CHAPTER 13 – STRUCTURE AND FUNCTION OF OSMOREGULATED ABC TRANSPORTERS

CHAPTER 13 – STRUCTURE AND FUNCTION OF OSMOREGULATED ABC TRANSPORTERS CHAPTER 13 – STRUCTURE AND FUNCTION OF OSMOREGULATED ABC TRANSPORTERS CHAPTER 13 – STRUCTURE AND FUNCTION OF OSMOREGULATED ABC TRANSPORTERS CHAPTER 13 – STRUCTURE AND FUNCTION OF OSMOREGULATED ABC TRANSPORTERS CHAPTER 13 – STRUCTURE AND FUNCTION OF OSMOREGULATED ABC TRANSPORTERS CHAPTER 13 – STRUCTURE AND FUNCTION OF OSMOREGULATED ABC TRANSPORTERS

263 13 CHAPTER STRUCTURE AND FUNCTION OF OSMOREGULATED ABC TRANSPORTERS BERT POOLMAN AND TIEMEN VAN DER HEIDE INTRODUCTION GENERAL In their natural habitats, microorganisms are often exposed to osmolality changes in the environment For instance, soil bacteria such as Bacillus subtilis are alternately exposed to periods of drought and rain, to which they have to adapt Since the cytoplasmic membrane of bacteria is highly permeable to water but forms an effective barrier for most solutes present in the medium and metabolites present in the cytoplasm, water will flow out of the cell when the outside osmolality increases (‘osmotic upshift’) As a consequence of an osmotic upshift, the turgor pressure will decrease and ultimately the cell may plasmolyze Upon osmotic downshift, water will flow into the cell and thereby increase the turgor pressure Maintenance of cell turgor is a prerequisite for almost any form of life, as it provides a mechanical force for the expansion of the cell envelope and regulates cell growth Generally, (micro)organisms respond to an osmotic upshift by rapidly accumulating compatible solutes to prevent the loss of water and loss of turgor pressure Upon osmotic downshift, the cells need to rapidly export the solutes to prevent the turgor pressure becoming too high, which, ultimately, may lead to breakage of the cells Since changes in protein expression (biosynthesis) are relatively slow, it ABC Proteins: From Bacteria to Man ISBN 0-12-352551-9 is evident that the primary response to osmotic stress needs to be one in which transport systems or channel proteins already present in the cytoplasmic membrane are activated in order to accumulate or release solutes (Glaasker et al., 1996a, 1996b) This implies that osmotic stress must be sensed by these systems and converted into a change in the appropriate activity such that the osmotic imbalance is rapidly restored Most osmotically controlled uptake systems are regulated at both the genetic (induction of gene expression) and the enzymatic level (direct ‘activation’ of the transport protein) The degree of induction can vary considerably (from 2- up to 500-fold), whereas in vivo activation of the transport protein by an osmotic upshift is usually in the range of 5- to 35-fold The transport systems known to be activated by osmotic upshift are either ATP-binding cassette (ABC) transporters or so-called secondary transporters, that is, transport proteins driven by an electrochemical ion gradient (Wood, 1999) The majority of both types of these energy coupling mechanisms have the quaternary ammonium compound glycine betaine as the preferred (high affinity) substrate Wellcharacterized osmotic downshift activated systems are the mechanosensitive channels, most notably the MscL protein, which open and thereby release osmolytes when the turgor pressure becomes too high This chapter only summarizes our knowledge of the ABC-type osmoregulated transporters Copyright 2003 Elsevier Science Ltd All rights of reproduction in any form reserved 264 ABC PROTEINS: FROM BACTERIA TO MAN BOX 13.1 DEFINITIONS The term hyperosmotic stress is often used to indicate an increased osmolality of the external medium This is somewhat confusing as the cytoplasm of growing cells in complete osmotic equilibrium is generally hyperosmotic relative to the outside, even after an osmotic upshift Therefore, we prefer to use the terms osmotic upshift and downshift for conditions that are often referred to as hyper- and hypo-osmotic stress Turgor pressure (⌬P) is the hydrostatic pressure difference that balances the difference in internal and external osmolyte concentration ⌬P ϭ (RT/Vw) ln(ao/ai) Ϸ RT(ci Ϫ co) in which Vw is the partial molal volume of water, a is the water activity, c is the total osmolyte concentration, and the subscripts i and o refer inside and outside, respectively A cell plasmolyzes when ⌬P becomes negative Osmolality is the osmotic pressure of a solution at a particular temperature, expressed as moles of solute per kilogram of solvent (osmol kgϪ1 or osmolal) The often used term osmolarity is an approximation for osmolality and is expressed as osmol lϪ1 or osmolar (for a more extensive overview of definitions, see Wood, 1999) BOX 13.2 COMPATIBLE SOLUTES Compatible solutes are molecules that can be accumulated to high cytoplasmic (often molar) concentrations without affecting vital cellular processes In many organisms, the compatible solutes glycine betaine and carnitine (and other quaternary ammonium compounds) offer the highest protection against osmotic stress Glycine betaine CH3 ϩ N CH3 CH3 Carnitine Ϫ COO CH3 ϩ N under high osmolality conditions, but it is difficult to derive a quantitative relationship between the internal concentrations of compatible solutes and the external osmolality as multiple (macro)molecules are involved The