The proximity between C‐termini of dimeric vacuolar H⁺‐pyrophosphatase determined using atomic force microscopy and a gold nanoparticle technique

Abstract

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.

Distance Variations between Active Sites of H+-Pyrophosphatase Determined by Fluorescence Resonance Energy Transfer

“…2D exhibits a prototypical globular structure of CtH ϩ -PPase under physiological conditions. The volume of CtH ϩ -PPase calculated is ϳ328.5 Ϯ 37.2 nm 3 (V; n ϭ 50), similar to mung bean H ϩ -PPase in a dimeric and symmetric structure (12).…”

“…Besides, the dimeric structure of H ϩ -PPase from Thermotoga maritima was shown by electron microscopy (11). Furthermore, the homodimeric mung bean H ϩ -PPase in the lipid bilayer had been visualized by atomic force microscopy to be larger in size at cytoplasmic protrusions but relatively smaller at luminal protrusions (12). After thermoinactivation or trypsin digestion, H ϩ -PPase displayed conformational changes upon substrate binding (13,14).…”

“…To further ensure the practicability of this technique for CtH ϩ -PPase, a technique combining monomaleimide gold nanoparticle (GNP) labeling with transmission electron microscopy (TEM) was employed to validate the distance measured from smFRET (12). GNPs (1.4 Ϯ 0.2 nm) were anchored to Cys-542 of the C80A CtH ϩ -PPase mutant.…”

“…2A). Subsequently, purified CtH ϩ -PPases were adsorbed randomly on the mica surface for atomic force microscopy imaging and subjected to analysis as described previously (12). Fig.…”

“…Fluorescence Spectroscopy-Fluorophore-labeled CtH ϩ -PPases (5 nM) were immobilized on the coverslips according to the previous protocol with minor modifications (12,18). Coverslips were cleaned and freshly coated with L-polylysine.…”