NMR structure of the enzyme GatB of the galactitol-specific phosphoenolpyruvate-dependent phosphotransferase system and its interaction with GatA

The phosphoenolpyruvate-dependent carbohydrate transport system (PTS) couples uptake with phosphorylation of a variety of carbohydrates in prokaryotes. In this multienzyme complex, the enzyme II (EII), a carbohydrate-specific permease, is constituted of two cytoplasmic domains, IIA and IIB, and a transmembrane channel IIC domain. Among the five families of EIIs identified in Escherichia coli, the galactitol-specific transporter (II(gat)) belongs to the glucitol family and is structurally the least well-characterized. Here, we used nuclear magnetic resonance (NMR) spectroscopy to solve the three-dimensional structure of the IIB subunit (GatB). GatB consists of a central four-stranded parallel beta-sheet flanked by alpha-helices on both sides; the active site cysteine of GatB is located at the beginning of an unstructured loop between beta1 and alpha1 that folds into a P-loop-like structure. This structural arrangement shows similarities with other IIB subunits but also with mammalian low molecular weight protein tyrosine phosphatases (LMW PTPase) and arsenate reductase (ArsC). An NMR titration was performed to identify the GatA-interacting residues.     

Nucleotide sequence of the gene encoding the Corynebacterium glutamicum mannose enzyme II and analyses of the deduced protein sequence

Abstract The complete nucleotide sequence of the gene encoding the Corynebacterium glutamicum mannose enzyme II (EII^Man) was determined. The gene consisted of 2052 base pairs encoding a protein of 683 amino acid residues; the molecular mass of the protein subunit was calculated to be 72570 Da. The N-terminal hydrophilic domain of EII^Man showed 39.7% homology with a C-terminal hydrophilic domain of Escherichia coli glucose-specific enzyme II (EII^Glc). Similar homology was shown between the C-terminal sequence of EII^Man and the E. coli glucose-specific enzyme III (EIII^Glc), or the EIII-like domain of Streptococcus mutans sucrose-specific enzyme II. Sequence comparison with other EIIs showed that EII^Man contained residues His-602 and Cys-28 which were homologous to the potential phosphorylation sites of EIII^Glc, or EIII-like domains, and hydrophilic domains (IIB) of several EIIs, respectively.     

Genetic dissection of specificity determinants in the interaction of HPr with enzymes II of the bacterial phosphoenolpyruvate:sugar phosphotransferase system in Escherichia coli

The histidine protein (HPr) is the energy-coupling protein of the phosphoenolpyruvate (PEP)-dependent carbohydrate:phosphotransferase system (PTS), which catalyzes sugar transport in many bacteria. In its functions, HPr interacts with a number of evolutionarily unrelated proteins. Mainly, it delivers phosphoryl groups from enzyme I (EI) to the sugar-specific transporters (EIIs). HPr proteins of different bacteria exhibit almost identical structures, and, where known, they use similar surfaces to interact with their target proteins. Here we studied the in vivo effects of the replacement of HPr and EI of Escherichia coli with the homologous proteins from Bacillus subtilis, a gram-positive bacterium. This replacement resulted in severe growth defects on PTS sugars, suggesting that HPr of B. subtilis cannot efficiently phosphorylate the EIIs of E. coli. In contrast, activation of the E. coli BglG regulatory protein by HPr-catalyzed phosphorylation works well with the B. subtilis HPr protein. Random mutations were introduced into B. subtilis HPr, and a screen for improved growth on PTS sugars yielded amino acid changes in positions 12, 16, 17, 20, 24, 27, 47, and 51, located in the interaction surface of HPr. Most of the changes restore intermolecular hydrophobic interactions and salt bridges normally formed by the corresponding residues in E. coli HPr. The residues present at the targeted positions differ between HPrs of gram-positive and -negative bacteria, but within each group they are highly conserved. Therefore, they may constitute a signature motif that determines the specificity of HPr for either gram-negative or -positive EIIs.     