compatible solutes to be accumulated to high intracellular levels are restricted to a few categories (for reviews see Csonka, 1989; Csonka and Hanson, 1991; Galinski and Trüper, 1994), and they include (i) potassium ions, (ii) amino acids (glutamate, alanine, proline), (iii) amino acid analogues (taurine, N-acetylglutaminylglutamine amide), (iv) methyl-amines and related compounds (glycine betaine, carnitine, ectoines), and (v) polyols and sugars (glycerol, sucrose, lactose) In general, compatible solutes should not interact specifically with the (mostly negatively charged) cellular macromolecules, nor should they perturb cytoplasmic solutes via (de)hydration, precipitation, or any other (charge) interaction Therefore, under steady state conditions most compatible solutes that are present in large amounts in the cytoplasm have no net charge The accumulation of potassium ions in response to an osmotic upshift in enteric bacteria is usually only transient Following the accumulation of potassium ions, other compatible solutes are synthesized (e.g trehalose), and the uptake systems for glycine betaine and proline are induced The accumulation of these solutes then eventually replaces potassium (see also Poolman and Glaasker, 1998) An exception to the rule of preferring neutral compatible solutes is found in thermophilic organisms (Bacteria and Archaea), where negatively charged compounds seem to be accumulated in response to osmotic upshifts These compounds include mannosylglycerate, glutamate, cyclic-2,3-bisphosphoglycerate, 1,3,4,6-tetracarboxyhexane, and myo-inositolphosphate derivates (Martins and Santos, 1995; Martins et al., 1996) Ϫ OH COO CH3 CH3 RATIONALE FOR THE ACCUMULATION OF COMPATIBLE SOLUTES After an osmotic upshift, cells benefit from the accumulation of compatible solutes by balancing the osmotic disturbance via an increase in cytoplasmic osmolality High intracellular concentrations of compatible solutes are common STRUCTURAL ANALYSIS OF OSMOREGULATED ABC TRANSPORTERS Typical ABC-type binding protein-dependent transporters are composed of five protein(s) (domains), that is, an extracellular binding protein (receptor), two ATP-binding subunits and two integral membrane subunits The integral membrane subunits provide the translocation STRUCTURE AND FUNCTION OF OSMOREGULATED ABC TRANSPORTERS Gram-negative bacteria X Gram-positive bacteria C X C Out W V W V B B A A BC A A ATP ATP ATP In ATP ATP ProUEc ATP OpuABS BC OpuALI Figure 13.1 Structural organization of osmoregulated ABC transporters Depicted are the ProU system from E coli, OpuABs from B subtilis and OpuALl from L lactis In Gram-negative bacteria, the binding protein is present in the periplasm, whereas in Gram-postive bacteria, the protein is anchored to the outer surface of the membrane via a lipid moiety (OpuAC) or fused to the translocator domain (OpuABC) For ProX and OpuAC, the equilibrium between unliganded and liganded binding protein is depicted pathway Except for the substrate-binding protein, the other subunits can be present as distinct polypeptides or fused to one another but always each entity is present twice The osmoregulated ABC transporters belong to the OTCN family (Dassa and Bouige, 2001), of which the members transport substrates as diverse as quaternary ammonium compounds (glycine betaine, carnitine), proline, alkylsulfonates and -phosphonates, phosphites, cyanate, and nitrate Although biochemical evidence is not available, the osmoregulated ABC transporters most probably have two identical copies of the ATPbinding subunit This suggestion is based on the finding that, in contrast to other families of ABC transporters, the operons specifying osmoregulated transporters only have a single gene for an ATP-binding subunit The integral membrane components are either present as two identical copies or two homologous proteins, but they are not fused to each other or to the ATP-binding subunit (Figure 13.1) The ligand-binding subunit is present as a free protein in the periplasm of Gram-negative bacteria, whereas in Gram-positive bacteria the protein can be anchored to the membrane through a fatty acid modification of the amino-terminal cysteine (Kempf et al., 1997; Sankaran and Wu, 1994) Recently, it has been shown that a subset of ABC transporters has the ligand-binding protein fused to the integral membrane subunit (Figure 13.1) OpuA1 of Lactococcus lactis is the only well-studied representative of this subset of binding protein-dependent ABC transporters, but database searches indicate that similar systems are present in Streptomyces coelicolor, Streptococcus pneumoniae, Streptococcus pyogenes, Chlamydia pneumoniae, Helicobacter pylori and Staphylococcus aureus By analogy with other ABC transporters, functional OpuA will most likely be composed of two integral membrane subunits and two ATP-binding subunits Since the translocator subunit is fused to the substrate-binding protein (OpuABC subunit), the oligomeric structure implies that two receptor domains are present per functional complex This raises questions about the observations that only a single substrate-binding protein interacts with the dimeric membrane complex of an ABC transporter, and that two lobes of a single substrate-binding protein interact with different integral membrane protein(s) (domains) (Ehrmann et al., 1998; Liu