Rapid Reconstitution of Tonoplast H+-Translocating Pyrophosphatase from Cultured Rice Cells into Liposomes

We report the rapid and functional reconstitution of H⁺-pyrophosphatase(H⁺-PPase) from the tonoplast of cultured rice (Oryza sativaL.) cells to proteoliposomes. The CHAPS-solubilized H⁺-PPasewas incorporated into liposomes by gel-filtration. Both theactivities of PP_i-hy-drolysis and H⁺-pumping were influencedby the lipid-pro-tein ratio and cholesterol. (Received June 10, 1996; Accepted January 9, 1997)     

Ion Effects on the H+-Translocating Adenosine Triphosphatase and Pyrophosphatase Associated with the Tonoplast of Chora corallina

H⁺-translocating ATPase and pyrophosphatase (PPase) associatedwith the tonoplast of Chara corallina were isolated with theaid of a perfusion technique, and the effects of ions on theiractivities were studied. All the alkali metal cations testedstimulated the ATPase and ATPdependent H⁺ pumping activitiesonly by 10 to 40%. Anions, on the other hand, strongly affectedthe activities. Potassium salts of Cl^- and Br^- stimulated them,while F^- and NO₃^- inhibited them. By contrast, the H⁺-translocatingPPase was insensitive to anions but sensitive to cations. Theorder of cation stimulation was Rb⁺=K⁺>Cs⁺>Na⁺=Li⁺>choline⁺.NO₃^- (50 mil), thought to be a specific inhibitor of the tonoplast-typeH⁺-ATPase, inhibited the ATPdependent H⁺ pumping almost completelybut the ATPase activity by only about 50%. Na⁺ inhibited thePP₁-dependent H⁺ pumping (I_5O=5OmM) in the presence of 50 mMKCl but not the ATP-dependent one. The PPase was more sensitiveto F^- (I₅₀=400µM) than the ATPase. Both the H⁺-ATPaseand the H⁺-PPase required Mg²⁺ for their activities, althoughan excess was inhibitory to both. The different sensitivitiesof the PP₁-dependent and the ATP-dependent H⁺- pumping enzymesto ions correspond to the tonoplast enzymes of higher plantsand may be used as "markers" to distinguish between these enzymesin characean cells (Received October 2, 1987; Accepted May 18, 1988)     

Hill Coefficient Analysis of Transmembrane Helix Dimerization

Sertoli cells are responsible for regulating a wide range of processes that lead to the differentiation of male germ cells into spermatozoa. Cytoplasmic pH (pH _i ) has been shown to be an important parameter in cell physiology, regulating namely cell metabolism and differentiation. However, membrane transport mechanisms involved in pH _i regulation mechanisms of Sertoli cells have not yet been elucidated. In this work, pH _i was determined using the pH-sensitive fluorescent probe 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). Addition of weak acids resulted in rapid acidification of the intracellular milieu. Sertoli cells then recovered pH _i by a mechanism that was shown to be sensitive to external Na⁺. pH _i recovery was also greatly reduced in the presence of 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and amiloride. These results point toward the action of an Na⁺-driven HCO₃⁻/Cl⁻ exchanger and/or an Na⁺/HCO₃⁻ cotransporter and the action of the Na⁺/H⁺ exchanger on pH _i regulation in the experimental conditions used. pH _i recovery was only slightly affected by ouabain, suggesting that the inhibition of Na⁺/K⁺-ATPase affects recovery indirectly, possibly via the shift on the Na⁺ gradient. On the other hand, recovery from the acid load was independent of the presence of concanamycin A, a specific inhibitor of the V-type ATPases, suggesting that these pumps do not have a relevant action on pH _i regulation in bovine Sertoli cells.     

Comparison of Plasma Membrane and Tonoplast H+-Translocating ATPases in Phaseolus mungo L. Roots

Two membrane fractions were obtained from 16%/26% and 34%/40%interfaces following discontinuous sucrose density gradientcentrifugation of a 10,000–80,000xg pellet from mung bean(Phaseolus mungo L.) roots. The ATPases in the fractions differedfrom each other in their sensitivity toward various inhibitors,activation with salts, dependence of activity on pH, and K_mfor ATP.Mg²⁺. Judging from their sensitivity toward inhibitors,the ATPases in the low and high density membranes are consideredmainly of tonoplast and plasma membrane origin, respectively.Both ATPases were activated by gramicidin D and nigericin. ATP-inducedquenching of quinacrine fluorescence in both fractions requiredMg²⁺ and permeant anions such as Cl^– and quenching wascollapsed by carbonylcyanide p-trifluoromethoxyphenyl hydrazone.The sensitivities of quenching to the inhibitors were essentiallythe same as those of ATPase activity in the membranes. Thesefindings suggest the involvement of ATPases in H⁺-pumping acrossa plasma membrane and tonoplast. (Received April 12, 1985; Accepted October 11, 1985)     

