MgATP hydrolysis destabilizes the interaction between subunit H and yeast V₁-ATPase, highlighting H's role in V-ATPase regulation by reversible disassembly

Abstract

Vacuolar H⁺-ATPases (V-ATPases; V₁V_o-ATPases) are rotary-motor proton pumps that acidify intracellular compartments and, in some tissues, the extracellular space. V-ATPase is regulated by reversible disassembly into autoinhibited V₁-ATPase and V_o proton channel sectors. An important player in V-ATPase regulation is subunit H, which binds at the interface of V₁ and V_o. H is required for MgATPase activity in holo-V-ATPase but also for stabilizing the MgADP-inhibited state in membrane-detached V₁. However, how H fulfills these two functions is poorly understood. To characterize the H–V₁ interaction and its role in reversible disassembly, we determined binding affinities of full-length H and its N-terminal domain (H_NT) for an isolated heterodimer of subunits E and G (EG), the N-terminal domain of subunit a (a_NT), and V₁ lacking subunit H (V₁ΔH). Using isothermal titration calorimetry (ITC) and biolayer interferometry (BLI), we show that H_NT binds EG with moderate affinity, that full-length H binds a_NT weakly, and that both H and H_NT bind V₁ΔH with high affinity. We also found that only one molecule of H_NT binds V₁ΔH with high affinity, suggesting conformational asymmetry of the three EG heterodimers in V₁ΔH. Moreover, MgATP hydrolysis–driven conformational changes in V₁ destabilized the interaction of H or H_NT with V₁ΔH, suggesting an interplay between MgADP inhibition and subunit H. Our observation that H binding is affected by MgATP hydrolysis in V₁ points to H's role in the mechanism of reversible disassembly.

Introduction

Vacuolar H⁺-ATPases (V-ATPases²; V₁V_o-ATPases) are ATP-dependent proton pumps present in all eukaryotic cells. Typically, the V-ATPase is localized on the endomembrane system where the enzyme acidifies intracellular compartments, a process essential for pH homeostasis, protein trafficking, endocytosis, hormone secretion, mTOR signaling, and lysosomal degradation (1). The V-ATPase is also present on the plasma membrane of certain specialized cells such as osteoclasts, renal cells, the vas deferens, and the epididymis where the enzyme acidifies the extracellular environment. V-ATPase's proton pumping activity has been linked to numerous human diseases including osteoporosis and -petrosis (2, 3), renal tubular acidosis (4), male infertility (5), neurodegeneration (6), diabetes (7), viral infection (8), and cancer (9), making the enzyme a valuable drug target (10, 11).

The V-ATPase shares a similar architecture and catalytic mechanism with the F-ATP synthase such that it consists of a water-soluble ATP-hydrolyzing machine (V₁) and a membrane-integral proton channel (V_o), which are structurally and functionally coupled via a central stalk and multiple peripheral stalks (12–14). The subunit composition of the V-ATPase from yeast is A₃B₃CDE₃FG₃H for the cytosolic V₁ (15) and ac₈c′c″def for the membrane-integral V_o (16, 17). The subunit architecture of the V-ATPase has been studied by electron microscopy (EM) and several low- to intermediate-resolution reconstructions of bovine, yeast, and insect V-ATPase are available, which, together with X-ray crystal structures of individual subunits and subcomplexes of yeast V-ATPase and bacterial homologs, have provided a detailed model of the subunit architecture of the eukaryotic V-ATPase complex (17–22) (see Fig. 1A). V-ATPase is a rotary-motor enzyme (14). ATP hydrolysis in the catalytic A₃B₃ hexamer is coupled to rotation of the proteolipid ring (c₈c′c″) via a central rotor made of subunits D, F, and d with concurrent proton translocation at the interface of the proteolipid ring and the C-terminal domain of subunit a (a_CT). During rotary catalysis, the motor is stabilized by a peripheral stator complex consisting of three peripheral stalks constituted by heterodimers of subunits E and G (hereafter referred to as EG1–3) that connect the A₃B₃ hexamer to the N-terminal domain of the membrane-bound a subunit (a_NT) via the single-copy “collar” subunits H and C (19, 21) (see Fig. 1A). Three intermediates (referred to as rotational states 1–3), in which the central rotor is spaced 120° relative to the catalytic hexamer and subunit a, have been visualized in the yeast enzyme by cryo-EM (21).

Figure 1.

Schematic of yeast V-ATPase regulation by reversible disassembly. A, subunit architecture of holo-V-ATPase. The V₁ and V_o sectors are assembled and form an active enzyme. B, upon glucose deprivation, the V₁ and V_o sectors disengage, and the activity of both sectors is silenced. The subunits of the peripheral stator that form the V₁–V_o interface are shown in color. prox., proximal; dist., distal.

Unlike the related F-ATP synthase, eukaryotic V-ATPase is regulated by a unique mechanism referred to as reversible disassembly wherein, upon receiving cellular signals, V₁ dissociates from V_o, and the activity of both sectors is silenced (22–26) (see Fig. 1B). Reversible disassembly was first observed in yeast (27) and insects (28), but the process has recently also been observed in higher animals including human (29–32). Studies in yeast have shown that, during enzyme disassembly, subunit C is released into the cytosol by an unknown mechanism and reincorporated during reassembly (27). Because of regulation of the V-ATPase by reversible disassembly, the peripheral stator subunit interactions at the V₁–V_o interface draw particular attention as they must be sufficiently strong to withstand the torque generated during ATP hydrolysis, but at the same time they must be vulnerable enough to allow breaking on a timescale for reversible disassembly to occur efficiently.

