Limits on the Tightness of Coupling in Active Transport

Control of the coupled reaction sequence in active transport depends on systematic changes in the properties of the carrier protein as the reaction proceeds. These changes would have to be brought about by specific interactions with the substrate, the binding forces being used to stabilize either (i) a carrier state with altered properties or (ii) the transition state in a carrier transformation. In the first case the tightness of coupling (the ratio of the coupled rate to slippage) will at first rise with the increment in binding energy in the altered state but will approach an upper limit when overly strong binding forces retard substrate dissociation in a subsequent step in the coupled reaction sequence. Primary and secondary active transport are subject to this limitation because the coupling mechanism necessarily involves intermediates in which the substrate is strongly bound. Exchange-only transport is not necessarily subject to the same limitation because the mechanism can involve only a substrate-catalyzed change in carrier state. The available data, although scant, agree with these conclusions. Received: 3 June 1998/Revised: 22 September 1998     

The structural basis of secondary active transport mechanisms

Secondary active transporters couple the free energy of the electrochemical potential of one solute to the transmembrane movement of another. As a basic mechanistic explanation for their transport function the model of alternating access was put forward more than 40 years ago, and has been supported by numerous kinetic, biochemical and biophysical studies. According to this model, the transporter exposes its substrate binding site(s) to one side of the membrane or the other during transport catalysis, requiring a substantial conformational change of the carrier protein. In the light of recent structural data for a number of secondary transport proteins, we analyze the model of alternating access in more detail, and correlate it with specific structural and chemical properties of the transporters, such as their assignment to different functional states in the catalytic cycle of the respective transporter, the definition of substrate binding sites, the type of movement of the central part of the carrier harboring the substrate binding site, as well as the impact of symmetry on fold-specific conformational changes. Besides mediating the transmembrane movement of solutes, the mechanism of secondary carriers inherently involves a mechanistic coupling of substrate flux to the electrochemical potential of co-substrate ions or solutes. Mainly because of limitations in resolution of available transporter structures, this important aspect of secondary transport cannot yet be substantiated by structural data to the same extent as the conformational change aspect. We summarize the concepts of coupling in secondary transport and discuss them in the context of the available evidence for ion binding to specific sites and the impact of the ions on the conformational state of the carrier protein, which together lead to mechanistic models for coupling.     

A functional-phylogenetic classification system for transmembrane solute transporters.

A comprehensive classification system for transmembrane molecular transporters has been developed and recently approved by the transport panel of the nomenclature committee of the International Union of Biochemistry and Molecular Biology. This system is based on (i) transporter class and subclass (mode of transport and energy coupling mechanism), (ii) protein phylogenetic family and subfamily, and (iii) substrate specificity. Almost all of the more than 250 identified families of transporters include members that function exclusively in transport. Channels (115 families), secondary active transporters (uniporters, symporters, and antiporters) (78 families), primary active transporters (23 families), group translocators (6 families), and transport proteins of ill-defined function or of unknown mechanism (51 families) constitute distinct categories. Transport mode and energy coupling prove to be relatively immutable characteristics and therefore provide primary bases for classification. Phylogenetic grouping reflects structure, function, mechanism, and often substrate specificity and therefore provides a reliable secondary basis for classification. Substrate specificity and polarity of transport prove to be more readily altered during evolutionary history and therefore provide a tertiary basis for classification. With very few exceptions, a phylogenetic family of transporters includes members that function by a single transport mode and energy coupling mechanism, although a variety of substrates may be transported, sometimes with either inwardly or outwardly directed polarity. In this review, I provide cross-referencing of well-characterized constituent transporters according to (i) transport mode, (ii) energy coupling mechanism, (iii) phylogenetic grouping, and (iv) substrates transported. The structural features and distribution of recognized family members throughout the living world are also evaluated. The tabulations should facilitate familial and functional assignments of newly sequenced transport proteins that will result from future genome sequencing projects.     

Active transport of l-sorbose and 2-deoxy-d-galactose in Saccharomyces fragilis

Sorbose and 2-deoxy-d-galactose are taken up in Saccharomyces fragilis by an active transport mechanism, as indicated by the energy requirement of the process and the accumulation of free sugar against the concentration gradient. There are no indications for transport-associated phosphorylation as mechanism of energy coupling with these two sugars.The measured sugar-proton cotransport and the influx inhibition by uncouplers suggest a chemiosmotic coupling mechanism. Thus there are at least two different active transport mechanisms operative in Saccharomyces fragilis: transport-associated phosphorylation in the case of 2-deoxy-d-galactose and chemiosmotic coupling in the case of sorbose and 2-deoxy-d-galactose. The difference between the two mechanisms are discussed.Uncouplers do not stimulate downhill sorbose transport in energy-depleted cells and evoke an almost complete inhibition of efflux and of exchange transport.The differences between this sugar-proton cotransport system and similar systems in bacteria and Chlorella are discussed.     

