Reaction of the glucose carrier of erythrocytes with sodium tetrathionate: Evidence for inward-facing and outward-facing carrier conformations

Summary Sodium tetrathionate reacts with the glucose carrier of human erythrocytes at a rate which is greatly altered in the presence of competitive inhibitors of glucose transport. Inhibitors bound to the carrier on the outer surface of the membrane, either at the substrate site (maltose) or at the external inhibition site (phloretin and phlorizin), more than double the reaction rate. Inhibitors bound at the internal inhibition site (cytochalasin B and androstenedione), protect the system against tetrathionate. After treatment with tetrathionate, the maximum transport rate falls to less than one-third, and the properties of the binding sites are modified in unexpected ways. The affinity of externally bound inhibitors rises: phloretin is bound up to seven times more strongly and phlorizin and maltose twice as strongly. The affinity of cytochalasin B, bound at the internal inhibition site, falls to half while that of androstenedione is little changed. The affinity of external glucose falls slightly. Androstenedione prevents both the fall in transport activity and the increase in phloretin affinity produced by tetrathionate. An inhibitor of anion transport has no effect on the reaction. The observations support the following conclusions: (1) Tetrathionate produces its effects on the glucose transport system by reacting with the carrier on the outer surface of the membrane. (2) The carrier assumes distinct inward-facing and outward-facing conformations, and tetrathionate reacts with only the outward-facing form. (3) The thiol group with which tetrathionate is presumed to react is not present in either the substrate site or the internal or external inhibitor site. (4) In binding asymmetrically to the carrier, a reversible inhibitor shifts the carrier partition between inner and outer forms and thereby raises or lowers the rate of tetrathionate reaction with the system. (5) Reaction with tetrathionate converts the carrier to an altered state in which the conformation at all three binding sites is changed and the rate of carrier reorientation is reduced.     

The choline carrier of erythrocytes: Location of the NEM-reactive thiol group in the inner gated channel

Summary Choline transport across the human erythrocyte membrane is irreversibly inhibited when N-ethylmaleimide (NEM) reacts with a carrier SH group which is located outside the substrate site, and which is exposed in the inward-facing form of the carrier but prevented from reacting in the outward-facing form. The location of the SH group with respect to the membrane has now been determined by studying the dependence of the NEM-alkylation rate on the intracellular and extracellular pH. The results show that the reactive SH group equilibrates with hydrogen ions in the cytoplasm, but is completely isolated from hydrogen ions in the external medium. With this added evidence it becomes possible to conclude that the SH group is located in the inner gated channel of the choline carrier.     

The choline transport system of erythrocytes. Distribution of the free carrier in the membrane

A method is described, based on the kinetics of transport, for determining the equilibrium distribution of the carrier site on the inner and outer surfaces of the cell membrane, and this method is applied to the choline carrier of human erythrocytes. This method depends on measurement of flux ratios for both entry and exit, i.e., the transport rates of a low concentration of labeled substrate into a solution which contains either no substrate or a saturating concentration of unlabeled substrate. The concentrations of inward-facing and outward-facing carrier are found to be nearly equal, and therefore the 5-fold difference in choline affinity on the inner and outer surfaces of the membrane cannot be explained by an unequal carrier distribution. It is also shown that both reorientation and dissociation of the carrier-substrate complex are far more rapid than reorientation of the free carrier.     

The comparative specificity of the inner and outer substrate transfer sites in the choline carrier of human erythrocytes

Summary The substrate specificities on the inner and outer surfaces of the cell membrane have been compared by determining the relative affinities, inside and outside, of a series of choline analogs. The results of two different methods were in agreement: (1) the carrier distribution was determined in the presence of a saturating concentration of an equilibrated analog, using N-ethylmaleimide as a probe for the inward-facing carrier; (2) the degree of competition was measured between an equilibrated analog and choline in the external solution. The carrier sites are found to have markedly different specificities: the outer site is more closely complementary to the structure of choline than is the inner, and even a slight enlargement of either the trimethylammonium or hydroxyethyl group gives rise to preferential binding inside. It is also found that a nonpolar binding region, which is adjacent to the outer site, is absent from the inner site. As the transport mechanism involves the exposure of only one site at a time, first on one surface and then the other, it follows that an extensive reorganization of the structure of the substrate site may occur during the carrier-reorientation step, or alternatively that two distinct sites may be present, only one of which is exposed at a time.     

