Diversity of transport mechanisms: common structural principles.

Traditionally, prokaryotic solute transport systems are classified into major groups based on the energetic requirement of the transport process. These include the secondary transporters that are driven by a proton or sodium motive force, and the ATP-binding cassette (ABC) primary transporters, which use the hydrolysis of ATP to fuel transport. These transporters are specified by entirely different architectures of polypeptides. Recently, transport systems have been discovered that are composed of combinations of distinct functional modules of both secondary and ABC transporters. These findings indicate that during evolution the combination of integral membrane transport proteins with either a periplasmic solute-binding protein or a cytosolic ATPase, or both, have resulted in distinct classes of transporters with unique architectures and properties.

Glutamate transporters combine transporter- and channel-like features.

Glutamate transporters in the mammalian central nervous system have a unique position among secondary transport proteins as they exhibit glutamate-gated chloride-channel activity in addition to glutamate-transport activity. In this article, the available data on the structure of the glutamate transporters are compared with high-resolution crystal structures of channel proteins. In addition, binding-site properties of glutamate transporters, and the ligand-binding site of an ionotropic glutamate receptor of which the crystal structure is known, are compared. Possible structural solutions for the combination of channel and transporter activity in one membrane protein are proposed.

Structural features of the glutamate transporter family.

Neuronal and glial glutamate transporters remove the excitatory neurotransmitter glutamate from the synaptic cleft and thus prevent neurotoxicity. The proteins belong to a large and widespread family of secondary transporters, including bacterial glutamate, serine, and C4-dicarboxylate transporters; mammalian neutral-amino-acid transporters; and an increasing number of bacterial, archaeal, and eukaryotic proteins that have not yet been functionally characterized. Sixty members of the glutamate transporter family were found in the databases on the basis of sequence homology. The amino acid sequences of the carriers have diverged enormously. Homology between the members of the family is most apparent in a stretch of approximately 150 residues in the C-terminal part of the proteins. This region contains four reasonably well-conserved sequence motifs, all of which have been suggested to be part of the translocation pore or substrate binding site. Phylogenetic analysis of the C-terminal stretch revealed the presence of five subfamilies with characterized members: (i) the eukaryotic glutamate transporters, (ii) the bacterial glutamate transporters, (iii) the eukaryotic neutral-amino-acid transporters, (iv) the bacterial C4-dicarboxylate transporters, and (v) the bacterial serine transporters. A number of other subfamilies that do not contain characterized members have been defined. In contrast to their amino acid sequences, the hydropathy profiles of the members of the family are extremely well conserved. Analysis of the hydropathy profiles has suggested that the glutamate transporters have a global structure that is unique among secondary transporters. Experimentally, the unique structure of the transporters was recently confirmed by membrane topology studies. Although there is still controversy about part of the topology, the most likely model predicts the presence of eight membrane-spanning alpha-helices and a loop-pore structure which is unique among secondary transporters but may resemble loop-pores found in ion channels. A second distinctive structural feature is the presence of a highly amphipathic membrane-spanning helix that provides a hydrophilic path through the membrane. Recent data from analysis of site-directed mutants and studies on the mechanism and pharmacology of the transporters are discussed in relation to the structural model.

Molecular properties of bacterial multidrug transporters.

One of the mechanisms that bacteria utilize to evade the toxic effects of antibiotics is the active extrusion of structurally unrelated drugs from the cell. Both intrinsic and acquired multidrug transporters play an important role in antibiotic resistance of several pathogens, including Neisseria gonorrhoeae, Mycobacterium tuberculosis, Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, and Vibrio cholerae. Detailed knowledge of the molecular basis of drug recognition and transport by multidrug transport systems is required for the development of new antibiotics that are not extruded or of inhibitors which block the multidrug transporter and allow traditional antibiotics to be effective. This review gives an extensive overview of the currently known multidrug transporters in bacteria. Based on energetics and structural characteristics, the bacterial multidrug transporters can be classified into five distinct families. Functional reconstitution in liposomes of purified multidrug transport proteins from four families revealed that these proteins are capable of mediating the export of structurally unrelated drugs independent of accessory proteins or cytoplasmic components. On the basis of (i) mutations that affect the activity or the substrate specificity of multidrug transporters and (ii) the three-dimensional structure of the drug-binding domain of the regulatory protein BmrR, the substrate-binding site for cationic drugs is predicted to consist of a hydrophobic pocket with a buried negatively charged residue that interacts electrostatically with the positively charged substrate. The aromatic and hydrophobic amino acid residues which form the drug-binding pocket impose restrictions on the shape and size of the substrates. Kinetic analysis of drug transport by multidrug transporters provided evidence that these proteins may contain multiple substrate-binding sites.