Second Transmembrane Helix (M2) and Long Range Coupling in Ca2+-ATPase

The actuator (A) domain of sarco(endo)plasmic reticulum Ca²⁺-ATPase not only plays a catalytic role but also undergoes large rotational movements that influence the distant transport sites through connections with transmembrane helices M1 and M2. Here we explore the importance of long helix M2 and its junction with the A domain by disrupting the helix structure and elongating with insertions of five glycine residues. Insertions into the membrane region of M2 and the top junctional segment impair Ca²⁺ transport despite reasonable ATPase activity, indicating that they are uncoupled. These mutants fail to occlude Ca²⁺. Those at the top segment also exhibited accelerated phosphoenzyme isomerization E1P → E2P. Insertions into the middle of M2 markedly accelerate E2P hydrolysis and cause strong resistance to inhibition by luminal Ca²⁺. Insertions along almost the entire M2 region inhibit the dephosphorylated enzyme transition E2 → E1. The results pinpoint which parts of M2 control cytoplasm gating and which are critical for luminal gating at each stage in the transport cycle and suggest that proper gate function requires appropriate interactions, tension, and/or rigidity in the M2 region at appropriate times for coupling with A domain movements and catalysis.     

Control of Transmembrane Helix Dynamics by Interfacial Tryptophan Residues

Transmembrane protein domains often contain interfacial aromatic residues, which may play a role in the insertion and stability of membrane helices. Residues such as Trp or Tyr, therefore, are often found situated at the lipid-water interface. We have examined the extent to which the precise radial locations of interfacial Trp residues may influence peptide helix orientation and dynamics. To address these questions, we have modified the GW^5,19ALP23 (acetyl-GGALW⁵(LA)₆LW¹⁹LAGA-[ethanol]amide) model peptide framework to relocate the Trp residues. Peptide orientation and dynamics were analyzed by means of solid-state nuclear magnetic resonance (NMR) spectroscopy to monitor specific ²H- and ¹⁵N-labeled residues. GW^5,19ALP23 adopts a defined, tilted orientation within lipid bilayer membranes with minimal evidence of motional averaging of NMR observables, such as ²H quadrupolar or ¹⁵N-¹H dipolar splittings. Here, we examine how peptide dynamics are impacted by relocating the interfacial Trp (W) residues on both ends and opposing faces of the helix, for example by a 100° rotation on the helical wheel for positions 4 and 20. In contrast to GW^5,19ALP23, the modified GW^4,20ALP23 helix experiences more extensive motional averaging of the NMR observables in several lipid bilayers of different thickness. Individual and combined Gaussian analyses of the ²H and ¹⁵N NMR signals confirm that the extent of dynamic averaging, particularly rotational “slippage” about the helix axis, is strongly coupled to the radial distribution of the interfacial Trp residues as well as the bilayer thickness. Additional ²H labels on alanines A3 and A21 reveal partial fraying of the helix ends. Even within the context of partial unwinding, the locations of particular Trp residues around the helix axis are prominent factors for determining transmembrane helix orientation and dynamics within the lipid membrane environment.     

Structural and Functional Analysis of Transmembrane Segment VI of the NHE1 Isoform of the Na+/H+ Exchanger

The Na⁺/H⁺ exchanger isoform 1 is a ubiquitously expressed integral membrane protein. It resides on the plasma membrane of cells and regulates intracellular pH in mammals by extruding an intracellular H⁺ in exchange for one extracellular Na⁺. We characterized structural and functional aspects of the transmembrane segment (TM) VI (residues 227–249) by using cysteine scanning mutagenesis and high resolution NMR. Each residue of TM VI was mutated to cysteine in the background of the cysteineless NHE1 protein, and the sensitivity to water-soluble sulfhydryl-reactive compounds (2-(trimethylammonium)ethyl)methanethiosulfonate (MTSET) and (2-sulfonatoethyl)methanethiosulfonate (MTSES) was determined for those residues with significant activity remaining. Three residues were essentially inactive when mutated to Cys: Asp²³⁸, Pro²³⁹, and Glu²⁴⁷. Of the remaining residues, proteins with the mutations N227C, I233C, and L243C were strongly inhibited by MTSET, whereas amino acids Phe²³⁰, Gly²³¹, Ala²³⁶, Val²³⁷, Ala²⁴⁴, Val²⁴⁵, and Glu²⁴⁸ were partially inhibited by MTSET. MTSES did not affect the activity of the mutant NHE1 proteins. The structure of a peptide representing TM VI was determined using high resolution NMR spectroscopy in dodecylphosphocholine micelles. TM VI contains two helical regions oriented at an approximate right angle to each other (residues 229–236 and 239–250) surrounding a central unwound region. This structure bears a resemblance to TM IV of the Escherichia coli protein NhaA. The results demonstrate that TM VI of NHE1 is a discontinuous pore-lining helix with residues Asn²²⁷, Ile²³³, and Leu²⁴³ lining the translocation pore.     