We previously characterized the interaction of the collar subunit C with EG and a_NT and found that although the head domain of C (C_head) binds EG with high affinity (C_head–EG3; see Fig. 1A), its foot domain (C_foot) and EG both interact weakly with a_NT, resulting in a high-avidity ternary interface (EG2–a_NT–C_foot) in holo-V-ATPase (33, 34). Another collar subunit at the V₁–V_o interface is subunit H, and although deletion of C prevents stable assembly of V₁ and V_o (35), deletion of H results in the formation of a V₁V_o(ΔH) complex that lacks MgATPase and proton-pumping activities (36, 37). Moreover, although C is released into the cytosol upon disassembly of V₁V_o, H remains stably associated with V₁ (Fig. 1B). The crystal structure of H revealed that it consists of a larger N-terminal (H_NT) and smaller C-terminal domain (H_CT) connected by a short linker (38). Previous functional characterization of H_NT and H_CT suggested that, although H_NT is required for MgATPase activity in V₁V_o, H_CT has a dual function in that it is required for both coupling of V₁'s ATPase activity to proton pumping in V₁V_o (37) and inhibition of MgATPase activity in membrane-detached V₁ (39). The dual role and functional separation of H_NT and H_CT along with differences in regulatory function compared with C are not well understood and prompted the analyses of the interactions of H, H_NT, and H_CT with its binding partners in V₁ and V_o. We therefore used recombinant H, H_NT, and H_CT for quantification of their interactions with purified EG, a_NT, and V₁ lacking subunit H (V₁ΔH) using isothermal titration calorimetry (ITC) and biolayer interferometry (BLI). We found that H_NT binds no more than one of the three EGs on V₁ΔH and that the affinity of this interaction is ∼40-fold higher than that between H_NT and isolated EG, suggesting that H_NT prefers a particular conformation of EG on V₁. We further found that full-length H interacts with V₁ΔH with an ∼70-fold higher affinity than H_NT, indicative of a significant contribution of H_CT to the binding energy. Furthermore, we show that MgATP hydrolysis–driven conformational changes in the catalytic A/B pairs, the central rotor (DF), and the peripheral stalks (EG) destabilize the V₁–H interaction until inhibitory MgADP is trapped in a catalytic site. The findings are discussed in context of the mechanism of V-ATPase regulation by reversible disassembly.

Results

Expression, purification, and biophysical characterization of H, H_NT, H_CT, and a_NT(1–372)

To understand the role of the V₁–V_o interface in the mechanism of reversible disassembly, our laboratory has previously characterized the interactions among C_head, C_foot, EG, and a_NT (33, 34). Interactions involving subunit H, however, are yet to be quantified. Pulldown and yeast two-hybrid assays have shown that H is able to bind the N-terminal region of subunit E (40). In addition, EM and crystal structures of V₁V_o and V₁, respectively, show H_NT bound to one of the three EG peripheral stalks (EG1; see Fig. 2, A and B), whereas H_CT is seen to either rest on the coiled-coil middle domain of a_NT (in V₁V_o; Fig. 2A) or at the bottom of the A₃B₃ hexamer (in autoinhibited V₁) (Fig. 2B) (21, 22). To analyze the interactions of H within the enzyme in more detail, we expressed H, H_NT, and H_CT separately and quantified their interactions with recombinant EG, a_NT, and V₁ΔH purified from yeast.

Figure 2.

Binding interactions of subunit H in holo-V₁V_o and autoinhibited V₁-ATPase. A, in V₁V_o (Protein Data Bank code 3J9U) (21), H_NT (dark green) is bound to EG1 (blue/orange), and H_CT (light green) is in contact with a_NT (purple). B, in membrane-detached and autoinhibited V₁ (Protein Data Bank code 5D80) (22), the contact between H_NT and EG1 is preserved, but H_CT undergoes a ∼150° rotation to bind the bottom of the A₃B₃ hexamer and the rotor subunit D. The large conformational change in H_CT is depicted by the positions of the C-terminal α-helix (magenta) and the inhibitory loop (red) in holo-V₁V_o versus autoinhibited V₁-ATPase.

Full-length H, H_NT (residues 1–354), H_CT (residues 352–478), and a_NT (residues 1–372) were expressed in Escherichia coli as N-terminal fusions with maltose-binding protein (MBP). After amylose affinity capture, fusions were cleaved, and MBP was removed by ion exchange and size exclusion chromatography, resulting in purified subunits and subunit domains (Fig. 3A). All proteins eluted near their expected molecular masses on size exclusion chromatography except a_NT(1–372), which exists in a dimer–monomer equilibrium as described previously (26, 34) (Fig. 3, B and C). Consistent with available structural information, circular dichroism (CD) spectroscopy revealed a high degree of α-helical secondary structure, suggesting proper folding of the recombinant polypeptides (Fig. 3D).

Figure 3.

Purification and characterization of recombinant V-ATPase subunits and their domains. A, SDS-polyacrylamide gel of purified recombinant proteins used in the interaction studies described here. B, gel filtration elution profiles of H, H_NT, H_CT, and EG on a 1.6 × 50-cm Superdex 75 column. H, H_NT, and H_CT elute in single symmetric peaks (with molecular weights indicated next to the peaks), suggesting monomeric and monodisperse proteins. The majority of EG elutes as a single peak with some residual excess G subunit at larger elution volumes. C, a_NT(1–372) elutes on a 1.6 × 50-cm Superdex 200 column in two overlapping peaks corresponding to the dimer and monomer as described before. D, CD spectra of H, H_NT, H_CT, a_NT, and the EGH_NT complex recorded from 250 to 195 nm. The minima at 222 and 208 nm indicate α-helical secondary structure. deg, degrees; mAU, milliabsorbance units.

Interaction of H_NT with EG

We previously established that binding of C (or C_head) to isolated EG occurs with high affinity and that the interaction greatly stabilizes EG (33). To further characterize the interactions at the V₁–V_o interface, we set out to determine the affinity of the H_NT–EG interaction using ITC. Titration of H_NT into EG was exothermic, and the binding curve was fit to a single-binding-site model, revealing a K_d of the interaction of 187 nm. The binding enthalpy (ΔH) and entropy (ΔS) were −8 kcal/mol and 2.5 cal/(mol·K), respectively, with a concomitant free energy change (ΔG) of ∼−36 kJ/mol (Fig. 4A). Consistent with the ITC titration, size exclusion chromatography of a mixture of EG and an excess of H_NT resulted in the formation of a ternary H_NT–EG complex (Fig. 4, B and C), and taken together, the data show that H_NT forms a stable complex with the EG heterodimer. Previously, we found that the EG's N-terminal right-handed coiled coil is thermally labile with a T_m of ∼25 °C (Fig. 4D, blue trace) (29) and that the T_m of EG is increased by about 10 °C upon complex formation with C_(head) (33). To test whether H_NT binding has a similar stabilizing effect on EG, thermal unfolding of the individual proteins and EGH_NT complex was monitored by recording the CD signal at 222 nm as a function of temperature. The data show that isolated H_NT unfolds with an apparent T_m of ∼63 °C (Fig. 4D, green trace). The thermal unfolding curve of the EGH_NT complex showed two transitions, one at 25 °C and one at 64 °C, suggesting that the stability of EG is not increased upon H_NT binding (Fig. 4D, black trace). Moreover, as also shown previously (33), isolated EG heterodimer dissociates during native agarose gel electrophoresis, whereas in presence of C_head the three proteins migrate as a heterotrimeric complex in the electric field. However, consistent with the thermal unfolding data, a complex of EGH_NT did not comigrate on the native gel but ran as three separate species (Fig. 4E). Therefore, although both C_head and H_NT form a stable complex with EG, the nature of the two interactions are strikingly different.