The conserved substrate binding site of mitochondrial carriers

Mitochondrial carriers transport nucleotides, co-factors and metabolic intermediates across the inner mitochondrial membrane permeability barrier. They belong to a family of transporters unique to eukaryotes and they differ in structure and transport mechanism from other secondary transporters. The main structural fold consists of a barrel of six transmembrane alpha-helices closed at the matrix side by a salt-bridge network at the bottom of the cavity. The significant sequence conservation in the mitochondrial carrier family suggests that specific recognition of substrates is coupled to a common mechanism of transport. We have identified a common substrate binding site comprising residues that are highly conserved and, as demonstrated by mutagenesis, are essential for function. The binding site explains substrate selectivity, ion coupling and the effects of the membrane potential on transport. The main contact points in the site are related by threefold symmetry like the common structural fold. The substrate is bound at the midpoint of the membrane and may function as a pivot point for the movements of the transmembrane alpha-helices as the carrier changes conformation. The trigger for the translocation event is likely to be the substrate-induced perturbation of the salt bridge network at the bottom of the cavity.     

Substrate specificity of the two mitochondrial ornithine carriers can be swapped by single mutation in substrate binding site

Mitochondrial carriers are a large family of proteins that transport specific metabolites across the inner mitochondrial membrane. Sequence and structure analysis has indicated that these transporters have substrate binding sites in a similar location of the central cavity consisting of three major contact points. Here we have characterized mutations of the proposed substrate binding site in the human ornithine carriers ORC1 and ORC2 by carrying out transport assays with a set of different substrates. The different substrate specificities of the two isoforms, which share 87% identical amino acids, were essentially swapped by exchanging a single residue located at position 179 that is arginine in ORC1 and glutamine in ORC2. Altogether the substrate specificity changes demonstrate that Arg-179 and Glu-180 of contact point II bind the C(α) carboxylate and amino group of the substrates, respectively. Residue Glu-77 of contact point I most likely interacts with the terminal amino group of the substrate side chain. Furthermore, it is likely that all three contact points are involved in the substrate-induced conformational changes required for substrate translocation because Arg-179 is probably connected with Arg-275 of contact point III through Trp-224 by cation-π interactions. Mutations at position 179 also affected the turnover number of the ornithine carrier severely, implying that substrate binding to residue 179 is a rate-limiting step of the catalytic transport cycle. Given that Arg-179 is located in the vicinity of the matrix gate, it is concluded that it is a key residue in the opening of the carrier to the matrix side.     

Asymmetric binding of steroids to internal and external sites in the glucose carrier of erythrocytes

Steroids inhibit glucose transport in erythrocytes by binding to sites in the carrier which are exposed on both the outer and inner surfaces of the cell membrane. Some steroids are bound almost exclusively at inner sites (androstendione and androstandione), while others are bound about as firmly on one side as the other (corticosterone). Still others exhibit a moderate preference for the internal site (deoxycorticosterone). The inhibition is in all cases competitive with respect to a substrate which is bound at the same surface of the membrane as the inhibitor. However, in experiments on substrate entry, internally bound inhibitors act in an apparently non-competitive fashion, as expected if the carrier model is valid. This behaviour explains the appearance of competitive, non-competitive and mixed inhibitions with different steroids (Lacko, L., Wittke, B. and Geck, P. (1975) J. Cell Physiol. 86, 673–680).     

Inhibition of choline transport in erythrocytes by n-alkanols

The choline transport system of erythrocytes is reversibly inhibited by ethanol, n-butanol, n-hexanol, n-octanol, and n-decanol, but not by n-dodecanol. Each methylene group in the alkyl chain contributes 560 cal/mol to the free energy of binding at the inhibitory site. Inhibition results from the cooperative binding of two molecules of an alcohol, judging by the Hill coefficient n of 1.7-1.9. The mechanism of inhibition is noncompetitive, and the partition of the carrier between inward-facing and outward-facing forms is unaffected by the alcohols; it follows that the four main carrier forms, the inner and outer free carrier, and the inner and outer carrier-substrate complex, are equally susceptible to inhibition. Hexanol and decanol accelerate the reaction of N-ethylmaleimide with a thiol group in the inner carrier channel, but ethanol and butanol, at concentrations that inhibit transport by 70%, do not. The disproportionate effects on substrate transport and the N-ethylmaleimide reaction are most simply explained as the direct result of binding of alcohol molecules in different regions of the carrier, rather than as the indirect result of a disturbance in the structure of the lipid bilayer induced by the alcohols.     

