Passive Proton Translocation Research Articles

Quantification of Hv1-induced proton translocation by a lipid-coupled Oregon Green 488-based assay

Passive proton translocation across membranes through proton channels is generally measured with assays that allow a qualitative detection of the H+-transfer. However, if a quantitative and time-resolved analysis is required, new methods have to be developed. Here, we report on the quantification of pH changes induced by the voltage-dependent proton channel Hv1 using the commercially available pH-sensitive fluorophore Oregon Green 488-DHPE (OG488-DHPE). We successfully expressed and isolated Hv1 from Escherichia coli and reconstituted the protein in large unilamellar vesicles. Reconstitution was verified by surface enhanced infrared absorption (SEIRA) spectroscopy and proton activity was measured by a standard 9-amino-6-chloro-2-methoxyacridine assay. The quantitative OG488-DHPE assay demonstrated that the proton translocation rate of reconstituted Hv1 is much smaller than those reported in cellular systems. The OG488-DHPE assay further enabled us to quantify the KD-value of the Hv1-inhibitor 2-guanidinobenzimidazole, which matches well with that found in cellular experiments. Our results clearly demonstrate the applicability of the developed in vitro assay to measure proton translocation in a quantitative fashion; the assay allows to screen for new inhibitors and to determine their characteristic parameters. Graphical abstract ᅟ.

Affinity Purification and Structural Features of the Yeast Vacuolar ATPase Vo Membrane Sector

The membrane sector (Vo) of the proton pumping vacuolar ATPase (V-ATPase, V1Vo-ATPase) from Saccharomyces cerevisiae was purified to homogeneity, and its structure was characterized by EM of single molecules and two-dimensional crystals. Projection images of negatively stained Vo two-dimensional crystals showed a ring-like structure with a large asymmetric mass at the periphery of the ring. A cryo-EM reconstruction of Vo from single-particle images showed subunits a and d in close contact on the cytoplasmic side of the proton channel. A comparison of three-dimensional reconstructions of free Vo and Vo as part of holo V1Vo revealed that the cytoplasmic N-terminal domain of subunit a (aNT) must undergo a large conformational change upon enzyme disassembly or (re)assembly from Vo, V1, and subunit C. Isothermal titration calorimetry using recombinant subunit d and aNT revealed that the two proteins bind each other with a Kd of ~5 μm. Treatment of the purified Vo sector with 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] resulted in selective release of subunit d, allowing purification of a VoΔd complex. Passive proton translocation assays revealed that both Vo and VoΔd are impermeable to protons. We speculate that the structural change in subunit a upon release of V1 from Vo during reversible enzyme dissociation plays a role in blocking passive proton translocation across free Vo and that the interaction between aNT and d seen in free Vo functions to stabilize the Vo sector for efficient reassembly of V1Vo.

Essential arginine residue of the Fo-a subunit in FoF1-ATP synthase has a role to prevent the proton shortcut without c-ring rotation in the Fo proton channel

In F(o)F(1) (F(o)F(1)-ATP synthase), proton translocation through F(o) drives rotation of the oligomer ring of F(o)-c subunits (c-ring) relative to F(o)-a. Previous reports have indicated that a conserved arginine residue in F(o)-a plays a critical role in the proton transfer at the F(o)-a/c-ring interface. Indeed, we show in the present study that thermophilic F(o)F(1s) with substitution of this arginine (aR169) to other residues cannot catalyse proton-coupled reactions. However, mutants with substitution of this arginine residue by a small (glycine, alanine, valine) or acidic (glutamate) residue mediate the passive proton translocation. This translocation requires an essential carboxy group of F(o)-c (cE56) since the second mutation (cE56Q) blocks the translocation. Rotation of the c-ring is not necessary because the same arginine mutants of the 'rotation-impossible' (c(10)-a)F(o)F(1), in which the c-ring and F(o)-a are fused to a single polypeptide, also exhibits the passive proton translocation. The mutant (aR169G/Q217R), in which the arginine residue is transferred to putatively the same topological position in the F(o)-a structure, can block the passive proton translocation. Thus the conserved arginine residue in F(o)-a ensures proton-coupled c-ring rotation by preventing a futile proton shortcut.