A model for coupling of H(+) and substrate fluxes based on "time-sharing" of a common binding site

Both prokaryotic and eukaryotic cells contain an array of membrane transport systems maintaining the cellular homeostasis. Some of them (primary pumps) derive energy from redox reactions, ATP hydrolysis, or light absorption, whereas others (ion-coupled transporters) utilize ion electrochemical gradients for active transport. Remarkable progress has been made in understanding the molecular mechanism of coupling in some of these systems. In many cases carboxylic residues are essential for either binding or coupling. Here we suggest a model for the molecular mechanism of coupling in EmrE, an Escherichia coli 12-kDa multidrug transporter. EmrE confers resistance to a variety of toxic cations by removing them from the cell interior in exchange for two protons. EmrE has only one membrane-embedded charged residue, Glu-14, which is conserved in more than 50 homologous proteins. We have used mutagenesis and chemical modification to show that Glu-14 is part of the substrate-binding site. Its role in proton binding and translocation was shown by a study of the effect of pH on ligand binding, uptake, efflux, and exchange reactions. The studies suggest that Glu-14 is an essential part of a binding site, which is common to substrates and protons. The occupancy of this site by H(+) and substrate is mutually exclusive and provides the basis of the simplest coupling for two fluxes.     

Active transport of L-sorbose and 2-deoxy-D-galactose in Saccharomyces fragilis.

Sorbose and 2-deoxy-D-galactose are taken up in Saccharomyces fragilis by an active transport mechanism, as indicated by the energy requirement of the process and the accumulation of free sugar against the concentration gradient. There are no indications for transport-associated phosphorylation as mechanism of energy coupling with these two sugars. The measured sugar-proton cotransport and the influx inhibition by uncouplers suggest a chemiosmotic coupling mechanism. Thus there are at least two different active transport mechanisms operative in Saccharomyces fragilis: transport-associated phosphorylation in the case of 2-deoxy-D-glucose and chemiosmotic coupling in the case of sorbose and 2-deoxy-D-galactose. The differences between the two mechanisms are discussed. Uncouplers do not stimulate downhill sorbose transport in energy-depleted cells and evoke an almost complete inhibition of efflux and of exchange transport. The differences between this sugar-proton cotransport system and similar systems in bacteria and Chlorella are discussed.     

Periplasmic binding protein-dependent transport systems: the membrane-associated components

Periplasmic binding protein-dependent transport systems are multicomponent, consisting of several inner membrane-associated proteins and a periplasmic component. The membrane-associated components of different systems are related in organization and function suggesting that, despite different substrate specificities, each transport system functions by a common mechanism. Current understanding of these components is reviewed. The nature of energy coupling to periplasmic transport systems has long been debated. Recent data now demonstrate that ATP hydrolysis is the primary source of energy for transport. The ATP-binding transport components are the best characterized of a family of closely related ATP-binding proteins believed to couple ATP hydrolysis to a variety of different biological processes. Intriguingly, systems closely related to periplasmic binding protein-dependent transport systems have recently been identified in several Gram-positive organisms (which lack a periplasm) and in eukaryotic cells. This class of transport system appears to be widespread in nature, serving a variety of important and diverse functions.     

Secondary active transport

Secondary active transport is defined as the transport of a solute in the direction of its increasing electrochemical potential coupled to the facilitated diffusion of a second solute (usually an ion) in the direction of its decreasing electrochemical potential. The coupling agents are membrane proteins (carriers), each of which catalyzes simultaneously the facilitated diffusion of the driving ion and the active transport of a given solute. The review starts with some considerations on the energetics followed by a presentation of the kinetics of secondary active transport. Examples of information which may be gained by such studies are discussed. In the second part, some examples of secondary transport are given; we also describe the characteristics of the corresponding carriers. The various transport systems presented are: the D-glucose/Na+ symport in brush-border membranes, the lactose/H+ symport in E. coli, the Na+/H+ antiport, the different transport systems in the inner mitochondrial membrane.     

