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On the osmotic signal and osmosensing mechanism of an ABC transport system for glycine betaine

2001; Springer Nature; Volume: 20; Issue: 24 Linguagem: Inglês

10.1093/emboj/20.24.7022

1460-2075

Tammo van der Heide,

RNA Interference and Gene Delivery

Article17 December 2001free access On the osmotic signal and osmosensing mechanism of an ABC transport system for glycine betaine Tiemen van der Heide Tiemen van der Heide Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands Search for more papers by this author Marc C.A. Stuart Marc C.A. Stuart Department of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands Search for more papers by this author Bert Poolman Corresponding Author Bert Poolman Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands Search for more papers by this author Tiemen van der Heide Tiemen van der Heide Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands Search for more papers by this author Marc C.A. Stuart Marc C.A. Stuart Department of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands Search for more papers by this author Bert Poolman Corresponding Author Bert Poolman Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands Search for more papers by this author Author Information Tiemen van der Heide1, Marc C.A. Stuart2 and Bert Poolman 1 1Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands 2Department of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:7022-7032https://doi.org/10.1093/emboj/20.24.7022 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The osmosensing mechanism of the ATP-binding cassette (ABC) transporter OpuA of Lactococcus lactis has been elucidated for the protein reconstituted in liposomes. Activation of OpuA by osmotic upshift was instantaneous and reversible and followed changes in volume and membrane structure of the proteoliposomes. Osmotic activation of OpuA was dependent on the fraction of anionic lipids present in the lipid bilayer. Also, cationic and anionic lipophilic amphiphiles shifted the activation profile in a manner indicative of an osmosensing mechanism, in which electrostatic interactions between lipid headgroups and the OpuA protein play a major role. Further support for this notion came from experiments in which ATP-driven uptake and substrate-dependent ATP hydrolysis were measured with varying concentrations of osmolytes at the cytoplasmic face of the protein. Under iso-osmotic conditions, the transporter could be activated by high concentrations of ionic osmolytes, whereas neutral ones had no effect, demonstrating that intracellular ionic strength, rather than a specific signaling molecule or water activity, signals osmotic stress to the transporter. The data indicate that OpuA is under the control of a mechanism in which the membrane and ionic strength act in concert to signal osmotic changes. Introduction Maintenance of cell turgor is a prerequisite for almost any form of life, as it provides the mechanical force for expansion of the cell wall. Generally, microorganisms respond to an osmotic upshift by accumulating kosmotropic organic solutes (compatible solutes) to counteract the loss of water and decrease in turgor pressure (Poolman and Glaasker, 1998). Upon osmotic downshift, the cells need to expel the solutes rapidly in order to prevent turgor pressure from becoming too high, which may lead to lysis of the cells. In order to cope with osmotic stress effectively, the primary response to this type of challenge must involve (in)activation of existing transporters, as synthesis of new enzyme systems would take too long to respond. A major topic in the field of cell volume regulation concerns the mechanism(s) underlying the osmotic activation of transport and channel molecules. The potential stimuli for such systems comprise changes in extra- or intracellular osmolality, ionic strength, turgor pressure, molecular crowding, osmolality gradient across the membrane or the physicochemical properties of the membrane (Wood, 1999). Recent work on the osmoregulated ATP-binding cassette (ABC) transporter OpuA from Lactococcus lactis, and the ion-linked