A superfamily of proteins involved in different secretion pathways in gram-negative bacteria: modular structure and specificity of the N-terminal domain

The family of PulD proteins, which has been characterized in a wide variety of microorganisms, comprises several membrane-associated proteins essential for the transport of macromolecules across bacterial membranes. These proteins are involved in the transport of complex structures (such as phage particles, DNA) or various proteins (such as extracellular enzymes and pathogenicity determinants). Amino acid sequence analysis revealed a possible modular organisation of proteins of this superfamily, with highly conserved C-terminal domains and dissimilar N-terminal domains. In the C-terminal domain, four highly conserved regions have been found, one of them containing a remarkable common motif: (V, I)PXL(S, G)XIPXXGXLF. Structural comparisons between the N-terminal domains indicate that proteins of this superfamily can be divided into at least two subgroups, probably reflecting the existence of distinct secretion mechanisms. This implies that members of the superfamily of PulD-related proteins are independently involved in (1) the general secretory pathway, (2) a new signal-peptide-independent secretion pathway found in several bacterial pathogens, and possibly in (3) the translocation of bacteriophage particles through the bacterial cell envelope.     

Essential cytoplasmic domains in the Escherichia coli TatC protein.

The twin-arginine translocation (Tat) system mediates the transport of proteins across the bacterial plasma membrane and chloroplast thylakoid membrane. Operating in parallel with Sec-type systems in these membranes, the Tat system is completely different in both structural and mechanistic terms, and is uniquely able to catalyze the translocation of fully folded proteins across coupled membranes. TatC is an essential, multispanning component that has been proposed to form part of the binding site for substrate precursor proteins. In this study we have tested the importance of conserved residues on the periplasmic and cytoplasmic face of the Escherichia coli protein. We find that many of the mutations on the cytoplasmic face have little or no effect. However, substitution at several positions in the extreme N-terminal cytoplasmic region or the predicted first cytoplasmic loop lead to a significant or complete loss of Tat-dependent export. The mutated strains are unable to grow anaerobically on trimethylamine N-oxide minimal media and are unable to export trimethylamine-N-oxide reductase (TorA). The same mutants are completely unable to export a chimeric protein, comprising the TorA signal peptide linked to green fluorescent protein, indicating that translocation is blocked rather than cofactor insertion into the TorA mature protein. The data point to two essential cytoplasmic domains on the TatC protein that are essential for export.     

ABC transporters and immunity: mechanism of self-defense

The transporter associated with antigen processing (TAP) is a prototype of an asymmetric ATP-binding cassette (ABC) transporter, which uses ATP binding and hydrolysis to translocate peptides from the cytosol to the lumen of the endoplasmic reticulum (ER). Here, we review molecular details of peptide binding and ATP binding and hydrolysis as well as the resulting allosteric cross-talk between the nucleotide-binding domains and the transmembrane domains that drive translocation of the solute across the ER membrane. We also discuss the general molecular architecture of ABC transporters and demonstrate the importance of structural and functional studies for a better understanding of the role of the noncanonical site of asymmetric ABC transporters. Several aspects of peptide binding and specificity illustrate details of peptide translocation by TAP. Furthermore, this ABC transporter forms the central part of the major histocompatibility complex class I (MHC I) peptide-loading machinery. Hence, TAP is confronted with a number of viral factors, which prevent antigen translocation and MHC I loading in virally infected cells. We review how these viral factors have been used as molecular tools to decipher mechanistic aspects of solute translocation and discuss how they can help in the structural analysis of TAP.     

Subunit interactions in ABC transporters: towards a functional architecture

The ABC superfamily is a diverse group of integral membrane proteins involved in the ATP-dependent transport of solutes across biological membranes in both prokaryotes and eukaryotes. Although ABC transporters have been studied for over 30 years, very little is known about the mechanism by which the energy of ATP hydrolysis is used to transport substrate across the membrane. The recent report of the high resolution crystal structure of HisP, the nucleotide-binding subunit of the histidine permease complex of Salmonella typhimurium, represents a significant breakthrough toward the elucidation of the mechanism of solute translocation by ABC transporters. In this review, we use data from the crystallographic structures of HisP and other nucleotide-binding proteins, combined with sequence analysis of a subset of atypical ABC transporters, to argue a new model for the dimerisation of the nucleotide-binding domains that embraces the notion that the C motif from one subunit forms part of the ATP-binding site in the opposite subunit. We incorporate this dimerisation of the ATP-binding domains into our recently reported beta-barrel model for P-glycoprotein and present a general model for the cooperative interaction of the two nucleotide-binding domains and the translocation of mechanical energy to the transmembrane domains in ABC transporters.     