et al., 1999) OpuA of L lactis is composed of two different types of subunits, that is, the ATPase subunit OpuAA and the chimeric ligand-binding/translocator protein OpuABC OpuA of B subtilis has three different subunits, OpuAA, OpuAB and OpuAC, and here the translocator (OpuAB) and binding protein (OpuAC) are separate polypeptides (see also Figure 13.1) To discriminate the L lactis system from OpuA of B subtilis, we use the subscripts Ll and Bs, respectively 265 266 ABC PROTEINS: FROM BACTERIA TO MAN THE ABC COMPONENT The ABC or ATPase component of known osmoregulated ABC transporters has a molecular mass of about 45 kDa Similar to the ATPase subunit (MalK) of the maltose transporters of Escherichia coli and Thermococcus litoralis (Diederichs et al., 2000), the ABC component of most osmoregulated systems consists of an amino-terminal ␣/␤ type ATPase domain (ϳ27 kDa) and a carboxyl-terminal regulatory domain (ϳ18 kDa) The carboxyl-terminal domain in MalK of E coli provides the system with a means to control the transport activity as well as the expression of the mal genes through interaction of MalK with regulatory proteins (Chapter 9) Whether the regulatory domain of the ATPase of the osmoregulated ABC transporters has such a role is not known Database searches indicate that some homologues of the family of osmoregulated ABC transporters have an ATPase component with a truncated regulatory domain These systems, e.g ChoQ of L lactis, have a molecular mass of about 35 kDa THE INTEGRAL MEMBRANE COMPONENT Hydropathy profiling of the sequences of the integral membrane components of the osmoregulated ABC transporters shows that the systems fall in four subfamilies (Figure 13.2) Each Subfamily I of the transporters has the conserved EAA motif in the equivalent cytoplasmic loop (loop V–VI in subfamilies I and III and loop III–IV in subfamilies II and IV, see Figure 13.2) Members of subfamily I, represented by ProW of the E coli ProU system and OpuAB of B subtilis OpuABs (Figure 13.1), are predicted to have seven transmembrane ␣-helical segments with the N-terminus facing the external surface of the membrane and the C-terminus facing the cytoplasm This membrane topology is supported by phoA and lacZ fusions, albeit a limited set of data (Haardt and Bremer, 1996) ProW differs from OpuABBs (and other subfamily I homologues) by having an unusually long amino-terminus of about 100 residues (depicted as dotted circle in Figure 13.2), which protrudes into the periplasmic space of E coli The members of subfamily II, represented here by OpuCB and OpuCD of the OpuC system of B subtilis, have five transmembrane ␣-helical segments with the N- and C-termini at the external and internal side of the membrane, respectively Thus, compared to the members of subfamily I, the first two transmembrane segments are missing Subfamily III, exemplified by OpuABC of the OpuA system of L lactis, is similar to subfamily I except that the member proteins have the ligand-binding domain fused to the integral membrane component (Figure 13.1) In this case, Subfamily II Out Out In In EAA I EAA II III IV V VI VII I Subfamily III II III IV V VI VII Subfamily IV Out Out In In EAA EAA I II III IV V VI VII VIII I II III IV V VI VII VIII Figure 13.2 Topology models of the members of the four subfamilies of osmoregulated ABC transporters The additional periplasmic domain in some members of subfamily I is indicated as a dotted circle The Pacman-like structure connected to the last transmembrane segment of the members of subfamilies III and IV represents the ligand-binding domain The EAA motif in the cytoplasmic loop is also depicted STRUCTURE AND FUNCTION OF OSMOREGULATED ABC TRANSPORTERS an eight transmembrane segment allows the binding domain to face the exterior of the cell It is worth noting that the majority of multidomain membrane transport proteins have the individual domains fused to another via so-called flexible linker regions (Sutrina et al., 1990) In the case of OpuA of L lactis, the predicted end of the eighth transmembrane segment is followed by a sequence that over its entire length is highly similar (Ͼ50% identity) to the ligand-binding protein ProX of the E coli ProU system On the assumption that the amino-terminus of ProX forms an intrinsic part of the binding protein, a flexible linker will not be present between the transmembrane and ligand-binding domains of the OpuABC polypeptide of the OpuA system Finally, members of subfamily IV, exemplified here by ProWX of H pylori, and the membrane component of the ChoQ complex of L lactis, are similar to those of subfamily II, except that also in this case the ligand-binding protein is fused to the integral membrane component This gives rise to a membrane protein with six predicted transmembrane ␣-helical segments and an amino- and carboxyl-terminus that are facing the outer surface of the membrane At present, it is unclear whether or not the smaller sizes of the proteins of subfamilies III and IV when compared to those of I and II are related to a different functioning of the systems THE SUBSTRATE-BINDING PROTEIN Members of subfamily I and II have a soluble substrate-binding protein that in Gram-negative bacteria resides in the periplasm In Grampositive bacteria, the ‘soluble’ binding protein of subfamily I and II type transporters is anchored