transporters ProP from Escherichia coli and BetP from Corynebacterium glutamicum showed that these systems act as both osmosensor and osmoregulator (Racher et al., 1999; Rübenhagen et al., 2000; van der Heide and Poolman, 2000b). These studies excluded turgor as the possible signal for osmotic activation. Activation of OpuA and BetP by charged amphipaths suggested that these transport systems sense osmotic stress via alterations in membrane properties/protein–lipid interactions, but direct effects via changes in the hydration state of the proteins could not be excluded. Several physicochemical properties of the membrane, such as membrane thickness, fluidity, interfacial polarity, membrane charge, hydration of lipid headgroups, acyl chain packing, but also the ‘macroscopic’ membrane folding, are affected upon osmolality shifts. Changes in one or more of these membrane parameters, sometimes described in terms of changes in the lateral pressure profile (Cantor, 1999), could influence the conformation of the transport proteins and thereby their activity. Lactococcus lactis responds to an osmotic upshift by accumulating glycine betaine via the ABC transporter OpuA, which is osmotically regulated at the level of both expression and transport activity (van der Heide and Poolman, 2000a). The protein is composed of two different polypeptides, i.e. the ATPase and a subunit that comprises both the translocator and ligand-binding domain (van der Heide and Poolman, 2000b). At present, OpuA is one of a few osmoregulated proteins, and the only osmotically activated ABC transporter, that has been characterized in a well-defined proteoliposomal system. Components other than the subunits of OpuA and a lipid membrane are not needed for osmotic regulation of the system. OpuA has the advantage over ion-linked transporters that both translocation and ATPase activity can be followed in a manner that allows discrimination between ‘external’ and ‘cytoplasmic’ hydration effects. In the present study, we show that the osmotic activation profile of OpuA is set by the bulk charge in the lipid headgroup region of the membrane, indicating that electrostatic interactions between lipids and the transporter are intrinsic to the osmosensing mechanism. It is also demonstrated that the ionic strength at the cytoplasmic face of the OpuA protein, rather than a specific signaling molecule or water activity, signals osmotic stress to the transporter. We thus conclude that cytoplasmic ionic strength serves as an osmotic signal, presumably by affecting lipid–protein interactions. Results The response of OpuA towards osmotic stress is instantaneous and reversible To assess the kinetics and reversibility of osmotic (in)activation of OpuA, the membrane-reconstituted protein was exposed alternately to osmotic up- and downshifts. The internal osmolality was varied between 190 and 380 mosmol/kg. Figure 1 shows that activation of OpuA by osmotic upshift is instantaneous and reversible upon returning to iso-osmotic conditions; the reversibility was not restricted to the particular lipid composition used in this experiment. OpuA was reactivated again by a second osmotic upshift, demonstrating that the integrity of the proteoliposomes was not compromised by the osmotic challenges. Maintenance of liposome integrity was confirmed by internal volume measurements, as shifts between hyper- and iso-osmotic conditions did not result in loss of the enclosed fluorescent dye calcein. It is important to stress here that the internal volume decreased in proportion to the osmotic upshift; the internal and external osmolalities became equal, within the time resolution of the experiment (∼1 s). Thus, upon osmotic upshift, the surface to volume ratio of the proteoliposome increased. Figure 1.Kinetics and reversibility of the osmotic activation of OpuA. Uptake of [14C]glycine betaine (final concentration, 76 μM) was assayed in 100 mM KPi pH 7.0 corresponding to 190 mosmol/kg. The proteoliposomes were composed of DOPC/DOPE/DOPG in a 2:1:1 mole ratio. At 105 (circles, squares and triangles) and 285 s (circles), the proteoliposomes were subjected to hyperosmotic