Role of transmembrane domain 8 in substrate selectivity and translocation of SteT, a member of the L-amino acid transporter (LAT) family

Abstract

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 alpha-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 EC(50) 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.

FIGURE 1.

Residue Cys-291 is the target of inactivation of SteT by MTSET in a manner protectable by substrate. a, l-serine/l-serine exchange in proteoliposomes containing wild-type (SteT) or the indicated SteT mutants, treated with 1 mm MTSET (closed bars) or DMSO (MTSET vehicle) (control; open bars) for 5 min. The five Cys residues of SteT were all simultaneously mutated to Ser (Cys-less) or individually mutated, as indicated. For the single Cys-291 (single-C291), the Cys at position 291 was reintroduced by mutation of the Cys-less mutant. MTSET inactivated l-serine/l-serine exchange in wild-type SteT and in all the mutants (p ≤ 0.001), except for the Cys-less and C291S mutants. b, protection of transport by l-serine from the inactivation by MTSET. Wild-type (squares) or single Cys-291 (circles) SteT proteoliposomes were treated for 5 min with the indicated concentrations of MTSET. This treatment was performed in the presence of 30 mm l-serine (closed squares and circles) or l-arginine (gray squares and circles) or in the absence of amino acids (open squares). Data are shown as the residual l-serine/l-serine exchange (%) with respect to proteoliposomes preincubated with DMSO. a and b, transport of 10 μm l-[³H]serine was measured for 10 min (linear conditions) in proteoliposomes containing 4 mm unlabeled l-serine or l-arginine (negative control of antiport). l-Serine/l-serine exchange was calculated by subtracting transport in proteoliposomes containing l-arginine from that in those containing l-serine. In no case was transport in proteoliposomes containing l-arginine affected by MTSET treatment (data not shown). Data (mean ± S.E.) correspond to representative experiments run in triplicate. When not visible, errors are smaller than symbols. prot, protein.

FIGURE 2.

Residue Cys-291 is the target of activation of SteT by DTT. Proteoliposomes containing wild-type SteT (a) or the indicated SteT mutants (b) were treated with 1 mm MTSET (a) or 10 mm DTT (a and b) for 5 min as indicated. Exchange of 10 μm l-[³H]serine, 4 mm l-serine was subsequently measured and calculated as indicated in the legend of Fig. 1. In no case was transport into proteoliposomes containing l-arginine affected by thiol reagent treatment (data not shown). Data (mean ± S.E.) correspond to representative experiments run in triplicate. a, DTT reverses inactivation of wild-type SteT by MTSET. l-Serine/l-serine exchange inactivated by MTSET (p ≤ 0.001) was reversed by DTT and reached an exchange activity higher than controls (proteoliposomes treated with DMSO) (p ≤ 0.01). DTT treatment alone also activated exchange (p ≤ 0.01). b, l-serine/l-serine exchange in proteoliposomes, containing the indicated mutated versions of SteT, treated (closed bars) or not (control; open bars) with DTT. Exchange activity was activated by DTT in all the mutants (at least p ≤ 0.01), except for Cys-less Stet and C291S. When not visible, error bars are smaller than lines. prot, protein.

FIGURE 3.