Bacterial solute uptake and efflux systems.

The recent discovery of binding protein dependent secondary transporters and the ever-growing family of membrane potential generating secondary transporters emphasize the diversity of transport systems in both the mechanistical and physiological sense. The vast amount of data on the lactose permease is now beginning to crystallize in a model that relates functional events to structural changes of the protein. Evidence has been presented that multidrug transporters pick up their substrates from the membrane, and the binding of a number of substrates to the binding-protein components of ATP-driven transporters is now understood in detail.

Lactic acid bacteria: the bugs of the new millennium.

Lactic acid bacteria (LABs) are widely used in the manufacturing of fermented food and are among the best-studied microorganisms. Detailed knowledge of a number of physiological traits has opened new potential applications for these organisms in the food industry, while other traits might be beneficial for human health. Important new developments have been made in the research of LABs in the areas of multidrug resistance, bacteriocins and quorum sensing, osmoregulation, proteolysis, autolysins and bacteriophages. Recently, progress has been made in the construction of food-grade genetically modified LABs.

Can the excretion of metabolites by bacteria be manipulated?

Bacteria can release metabolites into the environment by various mechanisms. Excretion may occur by passive diffusion or by the reversal of the uptake process when the internal concentration of the metabolite exceeds the thermodynamic equilibrium level. In other cases, solutes are excreted against the concentration gradient by special extrusion systems. Their mode of energy coupling is different to that of the well-studied group of uptake systems. A thorough understanding of the transport processes will help to improve the excretion of metabolites of commercial interest, allow a more efficient production of metabolites in bulk quantities, and permit their exploitation to establish new markets.

Bacterial solute transport proteins in their lipid environment.

The cytoplasmic membrane of bacteria is a selective barrier that restricts entry and exit of solutes. Transport of solutes across this membrane is catalyzed by specific membrane proteins. Integral membrane proteins usually require specific lipids for optimal activity and are inhibited by other lipid species. Their activities are also sensitive to the lipid bilayer dynamics and physico-chemical state. Bacteria can adapt to changes in the environments (respective temperature, hydrostatic pressure, and pH) by altering the lipid composition of the membrane. Homeoviscous adaptation results in the maintenance of the liquid-crystalline phase through alterations in the degree of acyl chain saturation and branching, acyl chain length and the sterol content of the membrane. Homeophasic adaptation prevents the formation of non-bilayer phases, which would disrupt membrane organization and increase permeability. A balance is maintained between the lamellar phase, preferring lipids, and those that adopt a non-bilayer organization. As a result, the membrane proteins are optimally active under physiological conditions. The molecular basis of lipid-protein interactions is still obscure. Annular lipids stabilize integral membrane proteins. Stabilization occurs through electrostatic and possibly other interactions between the lipid headgroups and the charged amino acid residues close to the phospholipid-water interface, and hydrophobic interactions between the fatty acyl chains and the membrane-spanning segments. Reconstitution techniques allow manipulation of the lipid composition of the membrane in a way that is difficult to achieve in vivo. The physical characteristics of membrane lipids that affect protein-mediated transport functions have been studied in liposomal systems that separate an inner and outer compartment. The activity of most transport proteins is modulated by the bulk physical characteristics of the lipid bilayer, while specific lipid requirements appear rare.

Mechanisms of multidrug transporters.

Drug resistance, mediated by various mechanisms, plays a crucial role in the failure of the drug-based treatment of various infectious diseases. As a result, these infectious diseases re-emerge rapidly and cause many victims every year. Another serious threat is imposed by the development of multidrug resistance (MDR) in eukaryotic (tumor) cells, where many different drugs fail to perform their therapeutic function. One of the causes of the occurrence of MDR in these cells is the action of transmembrane transport proteins that catalyze the active extrusion of a large number of structurally and functionally unrelated compounds out of the cell. The mode of action of these MDR transporters and their apparent lack of substrate specificity is poorly understood and has been subject to many speculations. In this review we will summarize our current knowledge about the occurrence, mechanism and molecular basis of (multi-)drug resistance especially as found in bacteria.