Figure 4.

Interaction of H_NT with the EG heterodimer and H with a_NT. A, isothermal titration calorimetry of the interaction between H_NT and EG. H_NT was titrated into EG (top panel, lower trace) or buffer only (top panel, top trace), and the heat associated with the interaction was measured at 10 °C in 20 mm Tris-HCl, 0.5 mm EDTA, 1 mm TCEP, pH 8. The area under the peaks in the top panel was integrated and plotted as kcal/mol of H_NT as a function of binding stoichiometry in the bottom panel. These data were fit using a one-site binding model resulting in a K_d of 187 nm with ΔH and ΔS values of −8 kcal/mol and 2.5 cal/(mol·K), respectively, resulting in a ΔG of the interaction of ∼−36 kJ/mol. A representative of three separate titrations is shown. B, the ITC cell content was subjected to gel filtration on a 1.6 × 50-cm Superdex 200 column (black trace). The individual gel filtration elution profiles of H_NT and EG using the same column are shown in green and blue, respectively. C, SDS-PAGE of gel filtration fractions from the ITC cell content. EGH_NT elutes at higher molecular mass with excess H_NT in a well separated peak. The shift of the EGH_NT peak (compared with EG or H_NT) toward higher molecular weight indicates complex formation. The inset in B shows the EGH_NT peak fraction (fraction 55) from the SDS-PAGE gel of the ITC cell content shown in C. D, thermal unfolding of H_NT, EG, and EGH_NT monitored by recording the CD signal at 222 nm with increasing temperature. H_NT shows highly cooperative unfolding with an apparent T_m of ∼63 °C (green). EG has an apparent T_m of ∼25 °C (data taken from Ref. 33). The EGH_NT complex shows two unfolding transition with T_m values similar to the those observed for the individual proteins, suggesting that H_NT binding to EG does not stabilize the EG heterodimer. E, native agarose gel electrophoresis of EG heterodimer, H_NT, and EGH_NT. 30 μg of purified EG, 30 μg of purified H_NT, and a mixture of equimolar amounts of H_NT and EG (total 60 μg) were loaded. Unlike binding of C_head to EG (33), binding of H_NT does not appear to stabilize EG under these conditions. F, isothermal titration calorimetry of the interaction between a_NT and H. H was titrated into a_NT (top panel, lower trace) or buffer (top panel, top trace), and the heat associated with the interaction was measured at 10 °C in 20 mm Tris-HCl, 0.5 mm EDTA, 1 mm TCEP, pH 8. Subtracting the heat of dilution of titration of H into buffer revealed an endothermic binding reaction. Fitting the data (bottom panel) to a one-site binding model with a fixed n = 1 allowed an estimate of the K_d of ∼130 μm. A representative titration from two repeats is shown. mAU, milliabsorbance units.

Interaction of H with a_NT

Prior work from our laboratory has shown that the EG2–a_NT–C_foot junction at the V₁–V_o interface (Fig. 1A) is formed by multiple low-affinity interactions, and we reasoned that the sum of these interactions provides a high-avidity binding site between V₁ and V_o that could be targeted for regulated disassembly (34). Another interaction that is seen in EM reconstructions of the intact V-ATPase, and that must be broken and reformed during reversible disassembly, is between H and a_NT (Figs. 1A and 2A). To estimate the affinity between H and a_NT, we performed ITC experiments by titrating H into a_NT(1–372) (Fig. 4F). Subtracting the heat generated from diluting H into buffer from the heat generated from titrating H into a_NT(1–372) revealed a weak endothermic binding reaction between the two proteins. Fitting the data to a one-site binding model revealed a K_d of 135 μm and ΔH, ΔS, and ΔG values of 4 kcal/mol, 36.1 cal/(mol·K), and −26 kJ/mol, respectively. Consistent with our ITC data, a mixture of H and a_NT eluted at the same volumes as the individual proteins on size exclusion chromatography (Fig. S1). The low-affinity interaction between H and a_NT supports our existing model that V₁ binds V_o via several low-affinity interactions that, taken together, result in a high-avidity interface in assembled V₁V_o.

Interaction of V₁ΔH with H, H_NT, and H_CT characterized by BLI

Previous experiments showed that H remains bound to V₁ even at the low concentrations used in enzyme essays (e.g. ∼15 nm) (22, 25, 39) and under the conditions of electrospray ionization used for native MS (15). Although the data so far have shown that the affinity of H_NT for EG as measured using ITC is moderately high, the observed K_d of ∼0.2 μm (Fig. 4A) could not explain the above observations, which means that the interaction of H with V₁ has to be much stronger (39). We therefore wished to determine the affinity of full-length H as well as H_NT and H_CT for V₁ΔH. The interaction between V₁ΔH and MBP-tagged H, H_NT, and H_CT was quantified using BLI. MBP-tagged proteins were immobilized on anti-mouse Fc capture (AMC) biosensors using an anti-MBP antibody, and the rate of association and dissociation of V₁ΔH was measured hereafter. The slow dissociation of MBP-tagged H, H_NT, and H_CT from the anti-MBP antibodies was subtracted from the V₁ΔH dissociation rates for analysis of the kinetic data. BLI experiments for measuring association and dissociation kinetics between V₁ΔH and MBP-H/H_NT were conducted at five different V₁ΔH concentrations, and the resulting association and dissociation curves were fit to a global single-site binding model (Fig. 5, A and B). Analysis of the data for MBP-H and MBP-H_NT revealed K_d values of ∼65 pm (Fig. 5A) and ∼4.5 nm (Fig. 5B), respectively. We also tested the binding of V₁ΔH to MBP-H_CT, but we were not able to determine a K_d as there was no measurable association at low V₁ΔH concentrations (<100 nm), and higher V₁ΔH concentrations (e.g. 1 μm) resulted in nonspecific binding to the BLI sensors (data not shown). Overall, the interaction of H_NT with EG as part of V₁ΔH was ∼40-fold tighter when compared with the interaction between H_NT and isolated EG (as measured using ITC; Fig. 4A), suggesting that the conformation of EG on V₁ΔH is more favorable for H_NT binding than the conformation(s) of isolated EG. In addition, although we could not detect an interaction between H_CT and V₁ΔH under the conditions of BLI, a ∼70-fold higher affinity of V₁ΔH for H as compared with H_NT suggests a significant contribution of H_CT to the V₁–H interaction. From our ITC and BLI experiments, we infer that the binding interaction between H_NT and EG allows H_CT to switch conformations so that it can either bind a_NT (in V₁V_o) or subunits B and D (in V₁) to efficiently carry out its dual role in reversible disassembly.

