably rotates the F1 motor in the reverse direction, reversing also the chemical reaction in F1 to let it synthesize ATP. Here we attempt to answer two related questions, How is free energy obtained by ATP hydrolysis converted to the mechanical work of rotation, and how is mechanical work done on F1 converted to free energy to produce ATP? After summarizing single-molecule observations of F1 rotation, we introduce a toy model and discuss its free-energy diagrams to possibly answer the above questions. We also discuss the efficiency of molecular motors in general.

Mechanically driven ATP synthesis by F1-ATPase.

ATP, the main biological energy currency, is synthesized from ADP and inorganic phosphate by ATP synthase in an energy-requiring reaction. The F1 portion of ATP synthase, also known as F1-ATPase, functions as a rotary molecular motor: in vitro its gamma-subunit rotates against the surrounding alpha3beta3 subunits, hydrolysing ATP in three separate catalytic sites on the beta-subunits. It is widely believed that reverse rotation of the gamma-subunit, driven by proton flow through the associated F(o) portion of ATP synthase, leads to ATP synthesis in biological systems. Here we present direct evidence for the chemical synthesis of ATP driven by mechanical energy. We attached a magnetic bead to the gamma-subunit of isolated F1 on a glass surface, and rotated the bead using electrical magnets. Rotation in the appropriate direction resulted in the appearance of ATP in the medium as detected by the luciferase-luciferin reaction. This shows that a vectorial force (torque) working at one particular point on a protein machine can influence a chemical reaction occurring in physically remote catalytic sites, driving the reaction far from equilibrium.

The ATP-waiting conformation of rotating F1-ATPase revealed by single-pair fluorescence resonance energy transfer.

F1-ATPase is an ATP-driven rotary motor in which a rod-shaped gamma subunit rotates inside a cylinder made of alpha3beta3 subunits. To elucidate the conformations of rotating F1, we measured fluorescence resonance energy transfer (FRET) between a donor on one of the three betas and an acceptor on gamma in single F1 molecules. The yield of FRET changed stepwise at low ATP concentrations, reflecting the stepwise rotation of gamma. In the ATP-waiting state, the FRET yields indicated a gamma position approximately 40 degrees counterclockwise (= direction of rotation) from that in the crystal structures of mitochondrial F1, suggesting that the crystal structures mimic a metastable state before product release.

Myosin V is a left-handed spiral motor on the right-handed actin helix.

Myosin V is a two-headed, actin-based molecular motor implicated in organelle transport. Previously, a single myosin V molecule has been shown to move processively along an actin filament in discrete approximately 36 nm steps. However, 36 nm is the helical repeat length of actin, and the geometry of the previous experiments may have forced the heads to bind to, or halt at, sites on one side of actin that are separated by 36 nm. To observe unconstrained motion, we suspended an actin filament in solution and attached a single myosin V molecule carrying a bead duplex. The duplex moved as a left-handed spiral around the filament, disregarding the right-handed actin helix. Our results indicate a stepwise walking mechanism in which myosin V positions and orients the unbound head such that the head will land at the 11th or 13th actin subunit on the opposing strand of the actin double helix.