conditions by the addition of 100 mM KCl (final osmolality corresponding to 380 mosmol/kg). Iso-osmotic conditions were restored at 180 s (circles) by dilution of the assay mixture with water (plus 76 μM [14C]glycine betaine). Download figure Download PowerPoint Proteoliposomes form ‘sickle-shaped’ structures when subjected to hyperosmotic stress To observe the changes in volume of the proteoliposomes upon osmotic upshifts, fluorophore (calcein) self-quenching and cryo-electron microscopy (cryo-EM) studies were performed. It was observed that, at trans-membrane osmotic gradients ranging from 0 to 535 mosmol/kg, the volume of the proteoliposomes decreased proportionally to the increase in the osmolality of the medium (data not shown), thereby dissipating the trans-membrane osmotic gradient. This osmometric behavior was accompanied by changes in ‘macroscopic’ folding of the membrane as visualized by cryo-EM (Figure 2). The proteoliposomes were converted from spherical into ‘sickle-shaped’ structures upon osmotic upshift. In principle, differences in the fraction of anionic lipids could affect the size and ‘macroscopic’ folding of the membrane, and thereby influence the activity of OpuA. The observed macromolecular structures, however, were the same for all lipid mixtures tested (Figure 2 and data not shown). The proteoliposomes with an average diameter of ∼150 nm have a low membrane curvature, but upon osmotic upshift highly curved regions appeared in the proteoliposomes. To exclude the possibility that initial differences in the ‘macroscopic’ curvature had an effect on the osmotic activation of OpuA, proteoliposomes obtained by extrusion through polycarbonate filters with a pore size of 200 and 400 nm were compared. The experiments showed that the dependence of OpuA on the external osmolality (osmotic activation profile) was the same for both preparations (data not shown). Figure 2.Morphology of proteoliposomes. Cryo-EM was used to study liposomes composed of mixtures of DOPC, DOPE and DOPG (20 mg/ml, prepared in 100 mM KPi pH 7.0). Samples were prepared under iso- and hyperosmotic conditions; the latter was effected by the addition of 200 mM KCl. The iso- and hyperosmotic conditions correspond to 190 and 535 mosmol/kg, respectively. (A and D) Liposomes composed of DOPC/DOPE in a 1:1 mole ratio, under iso- and hyperosmotic conditions, respectively. (B and E) Liposomes composed of DOPC/DOPE/DOPG in a 3:4:1 mole ratio, under iso- and hyperosmotic conditions, respectively. (C and F) Liposomes composed of DOPC/DOPE/DOPG in a 1:4:3 mole ratio, under iso- and hyperosmotic conditions, respectively. The vast majority of the proteoliposomes were unilamellar, and the appearance of a spherical shape inside a vesicle (E and F) generally represents a top view of an invaginated liposome. Download figure Download PowerPoint Membrane-permeable versus membrane-impermeable osmolytes It has been argued that membrane-impermeable osmolytes such as KCl and sucrose activate osmoregulated transporters by a mechanism different from that of membrane-permeable osmolytes, e.g. glycerol and low molecular weight polyethylene glycols (PEGs) (Racher et al., 2001). To test this suggestion experimentally, the effects of glycerol on the kinetics of osmotic activation of OpuA and on the changes in volume and macroscopic structure of the proteoliposomes were determined. In the presence of 270 mM glycerol, the amount of glycine betaine taken up after 30 s was 30% higher than that of the iso-osmotic control sample. The stimulation by glycerol was not observed when the osmolyte was added 1 min prior to the transport measurements (data not shown). Thus, glycerol elicited a transient activation of OpuA that paralleled the time dependence of the shrinkage of the (proteo)liposomes (Figure 3). The transient changes in liposome volume were confirmed by cryo-EM (data not shown). The kinetics of OpuA activation by glycerol reflect the transient osmotic stress imposed by this highly membrane-permeable osmolyte. We conclude that the mechanism underlying osmotic activation of OpuA is not dependent on the type of osmolyte used to challenge the system. Figure 