Figure 5.

Interaction of H and H_NT with V₁ΔH. A, the affinity of the interaction between H and V₁ΔH was determined using BLI at 22 °C in BLI buffer. Association and dissociation sensorgrams from five different concentrations of V₁ΔH (0.1, 0.3, 1, 3, and 9 nm) are shown in blue. The data were globally fit (traces in red) to reveal a K_d of ∼65 pm. B, a similar experiment was conducted to analyze the interaction between H_NT and V₁ΔH. H_NT was dipped in 10, 20, 40, 80, and 160 nm V₁ΔH followed by buffer to generate association and dissociation curves, respectively (blue). The data were globally fit (red traces) to reveal a K_d of ∼4.5 nm. C, V₁ΔH was incubated with a 5-fold excess of H_NT, and the mixture was resolved on a 1.6 × 50-cm Superdex 200 column. D, SDS-PAGE of gel filtration fractions. The higher molecular weight peak showed V₁ΔH in complex with H_NT with the lower molecular weight peak corresponding to excess H_NT. E, approximately equimolar amounts of V₁ΔH and V₁ΔH in complex with H_NT were resolved by SDS-PAGE. The staining intensity of the H_NT compared with the single-copy subunit D band in the V₁(ΔH)H_NT complex suggests that V₁ΔH binds no more than one copy of H_NT with high affinity. mAU, milliabsorbance units.

V₁ΔH binds no more than one H_NT

Because V₁ΔH contains three EG heterodimers, we wished to determine whether all three or only one of the EGs can bind H_NT. Purified V₁ΔH was mixed with a 5-fold molar excess of H_NT followed by size exclusion chromatography. Under these conditions, some H_NT coeluted with V₁ΔH with the excess H_NT eluting from the column as a separate, lower molecular weight peak (Fig. 5, C and D). The V₁(ΔH)H_NT complex was concentrated, and approximately equal amounts of V₁ΔH and V₁(ΔH)H_NT were resolved using SDS-PAGE. The staining of H_NT in the purified V₁(ΔH)H_NT complex was similar to that of single-copy subunits in the V₁ complex (for example subunit D), indicating that no more than one copy of H_NT bound to V₁ΔH (Fig. 5E). Therefore, although there are three EGs in V₁ΔH, only one of these is in a conformation that is able to bind H_NT with high affinity, highlighting the conformational asymmetry of the peripheral stalks.

The interaction of H with V₁ΔH is destabilized upon MgATP hydrolysis

The preference of H_NT for one of three EGs suggested that the asymmetry of the peripheral stalks originates in the catalytic core (A₃B₃DF) of V₁. Upon MgATP hydrolysis, however, the conformational changes of the catalytic sites from open to loose to tight drive counterclockwise rotation of the central rotor along with cyclic structural changes in the peripheral stalks from EG1 to EG3 to EG2 (41). In addition, based on the structure and nucleotide occupancy of the autoinhibited V₁ sector, our laboratory suggested that H_CT inhibits V₁-ATPase activity by preferentially binding to an open catalytic site, consequently maintaining inhibitory MgADP in the adjacent closed catalytic site (22). Taken together, H_NT's preference for EG1, as well as H_CT's role in MgADP inhibition, indicated a potential interplay between the nucleotide occupancy of the catalytic sites and the interaction of V₁ΔH with H. To probe the effect of nucleotides on the interaction of H with V₁ΔH, we again used BLI. V₁ΔH was bound to immobilized MBP-H, and the sensor was then dipped in wells containing buffer or buffer with 1 mm MgATP, MgADP + P_i, or MgAMPPNP. Interestingly, in the presence of MgATP, a biphasic dissociation curve was observed with an initial dissociation rate that was ∼6 times faster than the rate in buffer alone (Fig. 6, A and B). However, only ∼25% of the bound V₁ΔH dissociated with a fast rate with the remaining 75% coming off the sensor at a rate similar to the dissociation rate in buffer (Fig. 6, A and B). In contrast, a relatively slower rate of dissociation was observed in the presence of MgADP + P_i and MgAMPPNP.

Figure 6.

The interaction between subunit H and V₁ΔH is destabilized upon MgATP hydrolysis. A, sensorgrams of V₁ΔH dissociation from immobilized H subunit in the absence (blue) and presence of 1 mm nucleotides (MgADP + P_i, purple; MgAMPPNP, green; MgATP, orange). A representative from at least two independent experiments is shown. B, off-rates determined from fitting the sensorgrams from A to dual-exponential equations. C, sensorgrams of association and dissociation of NEM-inhibited V₁ΔH to and from immobilized H subunit in the absence or presence of 1 mm nucleotides (association of NEM-modified V₁ΔH in buffer/dissociation in MgADP + P_i, purple; association of NEM-modified V₁ΔH in buffer/dissociation in MgATP, red; both association and dissociation of NEM-modified V₁ΔH in buffer, blue; association of unmodified V₁ΔH in buffer/dissociation in MgATP, orange). A representative from at least two independent experiments is shown. D, observed on-rates (k_obs) and off-rates obtained from fitting the sensorgrams in C to single- and dual-exponential equations, respectively. E, sensorgrams of V₁ΔH dissociation from immobilized H_NT in the absence (blue) and presence of 1 mm nucleotides (MgADP + P_i, purple; MgAMPPNP, green; MgATP, orange). A representative from at least two independent experiments is shown. F, off-rates determined from fitting the sensorgrams from E to dual-exponential equations. G, average specific activities of V₁ΔH and V₁(ΔH)H plotted ±S.E. (error bars) from two independent purifications. Inset, raw data for activity measurement using an ATP-regenerating assay (22). V₁-ATPase activity is determined by monitoring a decrease in A₃₄₀ as a function of time. Traces for WT V₁, V₁ΔH, and V₁(ΔH)H are shown in dark green, blue, and light green, respectively. N/A, not applicable.