Phosphate transport in membrane vesicles from Escherichia coli.

Escherichia coli strain AN710 possesses only the PIT system for phosphate transport. Membrane vesicles from this strain, which contain phosphate internally, perform exchange and active transport of phosphate. The energy for active transport is supplied by the respiratory chain with ascorbate phenazine methosulphate as electron donor. To a lesser extent also the oxidation of D-lactate energizes phosphate transport; the oxidation of succinate is only marginally effective. Phosphate transport is driven by the proton-motive force and in particular by the pH gradient across the membrane. This view is supported by the observation that phosphate transport is stimulated by valinomycin, inhibited by nigericin and abolished by the uncoupler carbonyl cyanide m-chlorophenylhydrazone. Neither inhibitor affects phosphate exchange. The phosphate analogue arsenate inhibits both the exchange reaction and active transport. Both processes are stimulated by K+ and Mg2+, the highest activities being observed with both ions present. Membrane vesicles have also been isolated from Escherichia coli K10, a strain which possesses only a functional PST phosphate transport system. These vesicles perform neither exchange nor active transport of phosphate, although active transport of amino acids is observed in the presence of ascorbate-phenazine methosulphate or D-lactate.

Continuous measurement of the cytoplasmic pH in Lactococcus lactis with a fluorescent pH indicator.

The cytoplasmic pH of Lactococcus lactis was studied with the fluorescent pH indicator 2',7'-bis-(2-carboxyethyl)-5 (and-6)-carboxyfluorescein (BCECF). A novel method was applied for loading bacterial cells with BCECF, which consists of briefly treating a dense cell suspension with acid in the presence of the probe. This results in a pH gradient, which drives accumulation of the probe in the cytoplasm. After neutralization the probe was well retained in cells stored on ice. BCECF-loaded cells were metabolically active, and were able to generate a pH gradient upon energization. The probe leaks out slowly at elevated temperatures. Efflux is stimulated upon energization of the cells, and is most likely catalyzed by an active transport system. It is a first-order process, and the rate constant could be deduced from the decrease of the fluorescence signal in periods of constant intracellular pH. This allowed a correction of the fluorescence signal for efflux of the probe. After calibration the cytoplasmic pH could be calculated from efflux-corrected fluorescence traces.

The ABC family of multidrug transporters in microorganisms.

Multidrug transporters are membrane proteins that are able to expel a broad range of toxic molecules from the cell. In humans, the overexpression of the multidrug resistance P-glycoprotein (Pgp) and the multidrug resistance-associated protein MRP1 (MRP) is a principal cause of resistance of cancers to chemotherapy. These multidrug transporters belong to the ATP-binding cassette (ABC) family of transport proteins that utilize the energy of ATP hydrolysis for activity. In microorganisms, multidrug transporters play an important role in conferring antibiotic resistance on pathogens. In the last decade, homologs of human Pgp and MRP have been found in microorganisms such as Plasmodium falciparum, Candida albicans, Saccharomyces cerevisiae and, more recently, in Lactococcus lactis. In this review, we will summarize the current state of knowledge on three major aspects of microbial ABC-type multidrug transporters: (i) the functional and structural similarities among these proteins in prokaryotic and eukaryotic cells, (ii) the molecular mechanism of these transporters, and (iii) their potential physiological role.

What we can learn from the effects of thiol reagents on transport proteins.

Many secondary membrane transport systems contain reactive sulfhydryl groups. In this review the applications of SH reagents for analyzing the role of sulfhydryl groups in membrane transport systems will be discussed. First an overview will be given of the more important reagents, that have been used to study SH-groups in membrane transport systems, and examples will be given of transport proteins in which the role of cysteines have been analyzed. An important application of SH-reagents to label transport proteins using various SH-reagents modified with fluorescent- or spin-label moieties will be discussed. Two general models are shown which have been proposed to explain the role of sulfhydryl groups in some membrane transport systems.

Effect of the unsaturation of phospholipid acyl chains on leucine transport of Lactococcus lactis and membrane permeability.