3.The effect of membrane-permeable versus membrane-impermeable osmolytes on the internal volume of proteoliposomes. Calcein quenching was assayed in 100 mM KPi pH 7.0. The proteoliposomes were composed of 25 mol% DOPC, 50 mol% DOPE and 25 mol% DOPG. Iso-osmotic (1) and hyperosmotic conditions correspond to 190 and 390 mosmol/kg, respectively. To impose hyperosmotic conditions, 200 mM KCl (4), 270 mM glycerol (2) or 327 mM sucrose (3) were added to the sample after 1 min pre-incubation (indicated by an arrow). Download figure Download PowerPoint Effect of anionic lipids on the activation profile of OpuA To investigate whether osmotic activation of OpuA is dependent on a particular lipid composition, lipids were used that varied in headgroup size (lamellar versus non-lamellar lipids), charge (anionic versus zwitterionic/ neutral lipids), acyl chain length (14–22 carbon atoms) and position and configuration of the unsaturated bond in the acyl chain. The lipids predominantly used were phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylcholine (PC) and phosphatidylethanolamine (PE). To determine the specific effects of anionic lipids on OpuA activity, the protein was reconstituted in proteoliposomes composed of the dioleoyl (18:1 Δ9 cis) derivatives DOPC (zwitterionic, lamellar), DOPE (zwitterionic, non-lamellar) and DOPG or DOPS (anionic, lamellar) in the mole ratios 8:8:0, 7:8:1, 6:8:2, 4:8:4, 2:8:6 and 0:8:8, yielding proteoliposomes with 0, 6, 13, 25, 38 and 50 mol% of anionic lipid, respectively. Figure 4A shows that OpuA was active under iso-osmotic conditions and that an osmotic upshift did not stimulate the activity with 6–13 mol% of anionic lipid present (only the data for 13 mol% DOPG are shown). OpuA had low or no activity under iso-osmotic conditions when the membrane contained ≥25 mol% DOPG. From 25 to 50 mol% anionic lipid, the activation threshold (trans-membrane osmotic gradient needed for activation) of OpuA was shifted to higher values. The same trend was observed for the value of the trans-membrane osmotic gradient at which maximal activity was reached. The fact that these effects were observed with both DOPG and DOPS (data not shown) suggests that the activation threshold is determined by the fraction of anionic lipids rather than by a specific lipid requirement. No activity was observed in proteoliposomes composed of DOPC or DOPC plus DOPE. Figure 4.The effect of anionic lipids on the osmotic activation of OpuA. (A) Uptake of [14C]glycine betaine (final concentration, 76 μM) was assayed in 100 mM KPi pH 7.0. The proteoliposomes were composed of 50 mol% DOPE and 0 (circles), 13 (upright triangles), 25 (inverted triangles), 38 (diamonds) or 50 mol% DOPG (squares) plus 50, 37, 25, 12 or 0 mol% DOPC, respectively. (B) The proteoliposomes were composed of 50 mol% DOPE and 13 (upright triangles), 25 (inverted triangles) or 38 mol% DOPG (diamonds) plus 37, 25 or 12 mol% DOPC, respectively, and were obtained via fusion of proteoliposomes (DOPE/DOPG, 1:1 mole ratio) with liposomes of the appropriate composition in a 1:1 mole ratio. The osmolality of the medium was varied with KCl. Δ Osmolality refers to the difference in external and internal osmolality. Download figure Download PowerPoint To exclude the possibility that differences in activity were due to variations in reconstitution efficiencies, proteoliposomes consisting of DOPC/DOPE/DOPG (1:2:1 mole ratio) were fused in a 1:1 ratio with liposomes composed of DOPC/DOPE (1:1 mole ratio), DOPC/DOPE/DOPG (1:2:1 mole ratio) and DOPE/DOPG (1:1 mole ratio), resulting in proteoliposomes containing 50 mol% DOPE plus 13, 25 and 38 mol% DOPG, respectively. These fusion experiments yielded activation profiles similar to those observed with the membrane reconstitutions of OpuA in the separate lipid mixtures (Figure 4B). Fusion of proteoliposomes composed of DOPC or DOPC plus DOPE with DOPG liposomes did not result in significant transport activity despite the fact that all OpuA protein was associated with the vesicles. We conclude that the absence of anionic lipids in the reconstitution process results in trapping of the protein into an