Function and Subunit Interactions of the N-terminal Domain of Subunit a (Vph1p) of the Yeast V-ATPase

The vacuolar (H+)-ATPases (V-ATPases) are ATP-dependent proton pumps that operate by a rotary mechanism in which ATP hydrolysis drives rotation of a ring of proteolipid subunits relative to subunit a within the integral V(0) domain. In vivo dissociation of the V-ATPase (an important regulatory mechanism) generates a V(0) domain that does not passively conduct protons. EM analysis indicates that the N-terminal domain of subunit a approaches the rotary subunits in free V(0), suggesting a possible mechanism of silencing passive proton transport. To test the hypothesis that the N-terminal domain inhibits passive proton flux by preventing rotation of the proteolipid ring in free V(0), factor Xa cleavage sites were introduced between the N- and C-terminal domains of subunit a (the Vph1p isoform in yeast) to allow its removal in vitro after isolation of vacuolar membranes. The mutant Vph1p gave rise to a partially uncoupled V-ATPase complex. Cleavage with factor Xa led to further loss of coupling of proton transport and ATP hydrolysis. Removal of the N-terminal domain by cleavage with factor Xa and treatment with KNO3 and MgATP did not, however, lead to an increase in passive proton conductance by free V(0), suggesting that removal of the N-terminal domain is not sufficient to facilitate passive proton conductance through V(0). Photoactivated cross-linking using the cysteine reagent maleimido benzophenone and single cysteine mutants of subunit a demonstrated the proximity of specific sites within the N-terminal domain and subunits E and G of the peripheral stalk. These results suggest that a localized region of the N-terminal domain (residues 347-369) is important in anchoring the peripheral stator in V1V0.

Both Rotor and Stator Subunits Are Necessary for Efficient Binding of F1 to F0 in Functionally Assembled Escherichia coli ATP Synthase

In F1F0-ATP synthase, the subunit b2delta complex comprises the peripheral stator bound to subunit a in F0 and to the alpha3beta3 hexamer of F1. During catalysis, ATP turnover is coupled via an elastic rotary mechanism to proton translocation. Thus, the stator has to withstand the generated rotor torque, which implies tight interactions of the stator and rotor subunits. To quantitatively characterize the contribution of the F0 subunits to the binding of F1 within the assembled holoenzyme, the isolated subunit b dimer, ab2 subcomplex, and fully assembled F0 complex were specifically labeled with tetramethylrhodamine-5-maleimide at bCys64 and functionally reconstituted into liposomes. Proteoliposomes were then titrated with increasing amounts of Cy5-maleimide-labeled F1 (at gammaCys106 and analyzed by single-molecule fluorescence resonance energy transfer. The data revealed F1 dissociation constants of 2.7 nm for the binding of F0 and 9-10 nm for both the ab2 subcomplex and subunit b dimer. This indicates that both rotor and stator components of F0 contribute to F1 binding affinity in the assembled holoenzyme. The subunit c ring plays a crucial role in the binding of F1 to F0, whereas subunit a does not contribute significantly.

Direct Interaction of Subunits a and b of the F0 Complex of Escherichia coli ATP Synthase by Forming an ab2 Subcomplex

The addition of a His6 tag to the N terminus of subunit a of the F0 complex of the Escherichia coli ATP synthase allowed the purification of an ab2 subcomplex after solubilization of membranes with n-dodecyl-beta-d-maltoside and subsequent nickel-nitrilotriacetic acid affinity chromatography. After co-reconstitution of the ab2 subcomplex with purified subunit c, passive proton translocation rates as well as coupled ATPase activities after binding of F1 were measured that were comparable with those of wild type F0. The interaction between subunits a and b, which has been shown to be stoichiometric and functional, is not triggered by any cross-linking reagent and therefore reflects subunit interactions occurring within the F0 complex in vivo.