Kinetic asymmetry of renal Na+-L-lactate cotransport. Characteristic parameters and evidence for a ping pong mechanism of the trans-stimulating exchange by pyruvate

Brush border vesicles prepared from horse renal cortex were used to study the kinetic properties of the Na+-L-lactate carrier on the outer and inner faces of the membrane. Two methods were applied for these measurements (in the absence of an electrical gradient): a direct method using influx and efflux kinetics, and an indirect method applied to trans-stimulated influx kinetics using membrane vesicles preloaded with various pyruvate concentrations (the latter enabled us to observe simultaneously the inner and outer carrier properties). Kinetic parameters obtained by the first method have shown that under sodium lactate chemical gradient, the carrier efficiency (estimated by the ratio of k = Vm/Km) is higher for the influx than efflux, a mechanism indicating a kinetic asymmetry of the transport. This difference remains at chemical equilibrium of solute concentration. The similarity of outer and inner affinity of sodium permits one to conclude that the kinetic asymmetry of the sodium lactate transport is related to the lactate-carrier interaction and not to that of the sodium-carrier. The second method using the pyruvate trans-activation effect (under sodium chemical equilibrium) has shown an affinity of lactate (Kt(out) = 1.1 mM), about 15 times higher for the carrier in the extracellular orientation than that of pyruvate for the carrier in the intracellular orientation (Kt(pyr) = 36 mM). This method has demonstrated a ping pong mechanism for the trans-activation exchange which accounts for a selective pore carrier model like a gated channel. These asymmetric properties are related to the AS glide sequential model (A and S being Na+ and lactate, respectively) proposed previously for the Na-L-lactate cotransport and to a different accessibility of the organic solute but not of the sodium on the two membrane faces.     

Conversion of the FhuA transport protein into a diffusion channel through the outer membrane of Escherichia coli.

The FhuA receptor protein is involved in energy-coupled transport of Fe3+ via ferrichrome through the outer membrane of Escherichia coli. Since no energy source is known in the outer membrane it is assumed that energy is provided through the action of the TonB, ExbB and ExbD proteins, which are anchored to the cytoplasmic membrane. By deleting 34 amino acid residues of a putative cell surface exposed loop, FhuA was converted from a ligand specific transport protein into a TonB independent and nonspecific diffusion channel. The FhuA deletion derivative FhuA delta 322-355 formed stable channels in black lipid membranes, in contrast to wild-type FhuA which did not increase membrane conductance. The single-channel conductance of the FhuA mutant channels was at least three times larger than that of the general diffusion porins of E. coli outer membrane. It is proposed that the basic structure of FhuA in the outer membrane is a channel formed by beta-barrels. Since the loop extending from residue 316 to 356 is part of the active site of FhuA, it probably controls the permeability of the channel. The transport-active conformation of FhuA is mediated by a TonB-induced conformational change in response to the energized cytoplasmic membrane. The ferrichrome transport rate into cells expressing FhuA delta 322-355 increased linearly with increasing substrate concentration (from 0.5 to 20 microM), in contrast to FhuA wild-type cells, which displayed saturation at 5 microM. This implies that in wild-type cells ferrichrome transport through the outer membrane is the rate-limiting step and that TonB, ExbB and ExbD are only required for outer membrane transport.     

Molecular mechanisms of band 3 inhibitors. 2. Channel blockers

Band 3 is proposed to contain substrate channels that lead from the aqueous medium to a transport site buried within the membrane, and which can be blocked by inhibitors. The inhibitors 1,2-cyclohexanedione (CHD) and dipyridamole (DP) each inhibit the transport site 35Cl NMR line broadening, but neither competes with Cl- for binding. Thus these inhibitors do not occupy the transport site; instead they slow the migration of Cl- between the transport site and the medium. The simplest explanation for this behavior is that CHD and DP block one or more substrate channels. CHD is an arginine-specific covalent modification reagent, and its effectiveness as a channel blocker indicates that the channel contains arginine positive charges to facilitate the migration of anions through the channel. DP is a noncovalent channel blocker that binds with a stoichiometry of 1 molecule per band 3 dimer. DP binding is unaffected by CHD but is prevented by phenylglyoxal (PG), 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS), or niflumic acid. Thus the DP and CHD binding sites are distinct, with DP binding sufficiently close to the transport site to interact with PG and DNDS. It is proposed that substrate channels may be a general feature of transport proteins.     