Molecular mechanisms of band 3 inhibitors. 3. Translocation inhibitors

During the translocation of the band 3 transport site between the inward- and outward-facing orientations, the Cl- transport site complex passes through a transition state lying on the reaction pathway between the two extreme orientations. Niflumic acid, 2-[(7-nitrobenzofurazan-4-yl)amino]ethanesulfonate, and 2,4,6-trichlorobenzenesulfonate each are translocation blockers that can bind to both the inward- and outward-facing conformations of band 3. The principal mechanism of these inhibitors is a reduction in the translocation rate, since they have essentially no effect on the apparent KD for Cl- binding to the transport site and the migration of Cl- between the transport site and solution. Instead, these inhibitors raise the free energy of formation of the transition state during translocation and thereby can lock the transport site into either the inward- or outward-facing orientation. In contrast, 2,4-dinitrofluorobenzene (DNFB) appears to restrict the accessibility of the transport site to solution Cl-; also, the DNFB reaction rate is increased by Cl-, suggesting that DNFB modification may occur during translocation. Thus DNFB is proposed to trap the Cl--transport site complex site during translocation to yield a conformation intermediate to the inward- and outward-facing orientations. A model is presented for the molecular mechanism of transport across biological membranes. The transport machinery is proposed to contain greater than or equal to 6 transmembrane helices that surround a central channel containing a sliding hydrophobic barrier. The transport site lies between two of the channel-forming helices and remains stationary while the hydrophobic barrier slides from one end of the channel to the other, thereby exposing the transport site to the opposite solution compartment.     

The glucose transporter of Escherichia coli. Mutants with impaired translocation activity that retain phosphorylation activity.

The glucose transporter of the bacterial phosphotransferase system couples translocation with phosphorylation of the substrate in a 1:1 stoichiometry. It is a complex consisting of a transmembrane subunit (IIGlc) and a hydrophilic subunit (IIIGlc). Both subunits are transiently phosphorylated. IIIGlc is phosphorylated at a histidyl residue by the cytoplasmic phosphoryl carrier protein phospho-heat-stable phosphoryl carrier protein; IIGlc is phosphorylated at a cysteinyl residue by phospho-IIIGlc. The IIGlc subunit consists of two domains. The N-terminal hydrophobic domain is presumed to span the membrane several times; the C-terminal cytoplasmic domain includes the phosphorylation site. IIGlc phosphorylates glucose and methyl-alpha-D-glucopyranoside in transit across the inner membrane but can also phosphorylate intracellular glucose. Ten mutants resistant against extracellular toxic methyl-alpha-D-glucopyranoside yet capable of phosphorylating intracellular glucose were isolated. Strong impairment of transport activity in these mutants was accompanied by only a slight decrease of phosphorylation activity. Amino acid substitutions occurred at six sites that are clustered in three presumably hydrophilic loops in the transmembrane domain of IIGlc: M17T, M17I, G149S, K150E, S157F, H339Y, and D343G. We presume that the three polypeptide segments are directly involved in sugar translocation and/or binding but are of little importance for phosphorylation activity, folding, and membrane localization of IIGlc.     

Cooperativity in the inhibition of P-glycoprotein-mediated daunorubicin transport: evidence for half-of-the-sites reactivity

P-Glycoprotein (Pgp) is an important transport enzyme composed of two homologous domains and transports a wide range of structurally diverse xenobiotics from the cell. Recent studies have indicated that allosteric interactions occur between the nucleotide binding domains and between the substrate binding domains of the two halves, but the extent of this interaction as well as the means by which the enzyme can transport such a wide variety of substrates has not been elucidated. Herein, the Pgp-mediated transport of a marker substrate, daunorubicin (DNR), out of viable cells was examined in the presence of a variety of other known substrates of Pgp. For most of the typical Pgp substrates examined, the relationship between inhibition of DNR efflux and competing substrate concentration was sigmoidal and therefore not a simple mutually exclusive competitive inhibition of transport. The Hill coefficient ranged from about 3 to 5 for the inhibition of transport of DNR. This negative cooperativity in combination with recent evidence, including several examples of noncompetitive inhibition between the homologous halves of Pgp, indicates a "half-of-the-sites" reactivity. Our data support the mechanistic proposal that substrate binding at one putative transport binding site precludes activity at another unequal site; many of the substrates examined exert a negative allosteric effect on the other transport site (and vice versa). A half-of-the-sites reactivity model would account for many of these observations and may be critical to the efficiency of Pgp substrate transport of a broad spectrum of compounds.     

