Differential influence of the 4F2 heavy chain and the protein related to b(0,+) amino acid transport on substrate affinity of the heteromeric b(0,+) amino acid transporter

We provide evidence here that b(0,+) amino acid transporter (b(0, +)AT) interacts with 4F2 heavy chain (4F2hc) as well as with the protein related to b(0,+) amino acid transporter (rBAT) to constitute functionally competent b(0,+)-like amino acid transport systems. This evidence has been obtained by co-expression of b(0, +)AT and 4F2hc or b(0,+)AT and rBAT in human retinal pigment epithelial cells and in COS-1 cells. The ability to interact with 4F2hc and rBAT is demonstrable with mouse b(0,+)AT as well as with human b(0,+)AT. Even though both the 4F2hc x b(0,+)AT complex and the rBAT x b(0,+)AT complex exhibit substrate specificity that is characteristic of system b(0,+), these two complexes differ significantly in substrate affinity. The 4F2hc x b(0,+)AT complex has higher substrate affinity than the rBAT x b(0,+)AT complex. In situ hybridization studies demonstrate that the regional distribution pattern of mRNA in the kidney is identical for b(0,+)AT and 4F2hc. The pattern of rBAT mRNA expression is different from that of b(0,+)AT mRNA and 4F2hc mRNA, but there are regions in the kidney where b(0,+)AT mRNA expression overlaps with rBAT mRNA expression as well as with 4F2hc mRNA expression.     

The light subunit of system b(o,+) is fully functional in the absence of the heavy subunit

The heteromeric amino acid transporters are composed of a type II glycoprotein and a non-glycosylated polytopic membrane protein. System b(o,+) exchanges dibasic for neutral amino acids. It is composed of rBAT and b(o,+)AT, the latter being the polytopic membrane subunit. Mutations in either of them cause malfunction of the system, leading to cystinuria. b(o,+)AT-reconstituted systems from HeLa or MDCK cells catalysed transport of arginine that was totally dependent on the presence of one of the b(o,+) substrates inside the liposomes. rBAT was essential for the cell surface expression of b(o,+)AT, but it was not required for reconstituted b(o,+)AT transport activity. No system b(o,+) transport was detected in liposomes derived from cells expressing rBAT alone. The reconstituted b(o,+)AT showed kinetic asymmetry. Expressing the cystinuria-specific mutant A354T of b(o,+)AT in HeLa cells together with rBAT resulted in defective arginine uptake in whole cells, which was paralleled by the reconstituted b(o,+)AT activity. Thus, subunit b(o,+)AT by itself is sufficient to catalyse transmembrane amino acid exchange. The polytopic subunits may also be the catalytic part in other heteromeric transporters.     

Luminal heterodimeric amino acid transporter defective in cystinuria

Mutations of the glycoprotein rBAT cause cystinuria type I, an autosomal recessive failure of dibasic amino acid transport (b(0,+) type) across luminal membranes of intestine and kidney cells. Here we identify the permease-like protein b(0,+)AT as the catalytic subunit that associates by a disulfide bond with rBAT to form a hetero-oligomeric b(0,+) amino acid transporter complex. We demonstrate its b(0,+)-type amino acid transport kinetics using a heterodimeric fusion construct and show its luminal brush border localization in kidney proximal tubule. These biochemical, transport, and localization characteristics as well as the chromosomal localization on 19q support the notion that the b(0,+)AT protein is the product of the gene defective in non-type I cystinuria.     

The amino acid transport system b(o,+) and cystinuria

Amino acid transport in mammalian plasma membranes is mediated by a multiplicity of amino acid transport systems. Some of them (systems L, y+ L, x(c)- and b(o,+)) are the result of the activity of heteromeric amino acid transporters (HAT) (i.e. transport activity is elicited by the coexpression of a heavy and a light subunit). The two heavy subunits known today (HSHAT: rBAT and 4F2hc) were identified in 1992, and light subunits (LSHAT: LAT-1, LAT-2, asc-1, y+ LAT-1, y+ LAT-2, xCT and b(o,+)AT) have been cloned in the last 2 years. Defects in two genes of this family (SLC3A1, encoding rBAT and SLC7A9, encoding b(o,+)AT) are responsible for cystinuria, an inherited aminoaciduria of cystine and dibasic amino acids. This finding and functional studies of rBAT and b(o,+)AT suggested that these two proteins encompassed the high-affinity renal reabsorption system of cystine. In contrast to this view, immunofluorescence studies showed that rBAT is most abundant in the proximal straight tubule, and b(o,+)AT is most abundant in the proximal convoluted tubule of the nephron. The need for a new light subunit for rBAT and a heavy subunit for b(o,+)AT is discussed.     