The destabilization of the V₁–H interaction upon MgATP hydrolysis came as a surprise to us as the H subunit is known to inhibit V₁-ATPase activity (22, 25, 39). To confirm that the fast dissociation of V₁ΔH from MBP-H in wells containing MgATP was specifically due to MgATP binding to V₁'s catalytic sites, we conducted a similar BLI experiment using V₁ΔH treated with N-ethylmaleimide (NEM). It is known that NEM modification of a catalytic-site cysteine residue prevents binding of nucleotides (42). NEM-treated and untreated V₁ΔH were bound to MBP-H immobilized on sensors and then dipped in wells containing MgATP, MgADP + P_i, or buffer (Fig. 6, C and D). We found that NEM-treated V₁ΔH no longer showed a fast dissociation rate when dipped in MgATP-containing wells, suggesting that MgATP binding to the catalytic sites caused destabilization of the V₁–H interaction. However, if the above mentioned fast dissociation rate was a result of only MgATP binding, but not hydrolysis, we should have observed fast dissociation in the presence of MgAMPPNP, the nonhydrolyzable ATP analog. MgAMPPNP, however, had no effect on the V₁ΔH dissociation rate (Fig. 6A, green trace). Taken together, the BLI experiments with NEM-modified V₁ΔH and in the presence of MgAMPPNP suggest that it is MgATP binding and hydrolysis that destabilize the V₁–H interaction. Because both H_NT and H_CT contribute to the interaction of H with V₁ΔH, we also measured the off-rate of V₁ΔH from immobilized MBP-H_NT and found that the H_NT–V₁ΔH interaction was also destabilized upon MgATP hydrolysis as seen for H and V₁ΔH (Fig. 6, E and F).

To verify that V₁ΔH bound to H was capable of transient turnover, we purified V₁ΔH, incubated it with an excess of H, and resolved the mixture using size exclusion chromatography (Fig. S2). We found that V₁ΔH reconstituted with H (V₁(ΔH)H) showed ∼4.9 ± 0.55 units/mg of MgATPase activity, which was ∼30% of the activity of V₁ΔH (15.75 ± 1.7 units/mg) (Fig. 6G and Ref. 22). Considering the high-affinity interaction between V₁ΔH and H with a K_d of ∼65 pm, we expected stoichiometric amounts of H in the V₁(ΔH)H reconstituted complex. However, to exclude the possibility that the observed MgATPase activity in the V₁(ΔH)H complex was due to substoichiometric binding of H, we performed a pulldown experiment in which a 10-fold molar excess of MBP-H bound to amylose resin was used to capture V₁ΔH (Fig. S3). Although some MBP-H and V₁ΔH appeared in the supernatant and washes of the amylose resin, most of the MBP-H eluted in 10 mm maltose along with stoichiometrically bound V₁ΔH. Elution fraction E1 (Fig. S3A) exhibited significant MgATPase activity (Fig. S3B), indicating that a stoichiometric complex of V₁ΔH with MBP-H was capable of hydrolyzing MgATP.

A consistent feature of the dissociation curve of V₁ΔH from H in MgATP was its biphasic nature (Fig. 6A, orange trace), indicating that MgATP hydrolysis–dependent destabilization was transient. We found that, by using different concentrations of MgATP during dissociation, we were able to regulate the fast phase of the dissociation rate and consequently the duration of destabilization (Fig. 7). Not only does this experiment confirm MgATP hydrolysis as being the cause of V₁–H destabilization, it also explains why the destabilization is transient. Using P_i release–based ATPase assays, it has been shown that MgATPase activity of V₁ΔH subsides rapidly with time (25). This rapid decrease in activity, which is also observed in an ATP-regenerating assay (Fig. S4), has been attributed to the trapping of MgADP in a catalytic site, a phenomenon termed MgADP inhibition. We have observed that MgADP inhibition of V₁ΔH is more efficient under high Mg²⁺ (and by extension high MgATP) concentrations and that decreasing the initial concentration of MgATP results in delayed MgADP inhibition (Fig. S4). In the BLI experiment shown in Fig. 7, decreasing the concentration of MgATP to 100 μm (Fig. 7, red trace) and consequently decreasing the rate of MgATP hydrolysis led to a delay in the culmination of the fast dissociation phase. Taken together, these data suggest that MgATP hydrolysis causes transient destabilization of the V₁–H interaction until MgADP inhibition sets in.

Figure 7.

Modulation of the dissociation rate of V₁ΔH from H by varying the concentration of MgATP. A, sensorgrams of V₁ΔH dissociation from immobilized H subunit in the absence (blue) and presence of MgATP (10 μm, purple; 100 μm, red; 1 mm, orange; 4 mm, pink). A representative from two independent experiments is shown. B, off-rates determined by fitting the sensorgrams in A to dual-exponential equations.

Discussion

V-ATPase is regulated by reversible disassembly, a process that involves the breakage and reformation of several protein–protein interactions at the interface of V₁ and V_o. These interactions are mediated by the central rotor of V₁ (DF) binding to V_o's subunit d and the three peripheral stalks (EG1–3) that link the collar subunits H and C to a_NT (see Fig. 2A). Both H and C are two domain proteins, and we previously found that C_head binds EG with high affinity, whereas C_foot and EG bind a_NT weakly. Here, we analyzed binding of H and H_NT to isolated EG, a_NT, and V₁ΔH purified from yeast. We found that the majority of the binding energy between H and V₁ is contributed by the interaction between H_NT and EG and that binding of H_NT to EG is much stronger when EG is part of V₁ compared with isolated EG. However, only one copy of H_NT binds V₁ΔH with high affinity, indicating that the three EGs on V₁ are in different conformations and that only one of these conformations (EG1) is competent for H binding. The three different conformations of the EGs are evident from the crystal structure of autoinhibited V₁ (22) as well as the cryo-EM structures of V₁V_o (20, 21). The observation that H_NT binds only EG1 as part of V₁ with high affinity suggests that H_NT's preference is a result, and not the cause, of the conformational asymmetry of the peripheral stalks, which most likely originates in the catalytic core of V₁ (A₃B₃DF). Although we were not able to detect an interaction between isolated H_CT and V₁ΔH, the observation that intact H binds V₁ΔH with significantly higher affinity compared with H_NT suggests that the contact between H_CT and A₃B₃DF seen in the V₁ crystal structure (22) contributes to the avidity of the V₁–H interaction. In addition, much like the C_foot–a_NT–EG low-affinity (but high-avidity) ternary junction, we found that H and a_NT interact weakly. Taken together, the data support and extend our earlier findings that V₁ and V_o are held together by multiple weak interactions that allow rapid breaking and reforming in response to cellular needs.