Nuclear mRNA export requires specific FG nucleoporins for translocation through the nuclear pore complex

Trafficking of nucleic acids and large proteins through nuclear pore complexes (NPCs) requires interactions with NPC proteins that harbor FG (phenylalanine-glycine) repeat domains. Specialized transport receptors that recognize cargo and bind FG domains facilitate these interactions. Whether different transport receptors utilize preferential FG domains in intact NPCs is not fully resolved. In this study, we use a large-scale deletion strategy in Saccharomyces cerevisiae to generate a new set of more minimal pore (mmp) mutants that lack specific FG domains. A comparison of messenger RNA (mRNA) export versus protein import reveals unique subsets of mmp mutants with functional defects in specific transport receptors. Thus, multiple functionally independent NPC translocation routes exist for different transport receptors. Our global analysis of the FG domain requirements in mRNA export also finds a requirement for two NPC substructures-one on the nuclear NPC face and one in the NPC central core. These results pinpoint distinct steps in the mRNA export mechanism that regulate NPC translocation efficiency.     

Regulation of glucose uptake by stressed cells.

Lactate production by BHK cells is stimulated by arsenite, azide, or by infection with Semliki Forest virus (SFV). In the case of arsenite or SFV infection, the increase correlates approximately with the increase in glucose transport as measured by uptake of [3H] deoxy glucose (dGlc); in the case of azide, the increase in lactate production exceeds that of glucose transport. Hence glucose utilization by BHK cells and its stimulation by anaerobic and other types of cellular stress is controlled at least in part at the level of glucose transport. The glucose uptake by BHK cells is also stimulated by serum and by glucose deprivation. In these circumstances, as with arsenite, stimulation is reversible, with t1/2 of 1-2 hours; stimulation is compatible with a translocation of the glucose transporter protein between an intracellular site and the plasma membrane (shown here for serum and previously for arsenite). The surface binding and rate of internalization of [125I]-labelled transferrin and [125I] alpha 2-macroglobulin was studied to determine whether changes in glucose transport are accompanied by changes in the surface concentration or rate of internalization of membrane proteins. The findings indicate that changes in glucose transport do not reflect a consistent and general redistribution of membrane receptors. Taken together, the results are compatible with the proposal that BHK cells exposed to stimuli like insulin or serum, or to stresses like arsenite, azide, SFV infection, or deprivation of glucose, respond in the same manner: namely, by an increased capacity to transport glucose brought about by reversible and specific translocation of the transporter protein from an (inactive) intracellular site to the plasma membrane.     

The archaeal Sec-dependent protein translocation pathway

Over the past three decades, transport of proteins across cellular membranes has been studied extensively in various model systems. One of the major transport routes, the so-called Sec pathway, is conserved in all domains of life. Very little is known about this pathway in the third domain of life, archaea. The core components of the archaeal, bacterial and eucaryal Sec machinery are similar, although the archaeal components appear more closely related to their eucaryal counterparts. Interestingly, the accessory factors of the translocation machinery are similar to bacterial components, which indicates a unique hybrid nature of the archaeal translocase complex. The mechanism of protein translocation in archaea is completely unknown. Based on genomic sequencing data, the most likely system for archaeal protein translocation is similar to the eucaryal co-translational translocation pathway for protein import into the endoplasmic reticulum, in which a protein is pushed across the translocation channel by the ribosome. However, other models can also be envisaged, such as a bacterial-like system in which a protein is translocated post-translationally with the aid of a motor protein analogous to the bacterial ATPase SecA. This review discusses the different models. Furthermore, an overview is given of some of the other components that may be involved in the protein translocation process, such as those required for protein targeting, folding and post-translational modification.     

Energy Coupling Factor-Type ABC Transporters for Vitamin Uptake in Prokaryotes

Energy coupling factor (ECF) transporters are a subgroup of ATP-binding cassette (ABC) transporters involved in the uptake of vitamins and micronutrients in prokaryotes. In contrast to classical ABC importers, ECF transporters do not make use of water-soluble substrate binding proteins or domains but instead employ integral membrane proteins for substrate binding (named S-components). S-components form active translocation complexes with the ECF module, an assembly of two nucleotide-binding domains (NBDs, or EcfA) and a second transmembrane protein. In some cases, the ECF module is dedicated to a single S-component, but in many cases, the ECF module can interact with several different S-components that are unrelated in sequence and bind diverse substrates. The modular organization with exchangeable S-components on a single ECF module allows the transport of chemically different substrates via a common route. The recent determination of the crystal structures of the S-components that recognize thiamin and riboflavin has provided a first clue about the mechanism of S-component exchange. This review describes recent advances and the current views of the mechanism of transport by ECF transporters.     