to the outer surface of the cytoplasmic membrane via an amino-terminal lipid moiety Recently, ProX of E coli has been crystallized (Breed et al., 2001) and the structure, although not published, has been presented at scientific meetings As expected, ProX has an overall tertiary fold typical of ligand-binding proteins belonging to ABC transporters (Quiocho and Ledvina, 1996; see Chapter 10), which is indicative of a Venus fly-trap mechanism for substrate binding Interestingly, the substrate, glycine betaine, is bound to Trp residues via cation-␲ interactions similar to the observed binding of quaternary ammonium compounds to acetylcholine esterase of human brain (Bartolucci et al., 2001; Bremer and Welte, unpublished) The ␲-electrons interact with the quaternary ammonium group of glycine betaine One of the three Trp residues that interact with the substrate is highly conserved in the glycine betaine-binding proteins for which the primary sequences are available to date SPECIFICITY OF OSMOREGULATED ABC TRANSPORTERS The substrate specificity of osmoregulated (ABC) transporters has been investigated most extensively in B subtilis (Kempf and Bremer, 1998) Some systems seem very specific, e.g OpuB only selects choline, whereas others such as OpuABs accept a wide range of substrates It should be stressed, however, that in neither case has the specificity been determined directly, that is, as dissociation constants (Kd values) of ligand binding to the receptor The specificity of bacterial osmoregulated ABC transporters has been determined either as apparent affinity constants of transport (Km) or it has been inferred from the ability of a compound to offer protection during growth under hyperosmotic conditions Given our experience with this type of analysis for the oligopeptide-binding protein of the Opp ABC transporter (Detmers et al., 2000; Lanfermeijer et al., 1999, 2000), we expect that differences may be more subtle than suggested by the data presented in the literature Nevertheless, in terms of cell physiology, it is important to know if a compound does or does not offer protection against osmotic stress when taken up via a particular system The differences in narrow versus broad specificity are not readily revealed in the primary sequences of the ligandbinding proteins/domains when analyzed by multiple sequence alignments For Listeria monocytogenes and Lactobacillus plantarum, it has been shown that preaccumulated (trans) substrate inhibits the corresponding osmoregulated transport systems (Glaasker et al., 1998; Verheul et al., 1997) Upon raising the medium osmolality, the systems are rapidly activated through a diminished inhibition by trans substrate Once turgor pressure has been restored, the cells are in osmotic equilibrium again and the transporters need to be inactivated or switched off The so-called trans-inhibition may serve as a control mechanism to prevent the accumulation of these 267 268 ABC PROTEINS: FROM BACTERIA TO MAN compatible solutes to too high levels and thereby the turgor pressure from becoming too high In the case of L monocytogenes, carnitine is taken up via an ABC transporter that is specific for this substrate but is inhibited by high intracellular concentrations of both carnitine and glycine betaine (and perhaps other compatible solutes) depending on the osmotic status of the cells In kinetic terms, the osmotic activation of the system results in an increase in apparent inhibition constant (KI) for glycine betaine and carnitine at the inner surface of the membrane Apparently, as a consequence of the water efflux following an osmotic upshift, the internal binding site for glycine betaine and carnitine, of a system specific for carnitine in the uptake reaction, is altered Binding of compatible solutes to an internal site thus seems to represent a key step in the activation–inactivation mechanism There is yet no molecular data on the proposed regulatory binding site in the transporters, and it is not therefore clear whether this is in the translocator protein or the ATPase component OSMOTIC REGULATION OF EXPRESSION OF GENES ENCODING ABC TRANSPORTERS KINETICS OF OSMOTIC REGULATION AND OSMOTIC SIGNALS The ProU system of E coli is the best-studied ABC transporter in terms of osmotic regulation of gene expression, but despite intensive efforts, a full understanding of the molecular mechanism(s) underlying proU expression has not yet been achieved In a coupled transcription–translation system, the kinetics of induction of the proU operon involves a lag phase of 15 to 20 min, followed by a rapid increase in expression, and, subsequently, a slow decay in the expression rate (Jovanovich et al., 1989) This genetic regulation is slow in comparison to the activation of existing ProU (see below), which occurs within seconds following a change in osmolality Therefore, the increased expression of proU could be mediated by signaling molecules (second messengers) that have to build up in the cytoplasm, rather than by activation directly by signal transduction pathways Consistent with a role for specific second messenger molecules are observations that potassium glutamate is (at least partially) responsible for the induction of proU (Booth, 1992; Ramirez et al., 1989), but other studies have rejected these claims (Csonka et al., 1994; Jovanovich et al., 