Vacuolar H+‐translocating inorganic pyrophosphatase [vacuolar H+‐pyrophosphatase (V‐PPase); EC 3.6.1.1] is a homodimeric proton translocase; it plays a pivotal role in electrogenic translocation of protons from the cytosol to the vacuolar lumen, at the expense of PPi hydrolysis, for the storage of ions, sugars, and other metabolites. Dimerization of V‐PPase is necessary for full proton translocation function, although the structural details of V‐PPase within the vacuolar membrane remain uncertain. The C‐terminus presumably plays a crucial role in sustaining enzymatic and proton‐translocating reactions. We used atomic force microscopy to visualize V‐PPases embedded in an artificial lipid bilayer under physiological conditions. V‐PPases were randomly distributed in reconstituted lipid bilayers; approximately 43.3% of the V‐PPase protrusions faced the cytosol, and 56.7% faced the vacuolar lumen. The mean height and width of the cytosolic V‐PPase protrusions were 2.8 ± 0.3 nm and 26.3 ± 4.7 nm, whereas those of the luminal protrusions were 1.2 ± 0.1 nm and 21.7 ± 3.6 nm, respectively. Moreover, both C‐termini of dimeric subunits of V‐PPase are on the same side of the membrane, and they are close to each other, as visualized with antibody and gold nanoparticles against 6×His tags on C‐terminal ends of the enzyme. The distance between the V‐PPase C‐terminal ends was determined to be approximately 2.2 ± 1.4 nm. Thus, our study is the first to provide structural details of a membrane‐bound V‐PPase dimer, revealing its adjacent C‐termini.

An electrically reconfigurable liquid-core/liquid-cladding (L(2)) optical waveguide with core liquid γ-butyrolactone (GBL, ncore = 1.4341, εcore = 39) and silicone oil (ncladding = 1.401, εcladding = 2.5) as cladding liquid is accomplished using dielectrophoresis (DEP) that attracts and deforms the core liquid with the greater permittivity to occupy the region of strong electric field provided by Teflon-coated ITO electrodes between parallel glass plates. Instead of continuously flowing core and cladding liquids along a physical microchannel, the DEP-formed L(2) optical waveguide guides light in a stationary virtual microchannel that requires liquids of limited volume without constant supply and creates stable liquid/liquid interfaces for efficient light guidance in a simply fabricated microfluidic device. We designed and examined (1) stationary and (2) moving L(2) optical waveguides on the parallel-plate electromicrofluidic platform. In the stationary L-shaped waveguide, light was guided in a GBL virtual microchannel core for a total of 27.85 mm via a 90° bend (radius 5 mm) before exiting from the light outlet of cross-sectional area 100 μm × 100 μm. For the stationary spiral waveguide, light was guided in a GBL core containing Rhodamine 6G (R6G, 1 mM) and through a series of 90° bends with decreasing radii from 5 mm to 2.5 mm. With the stationary straight waveguide, the propagation loss was measured to be 2.09 dB cm(-1) in GBL with R6G (0.01 mM). The moving L-shaped waveguide was implemented on a versatile electromicrofluidic platform on which electrowetting and DEP were employed to generate a precise GBL droplet and form a waveguide core. On sequentially applying appropriate voltage to one of three parallel L-shaped driving electrodes, the GBL waveguide core was shifted; the guided light was switched at a speed of up to 0.929 mm s(-1) (switching period 70 ms, switching rate 14.3 Hz) when an adequate electric signal (173.1 VRMS, 100 kHz) was applied.

The vacuole is a fundamental and dominant organelle and occupies a large part of the total cell volume in most mature plant cells. The higher-plant vacuole contains two types of proton-translocating pumps, H+-ATPase (EC 3.6.1.3) and H+-pyrophosphatase (EC 3.6.1.1), residing on the same membrane. These two enzymes generate roughly equal proton gradients across the vacuolar membrane for the secondary transport of ions and metabolites. However, the pumps respond differentially to stress in order to maintain critical functions of the vacuole. In this work, tonoplasts from etiolated mung bean seedlings (Vigna radiata L.) were used to investigate the function of these two enzymes under high osmotic pressure. At high concentrations of sucrose or sorbitol, the light scattering and volume of isolated vesicles were progressively changed. Concomitantly, enzymatic activities, proton translocation, and coupling efficiencies of these two proton-pumping enzymes were inhibited to various extents under high osmotic pressure. No significant change in enzymatic activities of purified vacuolar H+-PPase and H+-ATPase under similar conditions was observed. We thus believe that the membrane structure is an important determinant for proper function of proton pumping systems of plant vacuoles. Furthermore, kinetic analysis shows different variation in apparent Vmax but not in KM values of vacuolar H+-PPase and H+-ATPase at high osmolarity of sucrose and sorbitol, respectively, suggesting probable alterations in substrate hydrolysis reactions but not substrate-binding affinity of the enzymes. A working model is proposed to interpret supplemental roles of vacuolar H+-PPase and H+-ATPase to maintain appropriate functions of plant tonoplasts.

No abstract