Structure of the ArsA ATPase: the catalytic subunit of a heavy metal resistance pump

Active extrusion is a common mechanism underlying detoxification of heavy metals, drugs and antibiotics in bacteria, protozoa and mammals. In Escherichia coli, the ArsAB pump provides resistance to arsenite and antimonite. This pump consists of a soluble ATPase (ArsA) and a membrane channel (ArsB). ArsA contains two nucleotide-binding sites (NBSs) and a binding site for arsenic or antimony. Binding of metalloids stimulates ATPase activity. The crystal structure of ArsA reveals that both NBSs and the metal-binding site are located at the interface between two homologous domains. A short stretch of residues connecting the metal-binding site to the NBSs provides a signal transduction pathway that conveys information on metal occupancy to the ATP hydrolysis sites. Based on these structural features, we propose that the metal-binding site is involved directly in the process of vectorial translocation of arsenite or antimonite across the membrane. The relative positions of the NBS and the inferred mechanism of allosteric activation of ArsA provide a useful model for the interaction of the catalytic domains in other transport ATPases.     

Two regions of EpsL involved in species-specific protein-protein interactions with EpsE and EpsM of the general secretion pathway in Vibrio cholerae

Extracellular secretion of proteins via the type II or general secretion pathway in gram-negative bacteria requires the assistance of at least 12 gene products that are thought to form a complex apparatus through which secreted proteins are translocated. Although this apparatus is specifically required only for the outer membrane translocation step during transport across the bacterial cell envelope, it is believed to span both membranes. The EpsE, EpsL, and EpsM proteins of the type II apparatus in Vibrio cholerae are thought to form a trimolecular complex that is required to either control the opening and closing of the secretion pore or to transduce energy to the site of outer membrane translocation. EpsL is likely to play an important role in this relay by interacting with both the cytoplasmic EpsE protein and the cytoplasmic membrane protein EpsM, which is predominantly exposed on the periplasmic side of the membrane. We have now extended this model and mapped the separate regions within EpsL that contain the EpsE and EpsM binding domains. By taking advantage of the species specificity of the type II pathway, we have used chimeric proteins composed of EpsL and its homologue, ExeL, from Aeromonas hydrophila together with either EpsE or its Aeromonas homologue, ExeE, to complement the secretion defect in both epsL and exeL mutant strains. These studies have mapped the species-specific EpsE binding site to the N-terminal cytoplasmic region between residues 57 and 216 of EpsL. In addition, the species-specific EpsM binding site was mapped to the C-terminal half of EpsL by coimmunoprecipitation of EpsM with different EpsL-ExeL chimeras. This site is present in the region between amino acids 216 and 296, which contains the predicted membrane-spanning segment of EpsL.     

Cation and harmaline interactions with Na(+)-independent dibasic amino acid transport system y+ in human erythrocytes and in erythrocytes from a primitive vertebrate the pacific hagfish (Eptatretus stouti).

Transport systems y+, asc and ASC exhibit dual interactions with dibasic and neutral amino acids. For conventional Na(+)-dependent neutral amino acid system ASC, side chain amino and guanido groups bind to the Na+ site on the transporter. The topographically equivalent recognition site on related system asc binds harmaline (a Na(+)-site inhibitor) with the same affinity as asc (apparent Ki range 1-4 mM), but exhibits no detectable affinity for Ha. Although also classified as Na(+)-independent, dibasic amino acid transport system y+ accepts neutral amino acids when Na+ or another acceptable cation is also present. This latter observation implies that the y+ translocation site binds Na+ and suggests possible functional and structural similarities with ASC/asc. In the present series of experiments with human erythrocytes, system y(+)-mediated lysine uptake (5 microM, 20 degrees C) was found to be 3-fold higher in isotonic sucrose medium than in normal 150 mM NaCl medium. This difference was not a secondary consequence of changes in membrane potential, but resulted from Na+ functioning as a competitive inhibitor of transport. Apparent Km and Vmax values for lysine transport at 20 degrees C were 15.2 microM and 183 mumol/l cells per h, respectively, in sucrose medium and 59.4 microM and 228 mumol/l cells per h in Na+ medium. Similar results were obtained with y+ in erythrocytes of a primitive vertebrate, the Pacific hagfish (Eptatretus stouti), indicating that Na(+)-inhibition is a general property of this class of amino acid transporter. At a permeant concentration of 5 microM, the IC50 value for Na(+)-inhibition of lysine uptake by human erythrocytes was 27 mM. Other inorganic and organic cations, including K+ and guanidinium+, also inhibited transport. In parallel with its actions on ASC/asc harmaline competitively inhibited lysine uptake by human cells in sucrose medium. As predicted from mutually competitive binding to the y+ translocation site, the presence of 150 mM Na+ increased the harmaline inhibition constant (Ki) from 0.23 mM in sucrose medium to 0.75 mM in NaCl medium. We interpret these observations as further evidence that y+, asc and ASC represent a family of closely related transporters with a common evolutionary origin.     