1989) It is now thought that the stimulation of transcription of proU in vitro by potassium glutamate is a manifestation of the generally favorable effect of these osmolytes on macromolecular function (e.g RNA polymerase–promoter interaction) and is not unique to osmotic regulation of the proU promoter (Csonka et al., 1994) Taken together, the data are consistent with changes in intracellular osmolality as the signal for proU transcription, which would lead to maximal levels of ProU at a time that the turgor has already (largely) been restored Such a signaling mechanism is in line with the observation that E coli responds to osmotic upshift by rapidly accumulating potassium glutamate and subsequent replacement of these ionic osmolytes for neutral ones such as the substrate of the ProU system TRANSCRIPTION FACTORS AND OSMOREGULATED PROMOTERS Contrary to what one would expect for a system that is tightly regulated by the osmolality of the medium (Ͼ500-fold induction), specific transcription factors not seem to be involved or at least they have not been discovered Genetic searches for such proteins have led to the isolation of mutants with defects in general DNA-binding proteins such as TopA, GyrAB, IHF, HU and H-NS (Kempf and Bremer, 1998) Mutations in these proteins have pleitropic effects on gene expression, for instance through alterations in DNA supercoiling, and it is unlikely that the tight osmotic control of the proU operon is solely mediated by these proteins On the basis of transcriptional analysis of the P1 promoter of the proP gene, which encodes an osmoregulated secondary proline transporter, the suggestion has been made that the cAMP receptor protein (CRP) could function as a general osmoregulator of transcription in E coli (Landis et al., 1999) Binding of CRP to a site within the proP P1 and some other promoters is destabilized after an osmotic upshift These studies imply that CRP could have a general osmoregulatory role in addition to its function in catabolite control Transcription of proU is effected via the promoters P1 (sigma factor ␴S) and P2 (sigma STRUCTURE AND FUNCTION OF OSMOREGULATED ABC TRANSPORTERS OSMOTIC REGULATION OF ACTIVITY OF ABC TRANSPORTERS KINETICS OF OSMOTIC (IN)ACTIVATION In order to cope effectively with osmotic stress, cells need osmotically controllable systems in the membrane at all times as de novo synthesis takes too long and is not adequate for a quick response In terms of an osmosensing/regulation mechanism, the only well-characterized ABC transport protein is OpuA of L lactis (van der Heide and Poolman, 2000b; van der Heide et al., 2001) The complete protein complex (OpuAA and OpuABC) has been purified and studied in proteoliposomes By including ATP plus an ATP-regenerating system inside the vesicle lumen (Figure 13.3), uptake of glycine betaine has been followed in response to osmotic shifts, as a function of membrane lipid AA AA ADP ATP ATP ADP AA ADP ATP AA factor ␴70) During exponential growth, transcription from P2 contributes most to the expression by employing ␴70 Sigma factor ␴S generally contributes to the expression of genes in the stationary phase of growth, but the transcription of proU is not significantly increased under these conditions The presence of potassium glutamate enhances transcription via ␴S and ␴70, and increases the selectivity of ␴S for P1 in vitro (Rajkumari et al., 1996) In organisms other than E coli, e.g B subtilis and L lactis, the expression of the genes specifying osmoregulated ABC transporters is also under osmotic control (Kempf and Bremer, 1998; van der Heide and Poolman, 2000a), but little is known about the signals and transcription factors that regulate expression In B subtilis, a general stress regulon is present, whose expression depends on the alternative sigma factor SigB (␴B) In addition to salt, heat, oxidative and pH stresses also affect the expression of the ␴B regulon (Wood et al., 2001) The induction of the ␴B regulon by osmotic upshift is only transient and ␴B-controlled proteins cannot adequately protect cells against prolonged high osmolality stress The structural genes for the glycine betaine (OpuD) and proline (OpuE) secondary transport proteins are members of the ␴B regulon, but there is no evidence that the osmoregulated ABC transporters (OpuABs, OpuB and OpuC) of B subtilis are under the control of ␴B The choline-specific transporter OpuB is under the control of the GbsR repressor, but this transcription factor is a choline sensor rather than an osmosensor/regulator (Bremer, 2002) Finally, it should be stressed that maximal rates of uptake via OpuABs and OpuC of B subtilis increase only 1.5- to 3-fold when 0.4 M NaCl is added to the growth medium (Kappes et al., 1996), indicating that the corresponding genes are not under tight osmotic control, as for example in the case of proU of E coli In L lactis IL1403, the glycine betaine uptake capacity increases more than 10fold when the cells are grown in the presence of 0.5 M KCl (van der Heide and Poolman, 2000a) The induction by osmotic stress, however, is only observed in chemically defined media and not in complex broth, suggesting that factors other than osmolality of the medium tune the expression levels to the needs of the cell A preliminary report on the regulation of expression of the ABC transporter OpuALl