Determinants of Cation Permeation and Drug Sensitivity in Predicted Transmembrane Helix 9 and Adjoining Exofacial Re-entrant Loop 5 of Na+/H+ Exchanger NHE1

Mammalian Na⁺/H⁺ exchangers (NHEs) regulate numerous physiological processes and are involved in the pathogenesis of several diseases, including tissue ischemia and reperfusion injuries, cardiac hypertrophy and failure, and cancer progression. Hence, NHEs are being targeted for pharmaceutical-based clinical therapies, but pertinent information regarding the structural elements involved in cation translocation and drug binding remains incomplete. Molecular manipulations of the prototypical NHE1 isoform have implicated several predicted membrane-spanning (M) helices, most notably M4, M9, and M11, as important determinants of cation permeation and drug sensitivity. Here, we have used substituted-cysteine accessibility mutagenesis and thiol-modifying methanethiosulfonate (MTS) reagents to further probe the involvement of evolutionarily conserved sites within M9 (residues 342–363) and the adjacent exofacial re-entrant loop 5 between M9 and M10 (EL5; residues 364–415) of a cysteine-less variant of rat NHE1 on its kinetic and pharmacological properties. MTS treatment significantly reduced the activity of mutants containing substitutions within M9 (H353C, S355C, and G356C) and EL5 (G403C and S405C). In the absence of MTS, mutants S355C, G403C, and S405C showed modest to significant decreases in their apparent affinities for Na⁺_o and/or H⁺_i. In addition, mutations Y370C and E395C within EL5, whereas failing to confer sensitivity to MTS, nevertheless, reduced the affinity for Na⁺_o, but not for H⁺_i. The Y370C mutant also exhibited higher affinity for ethylisopropylamiloride, a competitive antagonist of Na⁺_o transport. Collectively, these results further implicate helix M9 and EL5 of NHE1 as important elements involved in cation transport and inhibitor sensitivity, which may inform rational drug design.     

Structure of Transmembrane Helix 8 and Possible Membrane Defects in CFTR

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel that regulates the flow of anions across epithelia. Mutations in CFTR cause cystic fibrosis. CFTR belongs to the ATP-binding cassette transporter superfamily, and gating is controlled by phosphorylation and ATP binding and hydrolysis. Recently obtained ATP-free and ATP-bound structures of zebrafish CFTR revealed an unwound segment of transmembrane helix (TM) 8, which appears to be a unique feature of CFTR not present in other ATP-binding cassette transporter structures. Here, using μs-long molecular dynamics simulations, we investigate the interactions formed by this TM8 segment with nearby helices in both ATP-free and ATP-bound states. We highlight ATP-dependent interactions as well as the structural role of TM8 in maintaining the functional architecture of the pore via interactions common to both the ATP-bound and ATP-free state. The results of the molecular dynamics simulations are discussed in the context of the gating mechanism of CFTR.     

Relationship of K+-Uptaking System with H+-Translocating ATPase in Enterococcus hirae, Grown at a High or Low Alkaline pH