Expression of the activity of cystine/glutamate exchange transporter,system x(c)(-), by xCT and rBAT

The expression of the activity of cystine/glutamate exchange transporter, designated system x(c)(-), requires two components, xCT and 4F2 heavy chain (4F2hc) in Xenopus oocytes. rBAT (related to b(0,+) amino acid transporter) has a significant homology to 4F2hc and is known to be located in the apical membrane of epithelial cells. To determine whether xCT can associate with rBAT and express the activity of system x(c)(-), xCT, and rBAT were co-expressed in Xenopus oocytes and in mammalian cultured cells. In the oocytes injected with rBAT cRNA alone, the activities of cystine and arginine transport were induced, indicating that the system b(0,+)-like transporter was expressed by associating the exogenous rBAT with an endogenous b(0,+)AT-like factor as reported previously. In the oocytes injected with xCT and rBAT cRNAs, the activity of cystine transport was further induced. This induced activity of cystine transport was partially inhibited by glutamate or arginine and completely inhibited by adding both amino acids. In these oocytes, the activity of glutamate transport was also induced and it was strongly inhibited by cystine. In NIH3T3 cells transfected with xCT cDNA alone, the activity of cystine transport was significantly increased, and in the cells transfected with both xCT and rBAT cDNAs, the activity of cystine transport was further enhanced. The enhanced activity was Na(+)-independent and was inhibited by glutamate and homocysteate. These results indicate that rBAT can replace 4F2hc in the expression of the activity of system x(c)(-) and suggest that system x(c)(-) activity could be expressed in the apical membrane of epithelial cells.     

Identification of in vivo disulfide conformation of TRPA1 ion channel

TRPA1 (transient receptor potential ankyrin 1) is an ion channel expressed in the termini of sensory neurons and is activated in response to a broad array of noxious exogenous and endogenous thiol-reactive compounds, making it a crucial player in chemical nociception. A number of conserved cysteine residues on the N-terminal domain of the channel have been identified as critical for sensing these electrophilic pungent chemicals, and our recent EM structure with modeled domains predicts that these cysteines form a ligand-binding pocket, allowing for the possibility of disulfide bonding between the cysteine residues. Here, we present a comprehensive mass spectrometry investigation of the in vivo disulfide bonding conformation and in vitro reactivity of 30 of the 31 cysteine residues in the TRPA1 ion channel. Four disulfide bonds were detected in the in vivo TRPA1 structure: Cys-666-Cys-622, Cys-666-Cys-463, Cys-622-Cys-609, and Cys-666-Cys-193. All of the cysteines detected were reactive to N-methylmaleimide (NMM) in vitro, with varying degrees of labeling efficiency. Comparison of the ratio of the labeling efficiency at 300 μM versus 2 mM NMM identified a number of cysteine residues that were outliers from the mean labeling ratio, suggesting that protein conformation changes rendered these cysteines either more or less protected from labeling at the higher NMM concentrations. These results indicate that the activation mechanism of TRPA1 may involve N-terminal conformation changes and disulfide bonding between critical cysteine residues.     

Identification of an amino acid transporter associated with the cystinuria-related type II membrane glycoprotein.

We identified an amino acid transporter that is associated with the cystinuria-related type II membrane glycoprotein, rBAT (related to b(0,+) amino acid transporter). The transporter designated BAT1 (b(0, +)-type amino acid transporter 1) from rat kidney was found to be structurally related to recently identified amino acid transporters for system L, system y(+)L, and system x(-)C, which are linked, via a disulfide bond, to the other type II membrane glycoprotein, 4F2hc (4F2 heavy chain). In the nonreducing condition, a 125-kDa band, which seems to correspond to the heterodimeric complex of BAT1 and rBAT, was detected in rat kidney with anti-BAT1 antibody. The band was shifted to 41 kDa in the reducing condition, confirming that BAT1 and rBAT are linked via a disulfide bond. The BAT1 and rBAT proteins were shown to be colocalized in the apical membrane of the renal proximal tubules where massive cystine transport had been proposed. When expressed in COS-7 cells with rBAT, but not with 4F2hc, BAT1 exhibited a Na(+)-independent transport of cystine as well as basic and neutral amino acids with the properties of system b(0,+). The results from the present investigation were used to establish a family of amino acid transporters associated with type II membrane glycoproteins.     