suggests that a transcriptional regulator of the GnrR family acts as a repressor of the opuA operon (Obis et al., 2000), but the signal sensed by the protein is not known ATP Creatine phosphate ϩADP ADP Creatine kinase Creatine ϩ ATP Figure 13.3 Proteoliposomal system to measure the activity of OpuA from L lactis The purified protein complex was inserted into preformed liposomes, after which excess detergent was removed by adsorption onto polystyrene beads The ATP-regenerating system (ATP, creatine kinase plus creatine phosphate) was included in the vesicle lumen by freeze-thawing, followed by sizing of the proteoliposomes by extrusion through polycarbonate filters with 200 nm pores (for details see van der Heide and Poolman, 2000b; van der Heide et al., 2001) 269 270 ABC PROTEINS: FROM BACTERIA TO MAN composition and various water stress related parameters It has been shown that OpuALl is activated instantaneously upon raising the osmolality of the medium, that is, when the external medium is made hyperosmotic relative to the inside Activation has been measured as an increase in either the rate of ATP-driven substrate uptake or the rate of substratedependent ATP hydrolysis; for the latter measurements, ATP rather than the ATP-regenerating system was included in the proteoliposomes Activation is elicited by ionic and non-ionic osmolytes, provided the molecules not equilibrate across the membrane on the time scale of the transport measurements (van der Heide and Poolman, 2000b) Since (proteo)liposomes act as osmometers, that is, water diffuses across the membrane in response to the osmotic difference between the inner compartment and the outside medium, proteoliposomes are expected to decrease their volume to surface ratio when the outside osmolality is increased The changes in membrane structure and lumen contents (osmolyte concentration) in osmotically stressed (proteo)liposomes can be compared with those in cells that are in a state of plasmolysis Proteoliposomes with an average diameter of 200 nm change their shape from spherical to sickle-shaped as shown by cryo-electron microscopy (cryo-EM) These morphological changes occurred within milliseconds, i.e on a time scale much shorter than the interval over which transport was measured Upon lowering the outside osmolality to the initial value, yielding iso-osmotic conditions again, the vesicles regained their spherical shape and the transporter was deactivated (van der Heide et al., 2001) Thus, osmotic activation and inactivation of OpuALl is entirely reversible, occurs on a time scale of seconds or less, and follows the shape and volume changes of the liposomes (see below, for proposed mechanism of osmosensing) In contrast to the OpuA system from L lactis, the hyperosmotic activation of ProU from E coli takes several minutes (Faatz et al., 1988), which makes it unlikely that decreased external water activity or reduced turgor pressure triggers the activation A closer look at the data suggests that activation of ProU is also dependent on the presence of the transport substrate (glycine betaine), as the transport activity increases up to after an osmotic upshift in the absence but not in the presence of glycine betaine (Faatz et al., 1988) Overall, the regulation of ProU transport activity seems different from that of OpuA of L lactis and resembles more that of the transcription of proU, i.e both are delayed upon an upshift It should be stressed here that OpuALl has been studied in a well-defined proteoliposomal system whereas ProU has been analyzed in intact cells, which complicates a direct comparison of the data MECHANISM(S) OF OSMOSENSING Osmotic activation of membrane transporters may be triggered through a change in the hydration state of the transport protein complex, resulting from the altered water activity (aw), or the signal may be transmitted to the protein via a specific signaling molecule or a change in the physicochemical properties of the surrounding membrane Since OpuA of L lactis can be activated not only by osmotic upshift but also by the insertion of cationic amphipaths in the membrane, it seems plausible that the membrane transduces the activation (van der Heide and Poolman, 2000b) The observation that an osmotic upshift leads to sickle-shaped vesicle structures already indicates that at the macroscopic level major changes take place at the membrane These morphological changes, however, not occur upon insertion of amphipaths into the membrane, indicating that the macroscopic alterations in membrane folding are not intrinsic to the osmosensing/regulation mechanism of OpuALl At the molecular level, physical properties such as membrane fluidity, bilayer thickness, hydration state of lipid headgroups, interfacial polarity and charge, and/or lateral pressure may vary with changes in the osmolality of the medium To define the osmotic signaling process in more detail, OpuALl has been incorporated into liposomes of different lipid composition (van der Heide et al., 2001) It was found that the fraction of anionic (charged) lipids is of major importance for the osmosensing mechanism, whereas variations in acyl chain length, degree of fatty acid saturation, position of the cis/trans double bond, and the fraction of non-bilayer lipids have relatively minor effects By varying the fraction of anionic