Potassium ion pool was studied in glycolyzing Enterococcus hirae, grown at high or low alkaline pH (pH 9.5 and 8.0, respectively). Energy-dependent increase of K⁺ pool was lower for the wild-type cells, grown at pH 9.5, than that for the cells grown at pH 8.0. It was inhibited by N,N′-dicyclohexylcarbodiimide (DCCD). The stoichiometry of DCCD-inhibited K⁺ influx to DCCD-inhibited H⁺ efflux for the wild-type cells, grown at pH 9.5 or 8.0, was fixed for different K⁺ external activity. DCCD-inhibited ATPase activity of membrane vesicles was significantly stimulated by K⁺ for the wild-type cells grown at pH 9.5, and required K⁺ for the wild-type cells grown at pH 8.0, while the levels of α and β subunits of the F₁ and b subunit of the F₀ were lower for the cells grown at pH 9.5 than that for the cells grown at pH 8.0. Such an ATPase activity was residual in membrane vesicles from the atpD mutant with a nonfunctional F₀F₁. ATPase activity of membrane vesicles from the mutant with defect in Na⁺-ATPase was higher for the cells grown at pH 9.5 than that for the cells grown at pH 8.0, and was inhibited by DCCD. An energy-dependent increase of K⁺ pool in this bacterium, grown at a high or low alkaline pH, is assumed to occur through a K⁺ uptaking system, most probably the Trk. The latter functions in a closed relationship with the H⁺-translocating ATPase F₀F₁. Received: 30 June 1997 / Accepted: 4 August 1997     

Phosphatidylserine Asymmetry Promotes the Membrane Insertion of a Transmembrane Helix

The plasma membrane (PM) contains an asymmetric distribution of lipids between the inner and outer bilayer leaflets. A lipid of special interest in eukaryotic membranes is the negatively charged phosphatidylserine (PS). In healthy cells, PS is actively sequestered to the inner leaflet of the PM, but PS redistributes to the outer leaflet when the cell is damaged or at the onset of apoptosis. However, the influence of PS asymmetry on membrane protein structure and folding are poorly understood. The pH low insertion peptide (pHLIP) adsorbs to the membrane surface at a neutral pH, but it inserts into the membrane at an acidic pH. We have previously observed that in symmetric vesicles, PS affects the membrane insertion of pHLIP by lowering the pH midpoint of insertion. Here, we studied the effect of PS asymmetry on the membrane interaction of pHLIP. We developed a modified protocol to create asymmetric vesicles containing PS and employed Annexin V labeled with an Alexa Fluor 568 fluorophore as a new probe to quantify PS asymmetry. We observed that the membrane insertion of pHLIP was promoted by the asymmetric distribution of negatively charged PS, which causes a surface charge difference between bilayer leaflets. Our results indicate that lipid asymmetry can modulate the formation of an α-helix on the membrane. A corollary is that model studies using symmetric bilayers to mimic the PM may fail to capture important aspects of protein-membrane interactions.     

Role of Side-Chain Conformational Entropy in Transmembrane Helix Dimerization of Glycophorin A

Dimerization of the transmembrane domain of glycophorin A is mediated by a seven residue motif LIxxGVxxGVxxT through a combination of van der Waals and hydrogen bonding interactions. One of the unusual features of the motif is the large number of β-branched amino acids that may limit the entropic cost of dimerization by restricting side-chain motion in the monomeric transmembrane helix. Deuterium NMR spectroscopy is used to characterize the dynamics of fully deuterated Val80 and Val84, two essential amino acids of the dimerization motif. Deuterium spectra of the glycophorin A transmembrane dimer were obtained using synthetic peptides corresponding to the transmembrane sequence containing either perdeuterated Val80 or Val84. These data were compared with spectra of monomeric glycophorin A peptides deuterated at Val84. In all cases, the deuterium line shapes are characterized by fast methyl group rotation with virtually no motion about the C_α-C_β bond. This is consistent with restriction of the side chain in both the monomer and dimer due to intrahelical packing interactions involving the β-methyl groups, and indicates that there is no energy cost associated with dimerization due to loss of conformational entropy. In contrast, deuterium NMR spectra of Met81 and Val82, in the lipid interface, reflected greater motional averaging and fast exchange between different side-chain conformers.     

Reconstitution and Characterization of H+-Translocating ATPase from the Plasma Membrane of Phaseolus mungo L. Roots

Plasma membrane H⁺-translocating ATPase was partially purifiedfrom mung bean (Phaseolus mungo L.) roots and reconstitutedinto soybean phospholipid (asolectin) liposomes by the n-octylglucosidedilution method. The resulting proteoliposomes were mainly unilamellarvesicles ranging in size from 0.05 to 0.2 µm. The existenceof ATP-drived H⁺-pumping across the proteoliposomes was demonstratedby the quenching of quinacrine fluorescence in the presenceof Mg²⁺. The quenching could be abolished by an uncoupler, FCCP,and an inhibitor of H⁺-translocating ATPase, vanadate. The reconstitutedATPase consisted of three major polypeptides of 105 KDa, 67KDa and 57 KDa. Its pH optimum, divalent cation stimulationand vanadate sensitivity were similar to those of partiallypurified ATPase. However, the specificity toward ATP was muchgreater following reconstitution. Also reconstitution reducedthe degree of inhibition by DCCD. Local anesthetics (e.g. dibucaine)had no effect on H⁺-pumping activity but increased the ATPaseactivity when proteoliposomes were reconstituted in their presence. (Received May 2, 1986; Accepted October 17, 1986)     