Conserved intramolecular disulfide bond is critical to trafficking and fate of ATP-binding cassette (ABC) transporters ABCB6 and sulfonylurea receptor 1 (SUR1)/ABCC8

The ATP-binding cassette (ABC) transporter ABCB6 is a mitochondrial porphyrin transporter that activates porphyrin biosynthesis. ABCB6 lacks a canonical mitochondrial targeting sequence but reportedly traffics to other cellular compartments such as the plasma membrane. How ABCB6 reaches these destinations is unknown. In this study, we show that endogenous ABCB6 is glycosylated in multiple cell types, indicating trafficking through the endoplasmic reticulum (ER), and has only one atypical site for glycosylation (NXC) in its amino terminus. ABCB6 remained glycosylated when the highly conserved cysteine (Cys-8) was substituted with serine to make a consensus site, NXS. However, this substitution blocked ER exit and produced ABCB6 degradation, which was mostly reversed by the proteasomal inhibitor MG132. The amino terminus of ABCB6 has an additional highly conserved ER luminal cysteine (Cys-26). When Cys-26 was mutated alone or in combination with Cys-8, it also resulted in instability and ER retention. Further analysis revealed that these two cysteines form a disulfide bond. We discovered that other ABC transporters with an amino terminus in the ER had similarly configured conserved cysteines. This analysis led to the discovery of a disease-causing mutation in the sulfonylurea receptor 1 (SUR1)/ABCC8 from a patient with hyperinsulinemic hypoglycemia. The mutant allele only contains a mutation in a conserved amino-terminal cysteine, producing SUR1 that fails to reach the cell surface. These results suggest that for ABC transporters the propensity to form a disulfide bond in the ER defines a unique checkpoint that determines whether a protein is ER-retained.     

Cloning and expression of a b(0,+)-like amino acid transporter functioning as a heterodimer with 4F2hc instead of rBAT. A new candidate gene for cystinuria.

We have cloned a transporter protein from rabbit small intestine, which, when coexpressed with the 4F2 heavy chain (4F2hc) in mammalian cells, induces a b(0,+)-like amino acid transport activity. This protein (4F2-lc6 for the sixth member of the 4F2 light chain family) consists of 487 amino acids and has 12 putative transmembrane domains. At the level of amino acid sequence, 4F2-lc6 shows significant homology (44% identity) to the other five known members of the 4F2 light chain family, namely LAT1 (4F2-lc1), y(+)LAT1 (4F2-lc2), y(+)LAT2 (4F2-lc3), xCT (4F2-lc4), and LAT2 (4F2-lc5). The 4F2hc/4F2-lc6 complex-mediated transport process is Na(+)-independent and exhibits high affinity for neutral and cationic amino acids and cystine. These characteristics are similar to those of the b(0,+)-like amino acid transport activity previously shown to be associated with rBAT (protein related to b(0,+) amino acid transport system). However, the newly cloned 4F2-lc6 does not interact with rBAT. This is the first report of the existence of a b(0,+)-like amino acid transport process that is independent of rBAT. 4F2-lc6 is expressed predominantly in the small intestine and kidney. Based on the characteristics of the transport process mediated by the 4F2hc/4F2-lc6 complex and the expression pattern of 4F2-lc6 in mammalian tissues, we suggest that 4F2-lc6 is a new candidate gene for cystinuria.     

Expression and biochemical analysis of the entire HIV-2 gp41 ectodomain: determinants of stability map to N- and C-terminal sequences outside the 6-helix bundle core

The folding of HIV gp41 into a 6-helix bundle drives virus-cell membrane fusion. To examine the structural relationship between the 6-helix bundle core domain and other regions of gp41, we expressed in Escherichia coli, the entire ectodomain of HIV-2(ST) gp41 as a soluble, trimeric maltose-binding protein (MBP)/gp41 chimera. Limiting proteolysis indicated that the Cys-591-Cys-597 disulfide-bonded region is outside a core domain comprising two peptides, Thr-529-Trp-589 and Val-604-Ser-666. A biochemical examination of MBP/gp41 chimeras encompassing these core peptides indicated that the N-terminal polar segment, 521-528, and C-terminal membrane-proximal segment, 658-666, cooperate in stabilizing the ectodomain. A functional interaction between sequences outside the gp41 core may contribute energy to membrane fusion.     