Dissection of mechanistic principles of a secondary multidrug efflux protein

Multidrug transporters are ubiquitous efflux pumps that provide cells with defense against various toxic compounds. In bacteria, which typically harbor numerous multidrug transporter genes, the majority function as secondary multidrug/proton antiporters. Proton-coupled secondary transport is a fundamental process that is not fully understood, largely owing to the obscure nature of proton-transporter interactions. Here we analyzed the substrate/proton coupling mechanism in MdfA, a model multidrug/proton antiporter. By measuring the effect of protons on substrate binding and by directly measuring proton binding and release, we show that substrates and protons compete for binding to MdfA. Our studies strongly suggest that competition is an integral feature of secondary multidrug transport. We identified the proton-binding acidic residue and show that, surprisingly, the substrate binds at a different site. Together, the results suggest an interesting mode of indirect competition as a mechanism of multidrug/proton antiport.     

Cation-induced asymmetry of choline flux across presynaptic plasma membranes.

Highly cholinergic synaptosomes from the optic lobes of Sepia officinalis retain their ability to concentrate K+ and extrude Na+ sensitive but is not obligatorily coupled to choline metabolism, or an energy supply as shown by the action of metabolic and ion pump inhibitors. The influx and efflux and/or steady-state distributions of choline in the presence of Na+, Li+, Rb+, Cs+ and mannitol were studied. The influx studies at different cis-choline concentrations revealed two systems for choline influx with different monovalent cation sensitivity and suggested a 1 : 1 interaction of choline with both mechanisms. Choline efflux was stimulated by trans-choline. Calculations of the internal/external concentration ratio expected if choline transport were coupled to the Na+ gradient gave a maximal value of about 10(2). A secondary active transport of choline, where Na+ is the driver solute provides an explanation for the cation sensitivity of the mechanism as well as for the method of coupling of choline transport to the varying demands of the nervous system for acetylcholine.     

Genetics of lactose utilization in lactic acid bacteria

Abstract: Lactose utilization is the primary function of lactic acid bacteria used in industrial dairy fermentations. The mechanism by which lactose is transported determines largely the pathway for the hydrolysis of the internalized disaccharide and the fate of the glucose and galactose moieties. Biochemical and genetic studies have indicated that lactose can be transported via phosphotransferase systems, transport systems dependent on ATP binding cassette proteins, or secondary transport systems including proton symport and lactose-galactose antiport systems. The genetic determinants for the group translocation and secondary transport systems have been identified in lactic acid bacteria and are reviewed here. In many cases the lactose genes are organized into operons or operon-like structures with a modular organization, in which the genes encoding lactose transport are tightly linked to those for lactose hydrolysis. In addition, in some cases the genes involved in the galactose metabolism are linked to or co-transcribed with the lactose genes, suggesting a common evolutionary pathway. The lactose genes show characteristic configurations and very high sequence identity in some phylogenetically distant lactic acid bacteria such as Leuconostoc and Lactobacillus or Lactococcus and Lactobacillus . The significance of these results for the adaptation of lactic acid bacteria to the industrial milk environment in which lactose is the sole energy source is discussed.     

Bacterial transporters: Charge translocation and mechanism

A comparative review of the electrophysiological characterization of selected secondary active transporters from Escherichia coli is presented. In melibiose permease MelB and the Na⁺/proline carrier PutP pre-steady-state charge displacements can be assigned to an electrogenic conformational transition associated with the substrate release process. In both transporters cytoplasmic release of the sugar or the amino acid as well as release of the coupling cation are associated with a charge displacement. This suggests a common transport mechanism for both transporters. In the NhaA Na⁺/H⁺ exchanger charge translocation due to its steady-state transport activity is observed. A new model is proposed for pH regulation of NhaA that is based on coupled Na⁺ and H⁺ equilibrium binding.     

Uptake of glutamate in Corynebacterium glutamicum. 2. Evidence for a primary active transport system

The inducible glutamate uptake system in Corynebacterium glutamicum (Kr?mer, R., Lambert, C., Hoischen, C. & Ebbighausen, H., preceding paper in this journal) was characterized with respect to its mechanism and energy coupling. All possible secondary active uptake mechanisms can be excluded. Glutamate transport is not coupled to the translocation of H+, Na+ or K+ ions. Although changes in membrane potential and uptake activity cannot completely be separated, no correlation between these two parameters is observed. The uptake of glutamate resembles a primary active, ATP-dependent transport mechanism in several respects. (a) The substrate affinity is very high (1.3 microM). (b) Accumulation of glutamate reaches values of greater than 2.10(5), at least as high as those reported for binding-protein-dependent systems in Gram-negative bacteria. (c) The uptake is unidirectional. Even after complete deenergization, the accumulation ratio was not significantly reduced. (d) The rate of glutamate uptake is directly correlated to the cytosolic ATP content and also to the ATP/ADP ratio. This is shown by varying internal ATP by different procedures applying inhibitors (NaCN, dicyclohexyl carbodiimide), uncouplers (carbonyl m-chlorophenylhydrazone), ionophores (valinomycin), and even by shifting the cells to anaerobiosis. Uptake is not promoted by cytosolic ATP levels below 1.5 mM, the maximum uptake rate is reached at 4-5 mM ATP.     