F0 of ATP Synthase Is a Rotary Proton Channel: OBLIGATORY COUPLING OF PROTON TRANSLOCATION WITH ROTATION OF c-SUBUNIT RING

Coupling of proton flow and rotation in the F(0) motor of ATP synthase was investigated using the thermophilic Bacillus PS3 enzyme expressed functionally in Escherichia coli cells. Cysteine residues introduced into the N-terminal regions of subunits b and c of ATP synthase (bL2C/cS2C) were readily oxidized by treating the expressing cells with CuCl(2) to form predominantly a b-c cross-link with b-b and c-c cross-links being minor products. The oxidized ATP synthases, either in the inverted membrane vesicles or in the reconstituted proteoliposomes, showed drastically decreased proton pumping and ATPase activities compared with the reduced ones. Also, the oxidized F(0), either in the F(1)-stripped inverted vesicles or in the reconstituted F(0)-proteoliposomes, hardly mediated passive proton translocation through F(0). Careful analysis using single mutants (bL2C or cS2C) as controls indicated that the b-c cross-link was responsible for these defects. Thus, rotation of the c-oligomer ring relative to subunit b is obligatory for proton translocation; if there is no rotation of the c-ring there is no proton flow through F(0).

On the Role of Arg-210 and Glu-219 of Subunit a in Proton Translocation by the Escherichia coliF0F1-ATP Synthase

A strain of Escherichia coli was constructed which had a complete deletion of the chromosomal uncB gene encoding subunit a of the F0F1-ATP synthase. Gene replacement was facilitated by a selection protocol that utilized the sacB gene of Bacillus subtilis cloned in a kanamycin resistance cartridge (Ried, J. L., and Collmer, A. (1987) Gene (Amst.) 57, 239-246). F0 subunits b and c inserted normally into the membrane in the DeltauncB strain. This observation confirms a previous report (Hermolin, J., and Fillingame, R. H. (1995) J. Biol. Chem. 270, 2815-2817) that subunit a is not required for the insertion of subunits b and c. The DeltauncB strain has been used to characterize mutations in Arg-210 and Glu-219 of subunit a, residues previously postulated to be essential in proton translocation. The aE219G and aE219K mutants grew on a succinate carbon source via oxidative phosphorylation and membranes from these mutants exhibited ATPase-coupled proton translocation (i.e. ATP driven 9-amino-6-chloromethoxyacridine quenching responses that were 60-80% of wild type membranes). We conclude that the aGlu-219 residue cannot play a critical role in proton translocation. The aR210A mutant did not grow on succinate and membranes exhibited no ATPase-coupled proton translocation. However, on removal of F1 from membrane, the aR210A mutant F0 was active in passive proton translocation, i.e. in dissipating the DeltapH normally established by NADH oxidation with these membrane vesicles. aR210A membranes with F1 bound were also proton permeable. Arg-210 of subunit a may play a critical role in active H+ transport that is coupled to ATP synthesis or hydrolysis, but is not essential for the translocation of protons across the membranes.

Functional stability of the a-subunit of the F 0F 1-ATPase from Escherichia coli is affected by mutations in three proline residues