lipids (phosphatidylglycerol (PG) or phosphatidylserine (PS)) from to 13 to 25%, OpuALl is converted from an inactive or tense (T) to an intermediate (I) to an osmotically controllable (R) state (Figure 13.4) Moreover, at a given mol% of PG, OpuALl can be converted from R to I by adding cationic amphipaths and from I to R with anionic amphipaths This suggests that the overall charge of the headgroup STRUCTURE AND FUNCTION OF OSMOREGULATED ABC TRANSPORTERS R T Inactive “Low” Cytoplasmic Ionic Strength I R Active “High” [Anionic Lipids] [Cationic amphiphiles] [Anionic amphiphiles] Figure 13.4 Schematic presentation of the factors affecting the osmotic activation of the membrane-embedded OpuA complex A low fraction of anionic lipids converts OpuA from an inactive (T) to an intermediate state (C) A further increase of the anionic lipid fraction leads to an osmotically controlled (R) state At the level of the membrane, the effect of anionic lipids can be mimicked by the addition of anionic or cationic membrane-active compounds The effects of cytoplasmic ionic osmolytes, shifting the system from an inactive to an active state, is also depicted region of the membrane lipids determines the activity (kinetic state) of the transporter (van der Heide et al., 2001) The change in volume/surface ratio of the proteoliposomes upon osmotic upshift leads to an increase in the concentration of intravesicular osmolytes By varying the intravesicular composition, it was observed that ionic osmolytes rather than internal osmolality switch the system from an inactive to an active state In other words, the system can be activated at isoosmotic conditions simply by an increase in the internal ionic strength Because the effects of the ions vary with the fraction of anionic lipids in the membrane, and the threshold for osmotic activation is lowered by cationic and raised by anionic amphipaths, these experiments support the notion that osmotic signaling most probably occurs via the bilayer in which the protein complex is embedded (van der Heide et al., 2001) NATURE OF THE ACTIVATING SIGNAL Why would the cell use ionic strength rather than intracellular osmolality (affecting protein hydration) or a specific signaling molecule (allosteric regulatory site on the protein)? When the osmolality of the medium is raised, the initial change in cytoplasmic water activity depends on the elasticity of the cell wall Contrary to what is often thought, the cell wall of bacteria is not rigid but actually quite elastic (Csonka and Hanson, 1991; Doyle and Marquis, 1994) Consequently, even at turgor pressures above zero, the cytoplasmic volume decreases with increasing external osmolality, and the ion (osmolyte) concentrations increase accordingly The increase in ionic strength accompanying the volume decrease is undesirable as too high concentrations of electrolytes interfere with macromolecular functioning in eubacteria as well as in higher organisms (Yancey et al., 1982) As best documented for E coli (Higgins et al., 1987; Record et al., 1998), eubacteria expel ionic compounds if the electrolyte concentration becomes too high and replace these molecules with neutral osmolytes such as glycine betaine to balance the cellular osmolality The increase in electrolyte concentration (or ionic strength) upon a modest decrease in turgor pressure would thus represent an excellent trigger (‘osmotic signal’) for the activation of any osmoregulated transporter for neutral compatible solutes such as OpuALl Actually, it would prevent the osmotic stress from turning into ‘electrolyte stress’ Why is the increase in intracellular osmolality less suitable as osmotic signal? In order to maintain a relatively constant turgor pressure at different external osmolalities, the cell will have to switch on OpuALl and take up glycine betaine with maximal activity at different internal osmolalities In other words, the ability of (the majority of) microorganisms to grow at maximal rate over a wide range of osmolalities of the medium implies that cellular processes function optimally over wide range of intracellular osmolalities Finally, the cell could use the osmotic upshift-dependent change in concentration of a specific molecule as signal, but ionic strength seems a more general signal for osmoresponsive systems, including signal transduction pathways to control the expression of osmoregulated genes Although further work is needed to elucidate all the intricacies of the osmosensing mechanism of the ABC transporter for glycine betaine in L lactis, the in vitro studies with the purified protein complex indicate that all the regulatory properties observed in vivo are present in the two polypeptides comprising OpuALl The fact that osmotic activation of OpuALl in proteoliposomes mimics the regulation in vivo may seem surprising as the vesicles not withstand turgor, whereas the turgor pressure in cells is several atmospheres However, there is mounting evidence that in bacteria turgor pressure 271 272 ABC PROTEINS: FROM BACTERIA TO MAN exists only across the cell wall (and outer membrane in the case of Gram-negative bacteria) and not across the cytoplasmic membrane (Cayley et al., 2000) This implies that osmosensing devices present in the cytoplasmic membrane cannot repond to changes in turgor pressure; rather they must sense the consequences