Cryoenzymology: the use of sub-zero temperatures and fluid solutions in the study of enzyme mechanisms.

Behind the firm discrimination maintained between active and passive transport lies a definition of energetic coupling as a fusion between an exergonic chemical reaction and an uphill transport. In contrast, energetic coupling between paired chemical reactions tends. to be defined much more loosely, as if the term were merely equivalent to sequential linkage, even though the actual usage may parallel that in transport. This article argues for a sharpening of this definition through integrated consideration of chemi-chemical and chemi-osmotic coupling.Furthermore; it calls attention to the applicability of energetic coupling to both the backward and forward fluxes of the energized transport. When two parallel but distinct active transport systems act on the same solute, one is likely to operate more steeply uphill than the other. The situation then easily arises, and is probably widespread, whereby entry occurs largely by the first process and exodus by the reversal of the second, still energetically linked. In this way cases of chemi-osmoti-chemical coupling probably arise, beyond the one proposed by Mitchell. Presumably the term retention process has in the past unknowingly (and illogically) referred to the second transport process. The “uncoupling” of an active transport does not tend simply to convert it to a facilitated diffusion, and both fluxes are likely to be modified. Accordingly, measure of only one flux will not describe a change in energy transfer.     

Energy coupling mechanisms of MFS transporters

Major facilitator superfamily (MFS) is a large class of secondary active transporters widely expressed across all life kingdoms. Although a common 12‐transmembrane helix‐bundle architecture is found in most MFS crystal structures available, a common mechanism of energy coupling remains to be elucidated. Here, we discuss several models for energy‐coupling in the transport process of the transporters, largely based on currently available structures and the results of their biochemical analyses. Special attention is paid to the interaction between protonation and the negative‐inside membrane potential. Also, functional roles of the conserved sequence motifs are discussed in the context of the 3D structures. We anticipate that in the near future, a unified picture of the functions of MFS transporters will emerge from the insights gained from studies of the common architectures and conserved motifs.     

Energy-dependent masking of substrate binding sites of the lactose permease of Escherichia coli

A method was devised to measure the number of specific substrate binding sites of lactose permease in membrane preparations derived from mechanically disrupted Escherichia coli.The method consists of incubation with radioactive thiodigalactoside (galactosyl β-d-thiogalactoside, TDG) followed by precipitation with 80% saturated (NH₄)₂SO₄ and washing with the same solution.The measurement gave reproducible results, easy to correct for a moderate nonspecific binding, but active transport, when it occurred, resulted in excess counts.The radioactivity bound to the pellet was shown to depend on the presence of intact lac y gene product.Addition of ascorbate and phenazine methosulfate (PMS) stimulated active transport into the membrane vesicles. This could be inhibited by cyanide and by uncoupling agents and under these conditions the number of available binding sites was strongly diminished, while the inhibitors alone did not bring about a similar decrease.The decrease of available substrate binding sites was reversed by removal of oxygen or by washing out the respiratory substrates.The decrease in available binding sites is interpreted as reflecting one of the energy coupling steps which during in vivo active transport prevents the mobile carrier from being available for outflux, but the detailed interpretation of the reported results raises a number of problems connected with the energy cycle of active transport     

Basic Amino Acid Transport in Escherichia coli: Properties of Canavanine-Resistant Mutants

A mutant of Escherichia coli strain CanR 22 has been isolated which is resistant to growth inhibition by canavanine, an analogue of arginine. The properties of this strain and of another canavanine-resistant mutant, JC182-5 (isolated by Celis et al. [5]), were studied. The mutation is pleiotropic in that it results in a reduction in the activity of two distinct permeases, the arginine-specific and lysine-arginine-ornithine transport systems. The lesion maps at min 56 of the E. coli linkage map, at or near the argP locus. Although strain CanR 22 excretes arginine, this excretion appears to result from reduced ability to concentrate arginine, rather than the loss of transport ability being the result of excretion. This conclusion is based on findings with a canavanine-resistant strain auxotrophic for arginine, which exhibits transport properties similar to those of the prototrophic strains. Additionally, growth in the presence of arginine or ornithine results in a repression of the activity of the two basic amino acid transport systems. Neither the arginine-specific nor the lysine-arginine-ornithine binding proteins of the mutant cells show significant alterations in terms of amount, physical properties, or kinetic parameters. These observations lead to the proposal of a model for the two basic amino acid transport systems in which two carrier proteins with different specificities interact with a common energy coupling mechanism. A lesion in the gene (or one of the genes) for this coupling mechanism can confer canavanine resistance.     