Evidence for high affinity binding-protein dependent transport systems in gram-positive bacteria and in Mycoplasma

Gram-negative bacteria are surrounded by two membranes. In these bacteria, a class of high affinity transport systems for concentrating substrates from the medium into the cell, involves a binding protein located between the outer and inner membranes, in the periplasmic region. These 'periplasmic binding-proteins' are thought to bind the substrate in the vicinity of the inner membrane, and to transfer it to a complex of inner membrane proteins for concentration into the cytoplasm. We report evidence leading us to propose that a Gram-positive bacterium, Streptococcus pneumoniae, and a mycoplasma, Mycoplasma hyorhinis, which are surrounded by a single membrane and have therefore no periplasmic region, possess an equivalent to the high affinity periplasmic binding-protein dependent transport systems, i.e. extra-cytoplasmic binding lipoprotein dependent transport systems. The 'binding lipoproteins' would be maintained at proximity of the inner membrane by insertion of their N-terminal glyceride-cysteine into this membrane.     

FhuA, a transporter of the Escherichia coli outer membrane, is converted into a channel upon binding of bacteriophage T5.

The Escherichia coli outer membrane protein FhuA catalyzes the transport of Fe3+(-)ferrichrome and is the receptor of phage T5 and phi 80. The purified protein inserted into planar lipid bilayers showed no channel activity. Binding of phage T5 and FhuA resulted in the appearance of high conductance ion channels. The electrophysiological characteristics of the channels (conductance, kinetic behavior, substates, ion selectivity including the effect of ferrichrome) showed similarities with those of the channel formed by a FhuA derivative from which the 'gating loop' (delta 322-355) had been removed. binding of phage T5 to FhuA in E.coli cells conferred SDS sensitivity to the bacteria, suggesting that such channels also exist in vivo. These data suggest that binding of T5 to loop 322-355 of FhuA, which constitutes the T5 binding site, unmasks an inner channel in FhuA. Both T5 and ferrichrome bind to the closed state of the channel but only T5 can trigger its opening.     

Cytochalasin B and the kinetics of inhibition of biological transport: a case of asymmetric binding to the glucose carrier

Cytochalasin B inhibits glucose transport in human erythrocytes by competing with glucose for the carrier on the inner surface of the cell membrane, but there is no cytochalasin site associated with the outware-facing form of the carrier. Such asymmetry may be demonstrated by zero trans exit and entry experiments, whereas Sen-Widdas exit experiments are not easily interpretable. The orientation of the transport system appears to be reversed in certain other cell types: chich embryo fibroblasts, Novikoff hepatoma cells and HeLa cells. Here the cytochalasin site is present in the external but not internal carrier form.     

Cytochalasin B and the kinetics of inhibition of biological transport. A case of asymmetric binding to the glucose carrier

Cytochalasin B inhibits glucose transport in human erythrocytes by competing with glucose for the carrier on the inner surface of the cell membrane, but there is no cytochalasin site associated with the outward-facing form of the carrier. Such asymmetry may be demonstrated by zero trans exit and entry experiments, whereas Sen-Widdas exit experiments are not easily interpretable. The orientation of the transport system appears to be reversed in certain other cell types: chick embryo fibroblasts, Novikoff hepatoma cells and HeLa cells. Here the cytochalasin site is present in the external but not internal carrier form.     

Protein translocation across membranes.

Cellular membranes act as semipermeable barriers to ions and macromolecules. Specialized mechanisms of transport of proteins across membranes have been developed during evolution. There are common mechanistic themes among protein translocation systems in bacteria and in eukaryotic cells. Here we review current understanding of mechanisms of protein transport across the bacterial plasma membrane as well as across several organelle membranes of yeast and mammalian cells. We consider a variety of organelles including the endoplasmic reticulum, outer and inner membranes of mitochondria, outer, inner, and thylakoid membranes of chloroplasts, peroxisomes, and lysosomes. Several common principles are evident: (a) multiple pathways of protein translocation across membranes exist, (b) molecular chaperones are required in the cytosol, inside the organelle, and often within the organelle membrane, (c) ATP and/or GTP hydrolysis is required, (d) a proton-motive force across the membrane is often required, and (e) protein translocation occurs through gated, aqueous channels. There are exceptions to each of these common principles indicating that our knowledge of how proteins translocate across membranes is not yet complete.     