The effect of the degree of unsaturation of the phospholipid acyl chains on the branched-chain amino acid transport system of Lactococcus lactis was investigated by the use of a membrane fusion technique. Transport activity was analyzed in hybrid membranes composed of equimolar mixtures of synthetic unsaturated phosphatidylethanolamine (PE) and phosphatidylcholine (PC) in which the number of cis double bonds in the 18-carbon acyl chains was varied. The accumulation level and initial rate of both counterflow and protonmotive-force driven transport of leucine decreased with increasing number of double bonds. The reduction in transport activity with increasing number of double bonds correlated with an increase in the passive permeability of the membranes to leucine. The membrane fluidity was hardly affected by the double bond content. It is concluded that the degree of lipid acyl chain unsaturation is a minor determinant of the activity of the branched chain amino acid transport system, but effects strongly the passive permeability of the membrane.

Carbon-13 nuclear magnetic resonance studies of acetate metabolism in intact cells of rhodopseudomonas sphaeroides.

13C-nuclear magnetic resonance was used to study the metabolism of [2-(13)C]acetate in suspensions of Rhodopseudomonas sphaeroides. In the dark, in logarithmic-phase cells the 13C label appeared first in butyrate C-2 and C-4 and subsequently in glutamate C-4 and succinate C-2 and C-3. In the light, synthesis of poly(beta-hydroxybutyrate) (PHB) takes place. Butyrate synthesis seems to be independent of PHB synthesis or degradation activity. Starved, logarithmic-phase cells also show massive synthesis of PHB in the dark. Stationary-phase cells incorporate 13C predominantly into glutamate and succinate. No significant butyrate biosynthesis can be detected in the dark or during illumination. The incorporation of label in PHB is very slow in these cells and most probably originates from exchange of 12C for 13C into PHB. This might indicate slow turnover without net synthesis of the polymer occurring under these conditions. The results are discussed in relation to the redox state and the availability of metabolic energy for biosynthetic reactions in the dark and during illumination of cell suspensions of Rps. sphaeroides.

Spectral identification of the electrochromically active carotenoids of Rhodobacter sphaeroides in chromatophores and reconstituted liposomes.

Reaction centers with both light harvesting complexes I and II (B875 and B800/850; i.e., RCLHILHII complexes) have been isolated from Rhodobacter sphaeroides. These complexes have been incorporated into liposomes made from lipids purified from Escherichia coli. The electrochromic bandshift of carotenoids, present in these reconstituted complexes, shows shifted minima and maxima with respect to a similar spectrum in chromatophores of Rb. sphaeroides in a potassium diffusion potential induced difference spectrum (see also Crielaard, W., Hellingwerf, K.J. and Konings, W.N. (1989) Biochim. Biophys. Acta 973, 205-211). The absorbance spectrum, at room temperature or at 77 K, of both membrane preparations did not, however, reveal differences in the carotenoid region. The long-wavelength carotenoid peak in both preparations is located at 513 nm (77 K). A small difference could be observed between the 77 K excitation spectra of the B850 fluorescence. Reconstituted complexes show a carotenoid peak at 513 nm, whereas in chromatophores this peak is located at 514.5 nm. When fluorescence was recorded at 805 nm, to detect B800 excitation, there was a marked difference between both preparations. In liposomes the long wavelength B800-associated carotenoid peak is located at 512.5 nm, whereas in chromatophores this peak is located at 516 nm. These results explain the shifted minima and maxima in a potassium diffusion induced difference spectrum in proteoliposomes. The prediction of two carotenoid pools in chromatophores (De Grooth, B.G. and Amesz, J. (1977) Biochim. Biophys. Acta 462, 247-258) is confirmed, and the field sensitive carotenoids are identified as the pool that is associated with the B800 band (Kramer, H.J.M., Van Grondelle, R., Hunter, C.N., Westerhuis, W.H.J. and Amesz, J. (1984) Biochim. Biophys. Acta 765, 156-165).

Acidic phospholipids are required during solubilization of amino acid transport systems of Lactococcus lactis.

The branched-chain amino acid transport system of Lactococcus lactis was solubilized with n-octyl beta-D-gluco-pyranoside and reconstituted into proteoliposomes. Transport activity was recovered only when solubilization was performed in the presence of acidic phospholipids. Omission of acidic phospholipids during solubilization resulted in an inactive transport protein and the activity could not be restored in the reconstitution step. Similar results have been obtained for the arginine/ornithine exchange protein from Pseudomonas aeruginosa and L. lactis. Functional reconstitution of the transport protein requires the presence of aminophospholipids or glycolipids in the liposomes (Driessen, A.J.M., Zheng, T., In't Veld, G., Op den Kamp, J.A.F. and Konings, W.N. (1988) Biochemistry 27, 865-872). We propose that during the detergent solubilization the acidic phospholipids protect the transport systems against denaturation by preventing delipidation.