MgATP hydrolysis–dependent destabilization of the V₁–H interaction

Studies in yeast have shown that membrane-detached V₁ has no measurable MgATPase activity, a property of WT V₁ that has been attributed to the presence of the inhibitory H subunit (25). Therefore, our BLI data showing that the V₁–H interaction is destabilized in the presence of MgATP came as a surprise as we did not expect V₁(ΔH)H to be catalytically active. In contrast, previous biochemical studies had shown that V₁ΔH retained ∼20% MgATPase activity upon addition of an excess of recombinant H, an observation that, at the time, was attributed to substoichiometric binding of H (39). However, using pulldown assays, we here show that a stoichiometric complex of V₁ΔH with H has indeed transient MgATPase activity, indicating that WT V₁ isolated from yeast is not equivalent to reconstituted V₁(ΔH)H. One striking difference between V₁ and V₁(ΔH)H is that V₁ contains ∼1.3 mol/mol of tightly bound ADP, whereas V₁ΔH, and by extension V₁(ΔH)H, has only ∼0.4 mol/mol of ADP (22). This suggests that V₁'s ATPase activity is inhibited by tightly bound ADP and that the lack of ADP in V₁(ΔH)H allows transient MgATP hydrolysis with the associated conformational changes leading to destabilization of the V₁–H interaction on the BLI sensor.

MgADP inhibition is a conserved feature of the catalytic headpiece of rotary ATPases wherein, under ATP-regenerating conditions, the rate of MgATP hydrolysis decreases due to retention of tightly bound MgADP at a closed catalytic site. The MgADP-inhibited state is a conformation off-pathway from the catalytic cycle and associated with a structural change in the catalytic site (43). MgADP inhibition has been observed in both the F₁-ATPase (e.g. F₁ from bovine heart (44)) and Bacillus PS3 (45)) and the cytosolic A₁/V₁ sector from Thermus thermophilus (46). Parra et al. (25) reported a decrease in MgATPase activity of purified yeast V₁ΔH using P_i release assays, and we have observed a similar decrease in MgATPase activity of V₁ΔH using an ATP-regenerating assay system (22) (Fig. S4).

Interplay of MgADP inhibition with the V₁–H interaction

The structure of autoinhibited V₁ revealed an inhibitory loop in H_CT (amino acids 408–414) that mediates important contacts with V₁. First, H_CT binds to the C-terminal domain of subunit B, thereby stabilizing the corresponding catalytic A–B interface in its open conformation. Second, it interacts with two α-helical turns in the central rotor subunit D (residues 38–45) (22) (Fig. 2B). At any given rotational state of V₁, only one of the catalytic sites is in the open state with the two α-helical turns from the central rotor facing the open site (22, 47). It is therefore evident that H_CT preferentially binds to the open catalytic site with the central rotor in a particular conformation. Our data suggest that the peripheral stalks exhibit a conformational asymmetry, which most likely originates from the conformations of the catalytic core of the enzyme (A₃B₃DF). H_NT preferentially interacts with EG1, the peripheral stalk associated with the open catalytic site. Under the conditions of our BLI experiments, when V₁ΔH is bound to subunit H on the sensor, both H_NT and H_CT are associated with their binding partners at the open catalytic site, resulting in overall tight binding of H as evident from the observed slow dissociation of V₁ΔH from the BLI sensor (Fig. 8A). When sensors containing V₁ΔH bound to MBP-H are dipped into MgATP, the nucleotide binds to the open catalytic site and is hydrolyzed. Subsequent MgATP hydrolysis results in central stalk rotation, which destabilizes the interaction with H_CT. The conformational changes are also propagated to the peripheral stalks, resulting in destabilization, and ultimately breaking, of the interaction between H_NT and EG1 (Fig. 8B). However, MgATP hydrolysis on V₁(ΔH)H is transient and stops once inhibitory MgADP gets trapped in a tight catalytic site. All the V₁(ΔH)H complexes that withstood transient MgATP hydrolysis are now bound with high affinity because the binding site for H_CT is restored once the MgADP-inhibited conformation is obtained (Fig. 8C). Therefore, we conclude that the lack of inhibitory MgADP in V₁(ΔH)H allows transient MgATP hydrolysis and destabilization of the V₁–H interaction and that high-affinity binding of H is restored once inhibitory MgADP is trapped in a catalytic site.

Figure 8.

Model for the effect of MgATP hydrolysis on the V₁–subunit H interaction. Shown is a view of V₁ from the membrane with H shown in green, peripheral stalk subunits EG in blue and orange, central rotor subunits DF in yellow, catalytic A subunit in tan, and B subunit in gray. V₁ΔH binds subunit H with high affinity in the absence of nucleotide (A). However, when MgATP is added to the system, it binds to the empty catalytic sites of V₁, leading to MgATP hydrolysis (B). Cooperative and cyclical MgATP hydrolysis in V₁ leads to conformational changes in the three A/B pairs between open (O), loose (L), and tight (T) states. These cyclical switches of the A/B pairs are accompanied by structural changes in the associated EG heterodimers, which interchange between conformations denoted as EG1, EG2, and EG3. Because EG1 is the preferred binding site for H_NT, a change in conformation of EG1 to EG3 causes a destabilization of the EG–H_NT interaction. MgATP hydrolysis causes the counterclockwise rotation of the central rotor (DF) by ∼120°. Rotation of the central rotor along with conformational changes in the catalytic subunits causes destabilization of the V₁–H_CT interaction. As observed in the BLI experiments, only a subset of the V₁–H interactions are destabilized upon MgATP hydrolysis. The catalytic cycle stops once MgADP is bound in a tight site, causing MgADP inhibition. The V₁–H interactions that withstand MgATP hydrolysis subsequently remain bound with high affinity under ADP-inhibited conditions (C).

Considering the here observed MgATP hydrolysis–dependent destabilization of the interaction between H (and H_NT) with V₁ raises the question: how is this interaction maintained in V₁V_o during rotational catalysis? Between the three rotational states of the enzyme observed by cryo-EM (21), minor conformational differences are observed for the peripheral stalk bound to H (EG1 in V₁V_o). In V₁V_o, besides providing binding sites for both H_NT and H_CT, a_NT also interacts with and probably stabilizes the N termini of EG1 (Figs. 1A and 2A). The N-terminal region of the peripheral stalks have been described as unstable and flexible based on experiments conducted with isolated EG (33) and EG as part of A₁/V₁ (48) and as seen in the V₁ crystal structure (22). With EG1's N termini unsupported, as in membrane-detached V₁, expected conformational changes associated with rotary catalysis would be larger than those observed in V₁V_o. Hence, although multiple interactions with a_NT maintain the conformation of EG1 in V₁V_o, the lack of these interactions in V₁ enable rotary catalysis–driven conformational changes in EG1 with concomitant destabilization of the H_NT–EG1 interaction.