The molecular bases of cystinuria and lysinuric protein intolerance

Cystinuria and lysinuric protein intolerance are inherited aminoacidurias caused by defective amino-acid transport activities linked to a family of heteromeric amino-acid transporters (HATs). HATs comprise two subunits: co-expression of subunits 4F2hc and y(+)LAT-1 induces the efflux of dibasic amino acids from cells, whereas co-expression of subunits rBAT and b(o,+)AT induces the renal reabsorption and intestinal absorption of cystine and dibasic amino acids at the brush border of epithelial cells. Recently, the role of b(o,+)AT (SLC7A9) in cystinuria (non Type I) and the role of y(+)LAT-1 (SLC7A7) in lysinuric protein intolerance have been demonstrated.     

Distribution and abundance of nutrient transporter mRNA in the intestinal tract of the black bear, Ursus americanus

End products of digestion are absorbed by the body through the action of transporter proteins expressed on the apical membrane of intestinal epithelial cells. We investigated the mRNA abundance and distribution of a peptide transporter (PepT1), a glucose transporter (SGLT1), two amino acid transporters (NBAT and b(o,+)AT), and a digestive enzyme, aminopeptidase N (APN), in the intestinal tract of black bears (Ursus americanus). Intestinal total RNA was isolated from 10 bears and abundance of PepT1, SGLT1, NBAT, b(o,+)AT, and APN mRNA were determined by Northern blots. Abundance of PepT1 (P<0.05), APN (P<0.05), and SGLT1 (P<0.0001) changed quadratically from the proximal to distal intestine with abundance being greatest in the midregion. Abundance of b(o,+)AT mRNA increased linearly (P<0.05) from the proximal to distal intestine. The number of molecules of mRNA/ng of total RNA for each gene was determined using Real-Time PCR. PepT1 mRNA was present at 10-fold or greater levels than amino acid transporter mRNA in all segments of the intestine, suggesting that di- and tripeptides constitute a major form in which amino acids are absorbed in the black bear. The abundance of NBAT and b(o,+)AT mRNA was greater towards the distal intestine, suggesting a role in salvaging unabsorbed amino acids.     

Identification and characterization of a novel member of the heterodimeric amino acid transporter family presumed to be associated with an unknown heavy chain

We identified a novel amino acid transporter designated Asc-2 (for asc-type amino acid transporter 2). Asc-2 exhibited relatively low but significant sequence similarity to the members of the heterodimeric amino acid transporters. The cysteine residue responsible for the disulfide bond formation between transporters (light chains) and heavy chain subunits in the heterodimeric amino acid transporters is conserved for Asc-2. Asc-2 is, however, not colocalized with the already known heavy chains such as 4F2 heavy chain (4F2hc) or related to b(0,+) amino acid transporter (rBAT) in mouse kidney. Because Asc-2 solely expressed or coexpressed with 4F2hc or rBAT did not induce functional activity, we generated fusion proteins in which Asc-2 is connected with 4F2hc or rBAT. The fusion proteins were sorted to the plasma membrane and expressed the function corresponding to the Na(+)-independent small neutral amino acid transport system asc. Distinct from the already identified system asc transporter Asc-1 which is associated with 4F2hc, Asc-2-mediated transport is less stereoselective and did not accept some of the high affinity substrates of Asc-1 such as alpha-aminoisobutyric acid and beta-alanine. Asc-2 message was detected in kidney, placenta, spleen, lung, and skeletal muscle. In kidney, Asc-2 protein was present in the epithelial cells lining collecting ducts. In the Western blot analysis on mouse erythrocytes and kidney, Asc-2 was detected as multiple bands in the nonreducing condition, whereas the bands shifted to a single band at lower molecular weight, suggesting the association of Asc-2 with other protein(s) via a disulfide bond. The finding of Asc-2 would lead to the establishment of a new subgroup of heterodimeric amino acid transporter family which includes transporters associated not with 4F2hc or rBAT but with other unknown heavy chains.     