inactive state. Finally, comparable activation profiles of OpuA were observed with ionic (KCl, NaCl, KPi or K2SO4) and neutral osmolytes (sucrose) used to vary the external osmolality (data not shown). This indicates that the change in the external ionic strength is not responsible for the observed effects, but that changes in the activation of OpuA are due solely to osmotic effects. It is important to stress here that the majority of the experiments were performed at a relatively high ionic strength (iso-osmotic conditions correspond to 190 mosmol/kg or 100 mM KPi, pH 7.0, externally). Effect of non-bilayer lipids on the activation profile of OpuA To determine the effects of non-bilayer lipids on OpuA activity, the fraction of DOPE was varied between 0 and 50 mol% with DOPG at a constant 38 mol% (Figure 5). From these experiments, it is concluded that DOPE is essential for high activity of OpuA, but the non-bilayer lipid does not affect the activation threshold of OpuA or the value of the trans-membrane osmotic gradient at which maximal activity is reached. To assess further the requirement of OpuA for non-bilayer lipids, the effect of one (mono-methyl-DOPE) or two (di-methyl-DOPE) additional methyl groups on the ethanolamine headgroup of DOPE was investigated. The larger the size of the headgroup, the higher the propensity to form stable bilayer structures. It was observed that DOPE, mono-methyl and di-methyl DOPE, in combination with a fixed concentration of DOPG, are decreasingly effective in stimulating OpuA activity (data not shown). These observations confirm the notion that non-bilayer-forming lipids are needed for maximal activity of OpuA but do not affect the activation mechanism. Figure 5.The effect of non-lamellar lipids (DOPE) on the osmotic activation of OpuA. The proteoliposomes were composed of 38 mol% DOPG and 6 (circles), 13 (squares), 25 (upright triangles), 38 (inverted triangles) or 50 mol% DOPE (diamonds) plus 56, 49, 37, 24 or 12 mol% DOPC, respectively, and were obtained via fusion of proteoliposomes (DOPC/DOPE/DOPG, 2:1:1 mole ratio) with liposomes of the appropriate composition in a 1:3 mole ratio. Experimental details are as described in the legend to Figure 4. Download figure Download PowerPoint Effect of acyl chain length and configuration/position of the unsaturated bond on the activity of OpuA Besides the headgoup region, the hydrophobic core of the lipid bilayer could influence the activity and/or the osmotic activation profile of OpuA. To determine the effect of the acyl chain length (membrane thickness) on OpuA activity, membrane reconstitution was performed with liposomes consisting of 25 mol% DOPG, 25 mol% DOPE and 50 mol% PC with an acyl chain length of either 14, 16, 18, 20 or 22 carbon atoms (cis-unsaturated at the Δ9 position). The activation profiles of OpuA were not affected by the differences in acyl chain length, but the absolute activity reached a maximum in the composition with a PC acyl chain of 18 carbon atoms (data not shown). The osmotic activation profile of OpuA was also not significantly affected when PC (18:1 trans-unsaturated at the Δ9 position), PG (18:1 trans-unsaturated at the Δ9 position) or PE (18:1 cis-unsaturated at the Δ6 position) were used instead of the corresponding dioleoyl (all 18:1 cis-unsaturated at the Δ9 position) lipids. Taken together, this data set indicates that changes in the physicochemical properties of the hydrophobic core of the lipid bilayer are not a determining factor in the osmotic activation mechanism of OpuA. Effect of amphiphilic molecules on OpuA activity Since activation of OpuA is strongly dependent on the fraction of anionic lipids present in the membrane, experiments were performed with small charged amphiphilic molecules that partition into the lipid bilayer. Insertion of a cationic or anionic amphipath would lead to a decrease or increase, respectively, in the bulk negative charge at the membrane surface, while neutral amphipaths are predicted to have little effect. With proteoliposomes composed of DOPC/DOPE/DOPG in the mole ratios 2:1:1 and 0:1:1, low concentrations of the cationic amphipath tetracaine