of the water influx or efflux, e.g changes in ionic strength The consequences of osmotic shifts, experienced by proteins in membrane model systems such as proteoliposomses, may thus be similar to those in the in vivo situation Osmotic regulation of OpuALl requires the protein complex to be embedded in a membrane bilayer with the appropriate lipid composition (ϳ25% anionic and some non-bilayer lipids) and the appropriate concentration of small inorganic electrolytes in the vesicle lumen We cannot entirely rule out the possibility that additional proteinaceous factors influence the osmotic activation of OpuALl in vivo, as has been suggested for the proton motive force-driven proline transporter ProP (Kunte et al., 1999), but we not have any indications or need for such a factor to relate the in vitro and in vivo data CHALLENGES AND PERSPECTIVES From the studies presented in the previous section, it is evident that osmosensing and regulation, be it a transporter, channel or signaling pathway, can only be fully understood if the system can be analyzed not only in the intact cell but also in artificial membrane systems with defined components The ABC transporter for glycine betaine has some unique properties that make the protein complex ideally suitable for in vitro analyses Firstly, the system has the ligand-binding domain covalently linked to the translocator moiety, which simplifies purification of the system and results in a high efficiency of the transport reaction without having to use a large excess of (soluble) ligand-binding protein Secondly, the subunits of the OpuA complex (OpuAA and OpuABC) remain associated in the detergent-solubilized state, which greatly facilitates reconstitution in proteoliposmes Thirdly, the system has a high catalytic efficiency, which enables us to measure accurately activities even below 5% of Vmax The research on OpuA from L lactis work indicates that a change in intracellular ionic strength serves as primary signal of osmotic stress for this ABC transporter We propose that this signal is not sensed by the protein directly, i.e via changes in surface hydration of the protein or direct effects of the ions on the protein (allosteric site); rather the membrane in which the protein is embedded serves as a mediator Changes in cellular ionic strength are likely to alter specific interactions between (ionic) lipids and the protein, thereby affecting the transport activity The ATPase and translocation activity of the OpuA transporter are strictly coupled, and, at present, it is unclear whether the ATPase (OpuAA) or the membrane-embedded part (translocator) of OpuABC is the actual sensor A challenge will now be to assign specific regions or residues in the OpuALl protein complex as sites that actually sense the changes in membrane structure (e.g electrostatic interactions between protein and phospholipid molecules), and to translate the in vitro observations into proposals that can be experimentally tested in vivo Moreover, one may wish to study osmotic signaling pathways that affect the transcription of genes in a manner analogous to the studies described heretofore for the ABC transporter OpuALl Past research has been restricted to mutant selection and isolation, and construction of allelic strains defective in one or more transcription factors The proU regulatory circuit is clearly too complicated for a complete understanding of its osmoregulatory mechanism at this moment, but it is possible to devise protocols for the in vitro analysis of simpler pathways, e.g two-component regulatory systems One would also like to know how the cell responds to osmotic stress not only at the level of activation of transcription and transporter activity but also at the level of lipid synthesis The membrane bilayer composition is intrinsic to the osmosensing mechanism of OpuALl and most likely other transporters as well (Rübenhagen et al., 2000; Wood et al., 2001), and changes therein will on the longer time scales be important for volume control of the cell Preliminary studies have been reported on the changes in fatty acid composition of the membrane of L lactis in relation to osmotic stress (Guillot et al., 2000) Unfortunately, no information is available on variations in headgroup composition, e.g fractions of anionic lipids, as these factors seem far more important for the osmoregulation of OpuALl than acyl chain length, degree of saturation, etc Finally, the relationship between osmotic and other stresses needs to be evaluated more thoroughly at the level of transporter activity (in vitro and STRUCTURE AND FUNCTION OF OSMOREGULATED ABC TRANSPORTERS in vivo) and cellular glycine betaine accumulation levels Note added in proof: Recent analysis of genomic databases has indicated that some homologues of OpuA of L lactis have two substrate-binding domains fused in tandem to the translocator moiety of the ABC transporter These systems thus have four extracellular substrate-binding sites per functional complex For a full description of the newly discovered chimeric substrate-binding/translocator proteins, one is referred to van der Heide and Poolman (2002) EMBO reports, in press ACKNOWLEDGMENTS This work was supported by grants from the Netherlands Organization for Scientific Research (NWO) under 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