Hydrophobic membrane thickness and lipid-protein interactions of the leucine transport system of Lactococcus lactis.

The effect of the phospholipid acyl chain carbon number on the activity of the branched-chain amino acid transport system of Lactococcus lactis has been investigated. Major fatty acids identified in a total lipid extract of L. lactis membranes are palmitic acid (16:0), oleic acid (18:1) and the cyclopropane-ring containing lactobacillic acid (19 delta). L. lactis membrane vesicles were fused with liposomes prepared from equimolar mixtures of synthetic phosphatidylethanolamine (PE) and phosphatidylcholine (PC) with cis mono-unsaturated acyl chains. The activity of the branched-chain amino acid carrier is determined by the bulk properties of the membrane (Driessen, A.J.M., Zheng, T., In 't Veld, G., Op den Kamp, J.A.F. and Konings, W.N. (1988) Biochemistry 27, 865-872). PE acts as an activator and PC is ineffective. Counterflow and protonmotive-force driven transport of leucine is sensitive to changes in the acyl chain carbon number of both phospholipids and maximal with dioleoyl-PE/dioleoyl-PC. Above the gel to liquid-crystalline phase transition temperature of the lipid species, membrane fluidity decreased with increasing acyl chain carbon number. Our data suggest that the carbon number of the acyl chains of PE and PC determine to a large extent the activity of the transport system. This might be relevant for the interaction of PE with the transport protein. Variations in the acyl chain composition of PC exert a more general effect on transport activity. The acyl chain composition of phospholipids determines the membrane thickness (Lewis, B.A. and Engelman, D.M. (1983) J. Mol. Biol. 166, 211-217). We therefore propose that the degree of matching between the lipid-bilayer and the hydrophobic thickness of the branched-chain amino acid carrier is an important parameter in lipid-protein interactions.

Homeostasis of the membrane proton permeability in Bacillus subtilis grown at different temperatures.

Bacillus subtilis was grown at its growth temperature limits and at various temperatures in between the lower and upper growth temperature boundary. Liposomes were made of the extracted membrane lipids derived from these cells. The headgroup composition of the cytoplasmic membrane lipids did not differ significantly at the lower (13 degrees C) and upper (50 degrees C) temperature boundary. The averaged lipid acyl chain length, degree of saturation, and ratio of iso- and anteiso-branched fatty acids increased with the temperature. At the temperature of growth, the membranes were in a liquid-crystalline phase, but liposomes derived from cells grown at 13 degrees C were almost threefold more viscous than those derived from 50 degrees C grown cells. The temperature dependence of the proton permeability of the liposomes was determined using the acid-pulse method with monitoring of the outside pH with the fluorescent probe pyranine. The proton permeability of each liposome preparation increased with the temperature. However, the proton permeability of the liposomes at the growth temperature of the cells from which the lipids were derived was almost constant. These data indicate that the growth temperature dependent variation in lipid acyl chain composition permits maintenance of the proton permeability of the cytoplasmic membrane. This 'homeo-proton permeability adaptation' precludes futile cycling of protons at higher growth temperatures and allows cells to sustain the proton motive force as a driving force for essential energy transducing processes.

Stability and proton-permeability of liposomes composed of archaeal tetraether lipids.

Liposomes composed of tetraether lipids originating from the thermoacidophilic archaeon Sulfolobus acidocaldarius were analyzed for their stability and proton permeability from 20 degrees C up to 80 degrees C. At room temperature, these liposomes are considerably more stable and have a much lower proton permeability than liposomes composed of diester lipids originating from the mesophilic bacterium Escherichia coli or the thermophilic bacterium Bacillus stearothermophilus. With increasing temperature, the stability decreased and the proton permeability increased for all liposomes. Liposomes composed from tetraether lipids, however, remain the most stable. These data suggest these liposomes retain the rigidity of the cytoplasmic membrane of S. acidocaldarius needed to endure extreme environmental growth conditions.