in the assay medium indeed decreased the activation threshold of OpuA (Figure 6A and B; data from experiments with other lipid compositions and/or tetracaine concentrations not shown). The synergy between ionic strength and the presence of tetracaine was more pronounced at high than low mole fractions of DOPG (e.g. compare Figure 6A and B), suggesting that the effects of tetracaine on OpuA activity may be due not only to perturbation of the membrane surface charge. Figure 6B shows that pre-loading of the proteoliposomes with tetracaine resulted in a more pronounced shift of the activation threshold of OpuA than when the amphipath was added 1 min prior to the initiation of the transport reaction. This most probably reflects the time needed for tetracaine to equilibrate over the two membrane leaflets. Importantly, and opposite to the effects of tetracaine, the anionic amphipath capric acid (n-decanoic acid) shifted the activation threshold of OpuA to higher osmolalities (Figure 6A). Figure 6.The effect of membrane-active lipophilic compounds on OpuA activity. (A) Proteoliposomes were composed of DOPC/DOPE/DOPG in a 2:1:1 mole ratio. Transport activity was assayed without (circles) or with tetracaine (squares; cationic; 2 mM), decane (upright triangles; neutral; 4.2 mM) or capric acid (inverted triangles; anionic; 0.7 mM) present in the assay medium. (B) Proteoliposomes were composed of DOPE/DOPG in a 1:1 mole ratio. Transport activity was assayed in proteoliposomes, non-loaded (circles, squares) or pre-loaded (triangles) with 2 mM tetracaine, in the presence (squares, triangles) or absence (circles) of 2 mM tetracaine in the assay medium. In the case of the non-loaded proteoliposomes, the lipophilic compounds were added to the proteoliposomes 1 min prior to the initiation of the transport assay. Experimental details are as described in the legend to Figure 4. Download figure Download PowerPoint Tetracaine, at a concentration (2 mM) that increased the iso-osmotic activity ∼8-fold (Figure 6A), did not affect the morphology or surface to volume ratio of the proteoliposomes (data not shown). In these experiments, OpuA thus is not activated via direct changes in water activity, and vesicle shrinkage is not occurring. Since the cationic amphipath tetracaine remains mostly at the phospholipid headgroup level, it is conceivable that the charged form of the anesthetic altered the existing intra- and intermolecular electrostatic interactions (Boulanger et al., 1981). It is plausible that the same is true for the anionic amphipath n-decanoic acid, which shifted the activation profile to higher osmolalities, comparable with an increased fraction of anionic lipids in the membrane. The opposite effects, displayed by cationic and anionic amphipaths, support the view that electrostatic interactions between protein and membrane lipids are intrinsic to the mechanism of osmotic activation. The neutral amphipath lyso-PC and the aliphatic hydrocarbon n-decane (Figure 6A) did not affect the activation profile of OpuA. This suggests that the physical insertion of amphipaths in the lipid bilayer is not sufficient for osmotic activation of OpuA. Additionally, it is known that compounds such as tetracaine and n-decane increase the membrane fluidity (Salesse et al., 1982; Ueda and Yoshida, 1999), which on the basis of our work does not seem critical for osmotic activation of OpuA. Furthermore, the tetracaine-induced activation of glycine betaine transport is paralleled by a corresponding increase in the ATPase activity (data not shown), which indicates that the coupling between transport and ATP hydrolysis is maintained. High ionic strength at the cytoplasmic face of OpuA activates the transporter under iso-osmotic conditions Since the volume of proteoliposomes decreases upon osmotic upshift, the luminal concentrations of osmolytes increase. If ionic interactions between lipids and protein are intrinsic to the osmosensing mechanism of OpuA, then increases in the intravesicular concentration of ionic osmolytes should affect the activation profile. To discriminate between ionic strength, osmolality and specific osmolyte effects, the