Sequence-Specific Dimerization of a Transmembrane Helix in Amphipol A8-35

As traditional detergents might destabilize or even denature membrane proteins, amphiphilic polymers have moved into the focus of membrane-protein research in recent years. Thus far, Amphipols are the best studied amphiphilic copolymers, having a hydrophilic backbone with short hydrophobic chains. However, since stabilizing as well as destabilizing effects of the Amphipol belt on the structure of membrane proteins have been described, we systematically analyze the impact of the most commonly used Amphipol A8-35 on the structure and stability of a well-defined transmembrane protein model, the glycophorin A transmembrane helix dimer. Amphipols are not able to directly extract proteins from their native membranes, and detergents are typically replaced by Amphipols only after protein extraction from membranes. As Amphipols form mixed micelles with detergents, a better understanding of Amphipol-detergent interactions is required. Therefore, we analyze the interaction of A8-35 with the anionic detergent sodium dodecyl sulfate and describe the impact of the mixed-micelle-like system on the stability of a transmembrane helix dimer. As A8-35 may highly stabilize and thereby rigidify a transmembrane protein structure, modest destabilization by controlled addition of detergents and formation of mixed micellar systems might be helpful to preserve the function of a membrane protein in Amphipol environments.     

Functional Domains in the Retroviral Transmembrane Protein

The envelope glycoproteins of the mammalian type C retroviruses consist of two subunits, a surface (SU) protein and a transmembrane (TM) protein. SU binds to the viral receptor and is thought to trigger conformational changes in the associated TM protein that ultimately lead to the fusion of viral and host cell membranes. For Moloney murine leukemia virus (MoMuLV), the envelope protein probably exists as a trimer. We have previously demonstrated that the coexpression of envelope proteins that are individually defective in either the SU or TM subunits can lead to functional complementation (Y. Zhao et al., J. Virol. 71:6967–6972, 1997). We have now extended these studies to investigate the abilities of a panel of fusion-defective TM mutants to complement each other. This analysis identified distinct complementation groups within TM, with implications for interactions between different regions of TM in the fusion process. In viral particles, the C-terminal 16 amino acids of the MoMuLV TM (the R peptide) are cleaved by the viral protease, resulting in an increased fusogenicity of the envelope protein. We have examined the consequences of R peptide cleavage for the different TM fusion mutants and have found that this enhancement of fusogenicity can only occur in cis to certain of the TM mutants. These results suggest that R peptide cleavage enhances the fusogenicity of the envelope protein by influencing the interaction of two distinct regions in the TM ectodomain.     

Cysteine Scanning Mutagenesis of Transmembrane Helix 3 of a Brain Glutamate Transporter Reveals Two Conformationally Sensitive Positions

Glutamate transporters in the brain remove the neurotransmitter from the synapse by cotransport with three sodium ions into the surrounding cells. Recent structural work on an archaeal homolog suggests that, during substrate translocation, the transport domain, including the peripheral transmembrane helix 3 (TM3), moves relative to the trimerization domain in an elevator-like process. Moreover, two TM3 residues have been proposed to form part of a transient Na3′ site, and another, Tyr-124, appears close to both Na3′ and Na1. To obtain independent evidence for the role of TM3 in glutamate transport, each of its 31 amino acid residues from the glial GLT-1 transporter was individually mutated to cysteine. Except for six mutants, substantial transport activity was detected. Aqueous accessibility of the introduced cysteines was probed with membrane-permeant and membrane-impermeant sulfhydryl reagents under a variety of conditions. Transport of six single cysteine mutants, all located on the intracellular side of TM3, was affected by membrane-permeant sulfhydryl reagents. However, only at two positions could ligands modulate the reactivity. A120C reactivity was diminished under conditions expected to favor the outward-facing conformation of the transporter. Sulfhydryl modification of Y124C by 2-aminoethyl methanethiosulfonate, but not by N-ethylmaleimide, was fully protected in the presence of sodium. Our data are consistent with the idea that TM3 moves during transport. Moreover, computational modeling indicated that electrostatic repulsion between the positive charge introduced at position 124 and the sodium ions bound at Na3′ and Na1 underlies the protection by sodium.