Implications for the mechanism of reversible disassembly

Experiments conducted with yeast spheroplasts, isolated vacuoles, and purified V₁V_o have established that efficient disassembly of V-ATPase requires a catalytically active complex (49–51). From a structural comparison with the three rotational states of V₁V_o, we previously noted that upon disassembly of the holoenzyme autoinhibited V₁ and V_o end up in different rotational states: V₁ in state 2 and V_o in state 3 (22, 52, 53). This suggests that the MgATPase activity that is necessary for efficient disassembly serves to generate the rotational state mismatch associated with enzyme dissociation, a mismatch that likely functions to prevent rebinding of V₁ to V_o under cellular conditions that favor the disassembled state. It is well established that enzyme dissociation is accompanied by a release of subunit C into the cytosol, and it is possible that the energy from ATP hydrolysis also serves to break the high-affinity EGC_head interaction (33). Live cell imaging has captured V₁ on vacuolar membranes while C is released into the cytosol upon glucose removal, suggesting that C release may be one of the initial steps of disassembly. Our data suggest that, due to catalysis-driven conformational changes in EG, membrane-detached V₁ sector is incapable of binding H in a stable conformation while MgATP is being hydrolyzed at the catalytic sites. Therefore, it is possible that V₁ detaches from V_o on vacuolar membranes after its MgATPase activity is completely silenced. The specificity of H_NT and H_CT for their binding sites on V₁ supports a model wherein once V₁ is MgADP-inhibited in rotational state 2 the proximity of the open catalytic site and central rotor favor H_CT's conformational switch to its inhibitory position on V₁. At the same time, by binding and stabilizing the open catalytic site, H_CT facilitates the trapping of MgADP in the adjacent closed catalytic site (22). Hence, inhibitory MgADP and subunit H synergize by stabilizing each other to ensure that free V₁ remains in the autoinhibited state to prevent wasteful ATP hydrolysis when V-ATPase's proton-pumping activity is down-regulated.

The autoinhibited state of V₁ (with MgADP in a closed catalytic site stabilized by H_CT bound to an open catalytic site) is likely a low-energy state of V₁. For reassembly to occur, V₁ needs to be “reactivated” to allow H_CT to switch from its binding site on V₁ to bind a_NT in V₁V_o. Based on our observations in this study, we speculate that release of inhibitory MgADP and subsequent MgATP binding/hydrolysis induce structural changes in V₁ that detach H_CT, making it available to bind a_NT, in turn coupling V₁ to V_o. What then causes the required release of inhibitory MgADP from cytosolic V₁? Although this mechanism is currently not understood, it is possible that one of the protein factors that have been shown to be required for efficient reassembly, such as the regulator of the ATPase of vacuolar and endosomal membranes (RAVE) complex (54) or aldolase (55), plays a role in the release of inhibitory ADP, thereby allowing H_CT to assume its binding site on a_NT and restore MgATP hydrolysis–driven proton pumping.

Experimental procedures

Materials and methods

Plasmids encoding subunit H and its C-terminal domain (H_CT; residues 352–478) N-terminally tagged with a Prescission protease-cleavable maltose-binding protein (MBP-H and MBP-H_CT encoded by a pMalPPase vector derived from pMAL-c2E), a yeast strain deleted for subunits H and G (39), and a pRS315 vector containing FLAG-tagged subunit G (56) were kind gifts from Dr. Patricia Kane, SUNY Upstate Medical University.

Plasmid construction

The plasmid expressing the N-terminal domain of subunit H (H_NT; residues 1–354) was made using the above MBP-H pMalPPase vector as a template for QuikChange mutagenesis to delete the nucleotide sequence coding for amino acids 355–478 using the following primers: H_NT1–354 F, GGA AAT CCT AGA AAA CGA GTA CCA AGA ATT GAC CTA AAA GCT TGG CAC TGG CCG TCG TTT TAC AAC GTC G; H_NT1–354 R, GAC GGC CAG TGC CAA GCT TTT AGG TCA ATT CTT GGT ACT CGT TTT CTA GGA TTT CCT TG. The construction of the pMalPPase plasmid encoding N-terminally MBP-tagged a_NT(1–372) has been described (26).

Expression and purification of recombinant V-ATPase subunits

V-ATPase subunit constructs H_NT, EG, H_CT, H, and a_NT were expressed in E. coli strain Rosetta2, grown to midlog phase in rich broth (LB Miller plus 0.2% glucose) supplemented with ampicillin (100 μg/ml) and chloramphenicol (34 μg/ml). Protein expression was induced with 0.5 mm isopropyl β-d-thiogalactopyranoside (except for expression of EG where 1 mm isopropyl β-d-thiogalactopyranoside was used). Expression was induced at 30 °C for 6 h for H_NT, 20 °C for 6 h for H, 20 °C for ∼16 h for a_NT, 25 °C for ∼16 h for H_CT, and 30 °C for 6 h for EG. Cells were harvested by centrifugation, resuspended in amylose column buffer (CB; 20 mm Tris-HCl, 200 mm NaCl, 1 mm EDTA, pH 7.4), and stored at −20 °C until use. For purification, cells were treated with DNase (67 μg/ml), lysozyme (840 μg/ml), and PMSF (1 mm) before lysis by sonication. The lysate was then cleared by centrifugation at 12,000 × g for 30 min, the supernatant was diluted 1:4 with CB and applied to an amylose affinity column at a rate of 1 ml/min, and nonspecifically bound material was removed by washing with 12 column volumes of CB followed by 15 column volumes of CB without NaCl. Bound protein was eluted using 25 ml of 10 mm maltose in CB without salt. The MBP tag was cleaved using Prescission protease as described (34). H, H_CT, and a_NT were separated from MBP by anion exchange chromatography (Mono Q) using a linear gradient of 0–300 mm NaCl in 25 mm Tris-HCl, 1 mm EDTA, pH 7, for elution. Residual MBP was removed by a small amylose column, and the concentrated protein was subjected to size exclusion chromatography using a Superdex S75 column (1.6 × 50 cm) for H and H_CT and a Superdex 200 column of the same size for a_NT. H_NT was separated from MBP using a gravity DEAE (anion exchange) column. At a pH of 6.5, MBP was immobilized on the DEAE column, and the H_NT flowed through. The flow-through was collected, concentrated, and subjected to size exclusion chromatography using a Superdex S75 column (1.6 × 50 cm). EG heterodimer was purified as described (33).