intravesicular composition and concentration were varied with the compounds NaCl, KCl, sucrose, fructose, KPi (pH 7.0), NaPi (pH 7.0), K2SO4 and Na2SO4. Each of these osmolytes was included in the vesicle lumen at a concentration corresponding to 100 mosmol/kg and was present in addition to the standard components (50 mM KPi pH 7.0, plus the ATP-regenerating system), resulting in a total internal osmolality of 290 mosmol/kg. Figure 7A and B shows that each of the ions stimulated OpuA activity, whereas the neutral osmolytes had no effect. Representative data for the osmotic activation profiles of OpuA in proteoliposomes with 17 (Figure 7A) and 25 mol% (Figure 7B) DOPG are shown. Equi-osmolar concentrations of KPi and K2SO4 proved to be more effective in stimulating OpuA activity than the monovalent KCl and NaCl; K+ and Na+ proved to be equally effective as cation. To determine the effect of ionic strength on the iso-osmotic activity of OpuA, increasing concentrations of potassium phosphate were enclosed in the proteoliposomal lumen, thereby varying the internal osmolality from 170 to 290 mosmol/kg. Figure 7C shows the dependence of the iso-osmotic activity of OpuA on the internal potassium phosphate concentration. A number of conclusions can be drawn from these experiments. First, OpuA seems activated specifically by high ionic strength in the vesicle lumen, which indicates that OpuA does not sense water activity or a specific signaling. Secondly, the shifts in the activation profile of OpuA are similar to those observed when the surface charge of the membrane is altered by varying the fraction of anionic phospholipids or the insertion of charged amphiphiles, which strongly suggests that OpuA senses osmotic stress via alterations in the ionic interactions between protein and bilayer lipids. Figure 7.The effect of ionic strength on the osmotic activation of OpuA. Uptake of [14C]glycine betaine (final concentration, 76 μM) was assayed in 100 (172 mosmol/kg) or 150 mM (258 mosmol/kg) KPi pH 7.0. Proteoliposomes were composed of DOPC/DOPE/DOPG in a 58:25:17 (A) and 50:25:25 (B) mole ratio. The standard components (circles, 172 mosmol/kg) plus additional KPi (upright triangles), KCl (squares) or sucrose (inverted triangles) (total of 258 mosmol/kg) were enclosed in the proteoliposomal lumen. (C) Uptake of [14C]glycine betaine (final concentration, 76 μM) was assayed in KPi pH 7.0 (equi-osmolal to the intravesicular osmolality). Proteoliposomes were composed of DOPC/DOPE/DOPG in a 50:25:25 mole ratio. Potassium phosphate pH 7.0 was enclosed in the proteoliposomal lumen at the concentrations indicated. Download figure Download PowerPoint High ionic strength at the cytoplasmic face of OpuA increases the ATPase activity A caveat of the experiments in which the luminal contents of the proteoliposomes were varied is that separately prepared proteoliposome samples were compared. To characterize the ionic strength dependence of OpuA further, glycine betaine-dependent ATPase activity was measured. Since a fraction of OpuA is inserted into the proteoliposomes in the ‘inside-out’ orientation, we used this population of molecules to monitor the ATPase activity of the system as a function of external osmolality and ionic strength. Table I shows that the ATPase activity of OpuA, in proteoliposomes consisting of DOPC/DOPE/DOPG in a 2:1:1 mole ratio, required glycine betaine (in the vesicle lumen with this ‘inside-out’ system) and a relatively high ionic strength on the outside (the cytoplasmic face of the protein). The ATPase activity increased up to a KPi concentration of 200 mM (Table I). Above 200 mM, KPi became inhibitory in the ATPase assay, a phenomenon also observed when transport activity was measured (data not shown). The activation of ATPase by ionic osmolytes confirms the observations made with the transport assays, and indicates that in vivo osmotic stress is signaled to the protein via alterations in the intracellular ionic strength. A tight coupling between ATPase and transport activity is suggested by the requirement for (internal) glycine betaine when (external) ATPase activity was assayed. Table 1.