Site-directed mutagenesis was used to investigate the roles of three proline residues (Pro-103, Pro-122 and Pro-143) in the a-subunit of the E. coli F 0F 1-ATPase. All three were found to have a role in stabilizing the a-subunit structure in that removal of the F 1-ATPase from membranes prepared from each of the mutant strains resulted in the loss of passive proton translocation activity. Pro-103 is predicted to be within a transmembrane helix. Pro-122 and Pro-143 are located just outside the membrane and near two residues (Asp-124 and Arg-140) previously proposed to form a charge pair. The phenotype of mutants in which Pro-122 or Pro-143 were replaced by alanine was similar to previously isolated mutants affected in Asp-124 and Arg-140. This suggested that the main effect of the mutations was to destroy the charge pair between Asp-124 and Arg-140. Double mutants resulting from all possible combinations of these four mutations were constructed and, with the exception of P122A + D124A, had a similar phenotype to the single mutants. This is consistent with the idea that all four single changes had the same effect on a-subunit structure. In contrast, combining the P122A or P143A changes with another mutation which caused a similar phenotype (D44N) resulted in a complete loss of oxidative phosphorylation.

Pathways of water through erythrocyte membranes. Routes along defect structures

A new model for the passive (diffusional) permeation of water and for the transfer of protons across membranes from mammalian erythrocytes is presented. The proton transfer is anion-coupled through the action of the band3 protein. Inhibition studies reveal that the pathways for the anion- and water-exchange are independent. Dynamic grain boundaries and defect structures in thermodynamic equilibrium (steady state in the general case) with the membrane are considered to be the major routes for the water permeation. Modulators of membrane functions may either act only locally or may lead to a reordering of the membrane. The specific inhibitor of the anion-exchange, 4,4'-diisothiocyanatostilbene-2-2'-disulfonic acid (DIDS), is considered to induce only local effects by closing the anion-transfer gate. The more unspecific binding of the mercurial p-chloromercuribenzenesulfonic acid (pCMBS) should change the membrane aggregational states. Thus pCMBS can reduce the water permeation and at the same time stimulate the passive proton translocation as is experimentally observed.

57] Proton-conducting portion (F0) from Escherichia coli ATP synthase: Preparation, dissociation into subunits, and reconstitution of an active complex

Publisher Summary This chapter discusses the preparation, dissociation into subunits, and reconstitution of an active complex for the proton-conducting portion (F 0 ) from Escherichia coli ATP synthase. The F 0 part of the ATP synthase complex (F 1 F 0 ) of E. coli is composed of three different polypeptides and the complex serves as a proton channel. After reconstitution into liposomes a functional F 0 complex can be tested either by measuring passive proton translocation or by adding back F 1 . Passive proton translocation through F 0 is measured using K+-loaded liposomes. ATPase activity of reconstituted F 1 F 0 complexes is measured by determining the liberated orthophosphate. The chapter outlines the procedure for preparation of F 0 by hydrophobic interaction chromatography. Reconstitution of a functional F 0 complex is only achieved by incorporation of all three kinds of subunits into phospholipid vesicles.

On the mechanism of action of oligomycin and acidic uncouplers on proton translocation and energy transfer in "sonic" submitochondrial particles.

A study is presented of the effect of acidic uncouplers and oligomycin on energy-linked and passive proton translocation, oxidative phosphorylation, and energy-linked nicotinamide-adenine-nucleotide transhydrogenase in EDTA submitochondrial particles from beef-heart. A flow potentiometric technique has been applied to resolve the kinetics of the initial rapid phase of the redox proton pump. Rapid kinetics analysis shows that carbonyl-cyanide-p-trifluoromethoxyphenyl-hydrazone (FCCP) does not exert any direct effect on redox-linked active proton transport. The uncoupling action of FCCP on oxidative phosphorylation and energy-linked transhydrogenase is shown to be quantitatively accounted for by its promoting effect of passive proton-diffusion across the mitochondrial membrane. Oligomycin depresses passive proton diffusion in EDTA sonic particles and this effect accounts for the coupling action exerted by the antibiotic on oxidative phosphorylation and energy-linked transhydrogenase. In fact, rapid kinetic analysis demonstrates that oligomycin does not directly affect the redox-linked proton pump. The present results show that there does not exist any labile intermediate in the redox-linked proton pump which is sensitive to acidic uncouplers.