Purification of V₁ΔH

V₁-ATPase lacking subunit H was purified as described (22). Briefly, the yeast strain deleted for the genes encoding subunits H and G was transformed with a pRS315 plasmid encoding subunit G with an N-terminal FLAG tag (56). The cells were grown in synthetic defined medium lacking Leu to an OD of ∼4.0 and harvested by centrifugation, and the cell pellets were resuspended in TBSE (25 mm Tris-HCl, pH 7.2, 150 mm NaCl, 0.5 mm EDTA) and stored at −80 °C until use. Thawed cells were supplemented with 5 mm β-mercaptoethanol, 1 mm PMSF, and 2 μg/ml each of pepstatin and leupeptin before lysis by 15 passes through a microfluidizer with intermittent cooling on ice. The lysate was centrifuged at 4000 × g for 25 min, and the resultant supernatant was centrifuged again at 13,000 × g for 40 min. The cleared lysate was applied to a 5-ml FLAG column (Sigma) topped with Sephadex G50 and pre-equilibrated in TBSE. The column was washed with 10 column volumes of TBSE and eluted using 0.1 mg/ml FLAG peptide. V₁ΔH-containing fractions were pooled, concentrated, and resolved using a Superdex 200 1.6 × 50-cm column attached to an ÄKTA FPLC (GE Healthcare). Fractions were analyzed by SDS-PAGE and concentrated to 10 mg/ml, and the activity of the complex was measured using a coupled enzyme assay as described below (22).

CD spectroscopy

CD spectra were recorded on an Aviv 202 spectropolarimeter using a 2-mm–path length cuvette. CD spectra were recorded between 250 and 195 nm in 25 mm sodium phosphate, pH 7, and protein stability was monitored by recording the CD signal at 222 nm as a function of temperature. For cysteine-containing proteins, 0.3–1 mm TCEP was included in the buffer. Protein concentrations of H, H_NT, H_CT, and a_NT were 2, 2.25, 9.2, and 2.36 μm, respectively. The far-UV CD spectrum of 6.7 μm H_NT–EG complex was obtained with protein dissolved in 20 mm Tris-HCl, 1 mm TCEP, pH 8 buffer at 4 °C followed by monitoring the temperature dependence of the CD signal at 222 nm.

Isothermal titration calorimetry

The thermodynamic parameters of the interaction between H_NT and EG and between H_CT and a_NT were determined using a Microcal VP-ITC isothermal titration calorimeter. The interaction of H_NT and EG was monitored by titrating a stock of 0.278 mm H_NT into 0.0315 mm EG in 20 mm Tris-HCl, 0.5 mm EDTA, 1 mm TCEP, pH 8, at 10 °C. A total of 30 injections with 5% saturation per injection was carried out. The average value of signal postsaturation (last eight titration points) was subtracted from the H_NT into EG titration. Complex formation between a_NT and H was analyzed by titrating 0.3 mm H into 0.017 mm a_NT in 20 mm Tris-HCl, 0.5 mm EDTA, 1 mm TCEP, pH 8. A blank titration of H into buffer was subtracted from the a_NT into H titration using the curve fit option in OriginLab. ITC data were analyzed using VP-ITC programs in OriginLab.

Biolayer interferometry

BLI was used to measure the association and dissociation kinetics of interaction between V₁ΔH and MBP-tagged H, H_NT, and H_CT. An Octed-RED system and AMC–coated sensors (FortéBio, AMC biosensors, catalogue number 18-5088) were used to monitor protein–protein binding and dissociation for determination of binding affinities. Anti-MBP antibody (New England Biolabs) at 1 μg/ml was immobilized on the AMC biosensors. The anti-MBP antibody formed the bait for MBP-tagged H, H_NT, and H_CT (used at 5 μg/ml). Biosensors with immobilized H, H_NT, or H_CT were dipped in varying concentrations of V₁ΔH followed by buffer to measure association and dissociation rates. BLI buffer (20 mm Tris-HCl, pH 7.2, 150 mm NaCl, 1 mm EDTA, 0.5 mg/ml BSA) was used in all BLI experiments to reduce nonspecific binding to the biosensors except for experiments in the presence of nucleotides where the EDTA concentration was reduced to 0.5 mm. All steps were done at 22 °C with each biosensor agitated in 0.2 ml of sample at 1000 rpm and a standard measurement rate of 5 s⁻¹. Control experiments were performed to check for any nonspecific binding interaction between the antibodies and the proteins used. Reference sensors were used in each experiment with immobilized MBP-H/H_NT/H_CT but no V₁ΔH. FortéBio data analysis software (version 6.4) was used for subtraction of reference sensors, Savitzky-Golay filtering, and global fitting of the kinetic rates of V₁ΔH binding with H, H_NT, or H_CT.

ATPase activity assays

MgATPase activity of V₁, V₁ΔH, and V₁(ΔH)H was measured using an ATP-regenerating assay as described (22). Briefly, 10 μg of the V₁ mutant was added to an assay mixture containing 1 mm MgCl₂, 5 mm ATP, 30 units/ml each of lactate dehydrogenase and pyruvate kinase, 0.5 mm NADH, 2 mm phosphoenolpyruvate, 50 mm HEPES, pH 7.5, at 37 °C. The decrease of absorbance at 340 nm was measured in the kinetics mode on a Varian Cary Bio100 spectrophotometer.

Native gel electrophoresis

For native gel electrophoresis, purified V-ATPase subunits and subcomplexes were resolved using 2% agarose gels in 20 mm bis-Tris-acetic acid, pH 6, 1 mm TCEP. Gels were resolved for 1 h at 100 V, fixed in 25% isopropanol, 10% acetic acid for 30 minutes, rinsed in 95% ethanol, and dried on a slab dryer for 2 h at 80 °C. The dried gel was stained with Coomassie G and destained in fixing solution.

Author contributions

S. S., R. A. O., and S. W. conceptualization; S. S. data curation; S. S. formal analysis; S. S. validation; S. S. investigation; S. S. visualization; S. S. and R. A. O. methodology; S. S. writing-original draft; S. S., R. A. O., and S. W. writing-review and editing; R. A. O. and S. W. supervision; S. W. funding acquisition; S. W. project administration.