Comparison of the TIM and TOM channel activities of the mitochondrial protein import complexes

Water-filled channels are central to the process of translocating proteins since they provide aqueous pathways through the hydrophobic environment of membranes. The Tom and Tim complexes translocate precursors across the mitochondrial outer and inner membranes, respectively, and contain channels referred to as TOM and TIM (previously called PSC and MCC). In this study, little differences were revealed from a direct comparison of the single channel properties of the TOM and TIM channels of yeast mitochondria. As they perform similar functions in translocating proteins across membranes, it is not surprising that both channels are high conductance, voltage-dependent channels that are slightly cation selective. Reconstituted TIM and TOM channel activities are not modified by deletion of the outer membrane channel VDAC, but are similarly affected by signal sequence peptides.     

Substrate specificity within a family of outer membrane carboxylate channels

Many Gram-negative bacteria, including human pathogens such as Pseudomonas aeruginosa, do not have large-channel porins. This results in an outer membrane (OM) that is highly impermeable to small polar molecules, making the bacteria intrinsically resistant towards many antibiotics. In such microorganisms, the majority of small molecules are taken up by members of the OprD outer membrane protein family. Here we show that OprD channels require a carboxyl group in the substrate for efficient transport, and based on this we have renamed the family Occ, for outer membrane carboxylate channels. We further show that Occ channels can be divided into two subfamilies, based on their very different substrate specificities. Our results rationalize how certain bacteria can efficiently take up a variety of substrates under nutrient-poor conditions without compromising membrane permeability. In addition, they explain how channel inactivation in response to antibiotics can cause resistance but does not lead to decreased fitness.     

The carboxyl-terminal third of the dicarboxylate carrier is crucial for productive association with the inner membrane twin-pore translocase

The carrier proteins of the mitochondrial inner membrane consist of three structurally related tandem repeats (modules). Several different, and in some cases contradictory, views exist on the role individual modules play in carrier transport across the mitochondrial membranes and how they promote protein insertion into the inner membrane. Thus, by use of specific translocation intermediates, we performed a detailed analysis of carrier biogenesis and assessed the physical association of carrier modules with the inner membrane translocation machinery. Here we have reported that each module of the dicarboxylate carrier contains sufficient targeting information for its transport across the outer mitochondrial membrane. The carboxyl-terminal module possesses major targeting information to facilitate the direct binding of the carrier protein to the inner membrane twin-pore translocase and subsequent insertion into the inner membrane in a membrane potential-dependent manner. We concluded that, in this case, a single structural repeat can drive inner membrane insertion, whereas all three related units contribute targeting information for outer membrane translocation.     

Reconstituted TOM core complex and Tim9/Tim10 complex of mitochondria are sufficient for translocation of the ADP/ATP carrier across membranes

Precursor proteins of the solute carrier family and of channel forming Tim components are imported into mitochondria in two main steps. First, they are translocated through the TOM complex in the outer membrane, a process assisted by the Tim9/Tim10 complex. They are passed on to the TIM22 complex, which facilitates their insertion into the inner membrane. In the present study, we have analyzed the function of the Tim9/Tim10 complex in the translocation of substrates across the outer membrane of mitochondria. The purified TOM core complex was reconstituted into lipid vesicles in which purified Tim9/Tim10 complex was entrapped. The precursor of the ADP/ATP carrier (AAC) was found to be translocated across the membrane of such lipid vesicles. Thus, these components are sufficient for translocation of AAC precursor across the outer membrane. Peptide libraries covering various substrate proteins were used to identify segments that are bound by Tim9/Tim10 complex upon translocation through the TOM complex. The patterns of binding sites on the substrate proteins suggest a mechanism by which portions of membrane-spanning segments together with flanking hydrophilic segments are recognized and bound by the Tim9/Tim10 complex as they emerge from the TOM complex into the intermembrane space.