Conformational and functional analysis of the lipid binding protein Ag-NPA-1 from the parasitic nematode Ascaridia galli

Ag-NPA-1 (AgFABP), a 15 kDa lipid binding protein (LBP) from Ascaridia galli, is a member of the nematode polyprotein allergen/antigen (NPA) family. Spectroscopic analysis shows that Ag-NPA-1 is a highly ordered, alpha-helical protein and that ligand binding slightly increases the ordered secondary structure content. The conserved, single Trp residue (Trp17) and three Tyr residues determine the fluorescence properties of Ag-NPA-1. Analysis of the efficiency of the energy transfer between these chromophores shows a high degree of Tyr-Trp dipole-dipole coupling. Binding of fatty acids and retinol was accompanied by enhancement of the Trp emission, which allowed calculation of the affinity constants of the binary complexes. The distance between the single Trp of Ag-NPA-1 and the fluorescent fatty acid analogue 11-[(5-dimethylaminonaphthalene-1- sulfonyl)amino]undecanoic acid (DAUDA) from the protein binding site is 1.41 nm as estimated by fluorescence resonance energy transfer. A chemical modification of the Cys residues of Ag-NPA-1 (Cys66 and Cys122) with the thiol reactive probes 5-({[(2-iodoacetyl)amino]ethyl}amino) naphthalene-1-sulfonic acid (IAEDANS) and N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD), followed by MALDI-TOF analysis showed that only Cys66 was labeled. The observed similar affinities for fatty acids of the modified and native Ag-NPA-1 suggest that Cys66 is not a part of the protein binding pocket but is located close to it. Ag-NPA-1 is one of the most abundant proteins in A. galli and it is distributed extracellularly mainly as shown by immunohistology and immunogold electron microscopy. This suggests that Ag-NPA-1 plays an important role in the transport of fatty acids and retinoids.     

The effects of N-ethylmaleimide on active amino acid transport in Escherichia coli.

N-Ethylmaleimide (MalNEt) binds covalently and without specificity to accessible sulfhydryl residues in proteins. In some cases specificity has been imposed on this reaction by manipulating reaction conditions, yielding information concerning both enzyme mechanism and the identity of specific proteins (for example C.F. Fox and E.P. Kennedy (1965) Proc. Natl. Acad. Sci. u.s. 54, 891-899) and R.E. McCarty and J. Fagan (1973) Biochemistry 12, 1503-1507). We have examined the effects of MalNEt on the active accumulation of nine amino acids by Escherichia coli strains ML 308-225 and DL 54. Whole cells have been used in order that transport systems both dependent on and independent of periplasmic binding proteins could be studied under various conditions of energy supply for transport. Our results suggest that the systems transporting ornithine, phenylalanine and proline are those most likely to undergo inactivation by direct reaction of MalNEt with the transport apparatus, rather than merely via side effects such as interruption of their energy supply. The inhibition of proline transport is specifically enhanced by the presence of proline, competitive inhibitors of proline transport, or carbonylcyanide p-trifluoromethyoxyphenylhydrazone during MalNEt treatment. The other eight systems tested showed no analogous effects.     

A reaction-induced fourier transform-infrared spectroscopic study of the lactose permease. A transmembrane potential perturbs carboxylic acid residues

In chemiosmotic coupling, a transmembrane ion gradient is used as the source of energy to drive reactions. This process occurs in all cells, but the microscopic mechanism is not understood. Here, Escherichia coli lactose permease was used in a novel spectroscopic method to investigate the mechanism of chemiosmotic coupling in secondary active transporters. To provide a light-triggered electrochemical gradient, bacteriorhodopsin was co-reconstituted with the permease, and reaction-induced Fourier transform-infrared spectra were obtained from the co-reconstituted samples. The bacteriorhodopsin contributions were subtracted from these data to give spectra reflecting permease conformational changes that are induced by an electrochemical gradient. Positive bands in the 1765-1730 cm(-1) region are attributable to carboxylic acid residues in the permease and are consistent with changes of pK(a), protonation state, or environment. This is the first direct information concerning gradient-induced structural changes in the permease at the single amino acid level. Ultimately, these structural changes facilitate galactoside binding and may be involved in the storage of free energy.