Functional reconstitution of vacuolar H+-ATPase from Vo proton channel and mutant V1-ATPase provides insight into the mechanism of reversible disassembly

Abstract

The vacuolar H⁺-ATPase (V-ATPase; V₁V_o-ATPase) is an ATP-dependent proton pump that acidifies subcellular compartments in all eukaryotic organisms. V-ATPase activity is regulated by reversible disassembly into autoinhibited V₁-ATPase and V_o proton channel subcomplexes, a process that is poorly understood on the molecular level. V-ATPase is a rotary motor, and recent structural analyses have revealed different rotary states for disassembled V₁ and V_o, a mismatch that is likely responsible for their inability to reconstitute into holo V-ATPase in vitro Here, using the model organism Saccharomyces cerevisiae, we show that a key impediment for binding of V₁ to V_o is the conformation of the inhibitory C-terminal domain of subunit H (H_CT). Using biolayer interferometry and biochemical analyses of purified mutant V₁-ATPase and V_o proton channel reconstituted into vacuolar lipid-containing nanodiscs, we further demonstrate that disruption of H_CT's V₁-binding site facilitates assembly of a functionally coupled and stable V₁V_o-ATPase. Unlike WT, this mutant enzyme was resistant to MgATP hydrolysis-induced dissociation, further highlighting H_CT's role in the mechanism of V-ATPase regulation. Our findings provide key insight into the molecular events underlying regulation of V-ATPase activity by reversible disassembly.

Keywords: biolayer interferometry; biophysics; lipid nanodisc; membrane protein; molecular motor; protein-protein interaction; reversible disassembly; vacuolar ATPase.

Figure 1.

Purification and characterization of V_oND. A and B, schematic of V-ATPase regulation by reversible disassembly. C, purification strategy. Yeast vacuoles are isolated by flotation on a Ficoll gradient (panel i). Detergent-solubilized vacuolar proteins are mixed with biotinylated MSP (panel ii) and reconstituted into lipid nanodiscs followed by α-FLAG affinity capture of V_oND (panel iii). D, size-exclusion chromatography of V_oND. E, peak fractions were resolved using SDS-PAGE. F, negative stain EM of purified V_oND. Bar, 20 nm.

Figure 2.

Purification and characterization of V₁ mutants. A, schematic representation of the V₁ mutants. B, time-dependent MgATPase activities of the V₁ mutants measured in an ATP regenerating assay. C, specific activities of the V₁ mutants ± S.E. from at least two independent purifications per mutant. D, size-exclusion chromatography of H_chim (blue trace) and V₁ΔH reconstituted with H_chim (V₁H_chim, red trace). E and F, SDS-PAGE of column fractions of H_chim (E) and V₁H_chim (F).

Figure 3.

V₁H_chim and C associate with V_oND to form coupled V₁V_o-ATPase. A, V_oND was immobilized on streptavidin-coated BLI sensors via biotinylated MSP (step 1). Sensors were then dipped into 0.4 μm of V₁ mutants in presence of 1 μm C (association; step 2) followed by buffer (dissociation; step 3). Association with V_oND was most efficient with V₁H_chim (red trace). Sensors were then dipped in PreScission protease to verify that the BLI signal was not due to nonspecific binding (step 4). Inset shows an enlarged view of the association and dissociation steps. B, equimolar amounts of V₁H_chim and V_oND, and a 2-fold molar excess of C subunit were incubated at 22 °C, and the ConA-sensitive MgATPase activity was measured as a function of time. Each point represents the mean ± S.E. of two separate reconstitutions from two individual purifications. Inset, specific MgATPase activities of reconstituted V₁H_chimV_oND and V₁H_wtV_oND (± S.E. from two independent purifications) compared with purified V₁V_oND (29). C, following association of the V₁H_chimV_oND complex, sensors were dipped in wells containing buffer (green) or buffer + 1 mm MgATP (blue) for dissociation rate measurement. The dissociation phase of WT V₁V_oND in buffer (red) and buffer +1 mm MgATP (orange) is included for comparison (data from Ref. 29).

Figure 4.

Structural and functional characterization of the V₁H_chimV_oND complex. A, reconstituted V₁H_chimV_oND was subjected to glycerol gradient centrifugation, and the gradient fractions were analyzed by silver-stained SDS-PAGE. B, negative stain EM of V₁H_chimV_oND showing homogeneous and monodisperse dumbbell-shaped molecules. Inset in the top right shows 2× zoomed area highlighted in the bottom left. C, a data set of ∼5800 particle projections was subjected to reference-free alignment and classification, and selected class averages were overlaid with projections of the cryoEM model of yeast V₁V_o (Protein Data Bank code 3J9U). Bars in B, 50 nm.

Figure 5.

Model for reassembly of autoinhibited V₁ and V_o. A–C, our in vitro experiments have shown that although WT H containing V₁ does not readily bind V_oND (A), V₁H_chim spontaneously associates with V_oND (B) to form a structurally and functionally coupled V-ATPase, albeit at a slow rate. C, in vivo, however, V₁ exists in the autoinhibited conformation (A), and the rate of assembly with V_o is significantly faster (within 5 min). D–F, for in vivo (re)assembly, we propose that the following steps occur: step 1, recruitment of V₁ and subunit C to the vacuolar membrane (D); step 2, release of inhibitory MgADP (E); step 3, detachment of H_CT from its inhibitory position on V₁; and step 4, H_CT binding to a_NT (F). For further details, see text.