Role of substrate binding forces in exchange-only transport systems: I. Transition-state theory

Summary An analysis of transition-state models for exchange-only transport shows that substrate binding forces, carrier conformational changes, and coupled substrate flow are interrelated. For a system to catalyze exchange but not net transport, addition of the substrate must convert the carrier from an immobile to a mobile form. The reduction in the energy barrier to movement is necessarily paid for out of the intrinsic binding energy between the substrate and the transport site, and is dependent on the formation of two different types of complex: a loose complex initially and a tight complex in the transition state in carrier movement. Hence the site should at first be incompletely organized for optimal binding but, following a conformational change, complementary to the substrate structure in the transition state. The conformational change, which may involve the whole protein, would be induced by cooperative interactions between the substrate and several groups within the site, involving a chelate effect. The tightness of coupling, i.e., the ratio of exchange to net transport, is directly proportional to the increased binding energy in the transition state, a relationship which allows the virtual substrate dissociation constant in the transition state to be calculated from experimental rate and half-saturation constants. Because the transition state is present in minute amount, strong bonding here does not enhance the substrate's affinity, and specificity may, therefore, be expressed in maximum exchange rates alone. However, where substrates largely convert the carrier to a transport intermediate whose mobility is the same with all substrates, specificity is also expressed in affinity. Hence the expression of substrate specificity provides evidence on the translocation mechanism.     

Crystal structure of MalK, the ATPase subunit of the trehalose/maltose ABC transporter of the archaeon Thermococcus litoralis

The members of the ABC transporter family transport a wide variety of molecules into or out of cells and cellular compartments. Apart from a translocation pore, each member possesses two similar nucleoside triphosphate-binding subunits or domains in order to couple the energy-providing reaction with transport. In the maltose transporter of several Gram-negative bacteria and the archaeon Thermo coccus litoralis, the nucleoside triphosphate-binding subunit contains a C-terminal regulatory domain. A dimer of the subunit is attached cytoplasmically to the translocation pore. Here we report the crystal structure of this dimer showing two bound pyrophosphate molecules at 1.9 A resolution. The dimer forms by association of the ATPase domains, with the two regulatory domains attached at opposite poles. Significant deviation from 2-fold symmetry is seen at the interface of the dimer and in the regions corresponding to those residues known to be in contact with the translocation pore. The structure and its relationship to function are discussed in the light of known mutations from the homologous Escherichia coli and Salmonella typhimurium proteins.     

Role of substrate binding forces in exchange-only transport systems: II. Implications for the mechanism of the anion exchanger of red cells

Summary The transition-state theory of exchange-only membrane transport is applied to experimental results in the literature on the anion exchanger of red cells. Two central features of the system are in accord with the theory: (i) forming the transition state in translocation involves a carrier conformational change; (ii) substrate specificity is expressed in transport rates rather than affinities. The expression of specificity is consistent with other evidence for a conformational intermediate (not the transition state) formed in the translocation of all substrates. The theory, in conjunction with concepts derived from the chemistry of macrocyclic ion inclusion complexes, prescribes certain essential properties in the transport site. Separate substites are required for the preferred substrates. Cl^– and HCO ₃ ^– , to account for tight binding in the transition state (K _diss1m). Further, the following mechanism is suggested. A substrate anion initially forms a loose surface complex at one subsite, but in the transition state the subsites converge to form an inclusion complex in which the binding forces are greatly increased through a chelation effect. The conformational change at the substrate site, which is driven by the mounting forces of binding, sets in train a wider conformational change that converts the carrier from an immobile to a mobile form. Though simple, this composite-site mechanism explains many unsual features of the system. It accounts for substrate inhibition, partially noncompetitive inhibition of one substrate by another, and tunneling, which is net transport under conditions where exchange should prevail, according to other models. All three types of behavior result from the formation of a ternary complex in which substrate anions are bound at both subsites. The mechanism also accounts for the enormous range of substrate structures accepted by the system, for the complex inhibition by the organic sulfate NAP-taurine, and for the involvement of several cationic side chains and two different protein domains in the transport site.