Determination of disulfide array and subunit structure of taste-modifying protein, miraculin

The taste-modifying protein, miraculin (Theerasilp, S. et al. (1989) J. Biol. Chem. 264, 6655-6659) has seven cysteine residues in a molecule composed of 191 amino acid residues. The formation of three intrachain disulfide bridges at Cys-47-Cys-92, Cys-148-Cys-159 and Cys-152-Cys-155 and one interchain disulfide bridge at Cys-138 was determined by amino acid sequencing and composition analysis of cystine-containing peptides isolated by HPLC. The presence of an interchain disulfide bridge was also supported by the fact that the cystine peptide containing Cys-138 showed a negative color test for the free sulfhydryl group and a positive test after reduction with dithiothreitol. The molecular mass of non-reduced miraculin (43 kDa) in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was nearly twice the calculated molecular mass based on the amino acid sequence and the carbohydrate content of reduced miraculin (25 kDa). The molecular mass of native miraculin determined by low-angle laser light scattering was 90 kDa. Application of a crude extract of miraculin to a Sephadex G-75 column indicated that the taste-modifying activity appears at 52 kDa. It was concluded that native miraculin in pure form is a tetramer of the 25 kDa-peptide and native miraculin in crude state or denatured, non-reduced miraculin in pure form is a dimer of the peptide. Both tetramer miraculin and native dimer miraculin in crude state had the taste-modifying activity.     

Regulation of amino acid/carnitine transporter B 0,+ (ATB 0,+) in astrocytes by protein kinase C: independent effects on raft and non-raft transporter subpopulations

Neutral and basic amino acid transporter B(0,+) belongs to a Na,Cl-dependent superfamily of proteins transporting neurotransmitters, amino acids and osmolytes, known to be regulated by protein kinase C (PKC). The present study demonstrates an increased phosphorylation of B(0,+) on serine moiety after treatment of rat astrocytes with phorbol 12-myristate 13-acetate, a process correlated with an augmented activity of l-leucine transport and an enhanced presence of the transporter at the cell surface. After solubilization with Triton X-100 and sucrose gradient centrifugation, B(0,+) was detected in non-raft as well as in detergent-resistant raft fractions under control conditions, while phorbol 12-myristate 13-acetate treatment resulted in a complete disappearance of the transporter from the raft fraction. B(0,+) was observed to interact with caveolin-1 and flotillin-1 (reggie-2) proteins, the markers of detergent-resistant microdomains of plasma membrane. As verified in immunocytochemistry and immunoprecipitation experiments, modification of PKC activity did not affect these interactions. It is proposed that PKC reveals different effects on raft and non-raft subpopulations of B(0,+). Phorbol ester treatment results in trafficking of the transporter from the intracellular pool to non-raft microdomains and increased activity, while B(0,+) present in raft microdomains undergoes either internalization or is transferred laterally to non-raft domains.     

Identification of a novel Na+-independent acidic amino acid transporter with structural similarity to the member of a heterodimeric amino acid transporter family associated with unknown heavy chains

We identified a novel Na(+)-independent acidic amino acid transporter designated AGT1 (aspartate/glutamate transporter 1). AGT1 exhibits the highest sequence similarity (48% identity) to the Na(+)-independent small neutral amino acid transporter Asc (asc-type amino acid transporter)-2 a member of the heterodimeric amino acid transporter family presumed to be associated with unknown heavy chains (Chairoungdua, A., Kanai, Y., Matsuo, H., Inatomi, J., Kim, D. K., and Endou, H. (2001) J. Biol. Chem. 276, 49390-49399). The cysteine residue responsible for the disulfide bond formation between transporters (light chains) and heavy chain subunits of the heterodimeric amino acid transporter family is conserved for AGT1. Because AGT1 solely expressed or coexpressed with already known heavy chain 4F2hc (4F2 heavy chain) or rBAT (related to b(0,+)-amino acid transporter) did not induce functional activity, we generated fusion proteins in which AGT1 was connected with 4F2hc or rBAT. The fusion proteins were sorted to the plasma membrane and expressed the Na(+)-independent transport activity for acidic amino acids. Distinct from the Na(+)-independent cystine/glutamate transporter xCT structurally related to AGT1, AGT1 did not accept cystine, homocysteate, and l-alpha-aminoadipate and exhibited high affinity to aspartate as well as glutamate, suggesting that the negative charge recognition site in the side chain-binding site of AGT1 would be closer to the alpha-carbon binding site compared with that of xCT. The AGT1 message was predominantly expressed in kidney. In mouse kidney, AGT1 protein was present in the basolateral membrane of the proximal straight tubules and distal convoluted tubules. In the Western blot analysis, AGT1 was detected as a high molecular mass band in the nonreducing condition, whereas the band shifted to a 40-kDa band corresponding to the AGT1 monomer in the reducing condition, suggesting the association of AGT1 with other protein via a disulfide bond. The finding of AGT1 and Asc-2 has established a new subgroup of the heterodimeric amino acid transporter family whose members associate not with 4F2hc or rBAT but with other unknown heavy chains.     

In vitro folding and assembly of the Escherichia coli ATP-binding cassette transporter, BtuCD

Studies on membrane protein folding have focused on monomeric α-helical proteins and a major challenge is to extend this work to larger oligomeric membrane proteins. Here, we study the Escherichia coli (E. coli) ATP-binding cassette (ABC) transporter that imports vitamin B(12) (the BtuCD protein) and use it as a model system for investigating the folding and assembly of a tetrameric membrane protein complex. Our work takes advantage of the modular organization of BtuCD, which consists of two transmembrane protein subunits, BtuC, and two cytoplasmically located nucleotide-binding protein subunits, BtuD. We show that the BtuCD transporter can be re-assembled from both prefolded and partly unfolded, urea denatured BtuC and BtuD subunits. The in vitro re-assembly leads to a BtuCD complex with the correct, native, BtuC and BtuD subunit stoichiometry. The highest rates of ATP hydrolysis were achieved for BtuCD re-assembled from partly unfolded subunits. This supports the idea of cooperative folding and assembly of the constituent protein subunits of the BtuCD transporter. BtuCD folding also provides an opportunity to investigate how a protein that contains both membrane-bound and aqueous subunits coordinates the folding requirements of the hydrophobic and hydrophilic subunits.     

Three-dimensional structure of the kringle sequence: structure of prothrombin fragment 1

The three-dimensional structure of bovine prothrombin fragment 1 has been solved at 2.8-A resolution. The electron density clearly reveals four disulfide bridges along with more than 80% of the side chains completely in density, which correspond faithfully to the kringle sequence, its preceding 30 residues, and the dodecapeptide carboxy terminal; the polysaccharide and the first 35 residues of the amino terminal of fragment 1 are disordered or about 40% of the structure. The folding of the kringle sequence is based upon close disulfide van der Waals contacts between Cys-87-Cys-127 and Cys-115-Cys-139 (4.1 A between midpoints of the bridges), two antiparallel strands of highly conserved (113-118, 124-129) beta-structure, and the stacking of some conserved aromatic residues, all near the center of the folded structure. Moreover, the overall folding appears to be duplicated as a pair of stacked duplex loops with an antiparallel open loop. The overall shape of the kringle structure approximates an eccentric oblate ellipsoid of dimensions 11 X 28 X 30 A. The residues immediately preceding the kringle are dominated by alpha-helical structure (Phe-41-Cys-48; Leu-56-Glu-63). Residues Phe-41-Trp-42 and Tyr-45, which are conserved in factor IX, factor X, protein C, and protein Z, form another aromatic stacked cluster while the Cys-48-Cys-61 disulfide loop corresponds to the well-known alpha/beta structural unit. The dodecapeptide carboxy-terminal interkringle chain extends along the periphery of the kringle in its plane and forms a beta-structure with the kringle-closing Ser-140-Val-143 tetrapeptide.     

Role of Transmembrane Domain 8 in Substrate Selectivity and Translocation of SteT,a Member of the l-Amino Acid Transporter (LAT) Family

System l-amino acid transporters (LAT) belong to the amino acid, polyamine, and organic cation superfamily of transporters and include the light subunits of heteromeric amino acid transporters and prokaryotic homologues. Cysteine reactivity of SteT (serine/threonine antiporter) has been used here to study the substrate-binding site of LAT transporters. Residue Cys-291, in transmembrane domain 8 (TM8), is inactivated by thiol reagents in a substrate protectable manner. Surprisingly, DTT activated the transporter by reducing residue Cys-291. Cysteine-scanning mutagenesis of TM8 showed DTT activation in the single-cysteine mutants S287C, G294C, and S298C, lining the same α-helical face. S-Thiolation in Escherichia coli cells resulted in complete inactivation of the single-cysteine mutant G294C. l-Serine blocked DTT activation with an EC₅₀ similar to the apparent K_M of this mutant. Thus, S-thiolation abolished substrate translocation but not substrate binding. Mutation of Lys-295, to Cys (K295C) broadened the profile of inhibitors and the spectrum of substrates with the exception of imino acids. A structural model of SteT based on the structural homologue AdiC (arginine/agmatine antiporter) positions residues Cys-291 and Lys-295 in the putative substrate binding pocket. All this suggests that Lys-295 is a main determinant in the recognition of the side chain of SteT substrates. In contrast, Gly-294 is not facing the surface, suggesting conformational changes involving TM8 during the transport cycle. Our results suggest that TM8 sculpts the substrate-binding site and undergoes conformational changes during the transport cycle of SteT.