Cysteine scanning mutagenesis of TM8. Proteoliposomes with wild-type SteT (SteT), a version devoid of Cys residues (Cysless), as in Fig. 1b or different mutants with a single Cys mutation introduced into Cys-less SteT at different positions in TM8 were used as indicated. Data (mean ± S.E.) correspond to representative experiments run in triplicate. a, transport of 10 μm l-[³H]serine was measured for 10 min in proteoliposomes containing 4 mm l-serine (closed bars) or l-arginine (open bars). l-Serine/l-serine exchange was detected (i.e. higher transport in l-serine- than in l-arginine-containing proteoliposomes) for wild-type SteT, its Cys-less mutant, and for the single-Cys mutants I288C, C291, L292C, K295C, L297C, S298C, and F299C (at least p ≤ 0.05). *, transport in single-Cys SteT mutant K295C proteoliposomes containing l-arginine was higher than for all other forms of SteT investigated (p ≤ 0.001). b, impact of MTSET and DTT treatment on l-serine/l-serine exchange. Proteoliposomes were treated with 1 mm MTSET ((gray bars) or 10 mm DTT (open bars) or DMSO (closed bars) for 5 min. Transport of 10 μm l-[³H]serine was subsequently measured for 10 min in proteoliposomes containing or lacking 4 mm l-serine. l-Serine/l-serine exchange was calculated by subtracting transport in proteoliposomes containing no amino acid from that in those containing l-serine. *, significant effect of MTSET treatment (at least p ≤ 0.05). DTT activated exchange in wild-type SteT (*; at least p ≤ 0.01), and in the single-Cys SteT mutants S287C, C291, G294C, and S298C (inverted triangles) (at least p ≤ 0.01). DTT inhibited exchange in I288C and L297C (*; at least p ≤ 0.01). c, expression levels in E. coli of the different SteT variants. Western blot was performed using 1% SDS-solubilized membranes (10 μg of total protein per variant) and detected with the anti-His tag probe HisProbe-HRP. prot, protein.

FIGURE 4.

S-thiolation of the single-Cys SteT mutant G294C in E. coli abolished transport activity. a, G294C mutant is oxidized by S-thiolation. The purified mutant SteT was treated or not (none) with 1 mm DTT, 20 mm ascorbate, 1 mm dimedone followed by 1 mm DTT or 20 mm arsenite as indicated. Each sample was subsequently labeled with biotin-HPDP and subjected to SDS-PAGE (1 μg of protein/sample). Coomassie Blue staining shown in upper panel. Biotin labeling was analyzed by blotting with avidin-HRP, as shown in lower panel. The single-Cys SteT mutant G294C has the typical mobility of the SteT monomer (∼40 kDa), and only DTT or dimedone plus DTT treatment increased biotin labeling, indicating that Cys-294 is S-thiolated by a small molecular weight compound. b, DTT treatment activated the single-Cys SteT mutant G294C in E. coli. An E. coli strain devoid of the serine transporters LIV1, SstT, and TdcC was transformed with the mutant (squares) or with nonrecombinant vector (circles) and transport of 10 μm l-[³H]serine was measured for the indicated time in the absence (control; open symbols) or presence (closed symbols) of 5 mm DTT. Only cells expressing the mutant and treated with DTT showed l-serine transport greater than background (that seen in cells transformed with nonrecombinant vector, in the absence or presence of DTT) (at least p ≤ 0.05). Transport is expressed in picomoles of l-serine/mg of protein. Data (mean ± S.E.) correspond to a representative experiment with four replicates. When not visible, error bars are smaller than symbols. A second experiment gave similar results.

FIGURE 5.

l-Serine blocked activation by DTT of the single-Cys SteT mutant G294C. a, proteoliposomes harboring mutant G294C were incubated without (open circles) or with 100 μm DTT (closed squares) for 5 min in the presence of the indicated concentrations of l-serine. Transport of 10 μm l-[³H]serine was subsequently measured for 10 min in proteoliposomes containing 4 mm l-serine or l-arginine. l-Serine/l-serine exchange was calculated as in Fig. 1 and is expressed as pmol/μg of protein in 10 min. Activation of l-serine/l-serine exchange by DTT was progressively blocked by increasing concentrations of l-serine (EC₅₀: 1.1 ± 0.2 mm; r = 0.95). In contrast, preincubation with 30 mm l-serine did not significantly affect l-serine/l-serine exchange in proteoliposomes not treated with DTT. Data (mean ± S.E.) correspond to a representative experiment run in triplicate. When not visible, error bars are smaller than symbols. b, kinetic analysis of the single-Cys G294C mutant after DTT activation. Proteoliposomes harboring the mutant were treated with 100 μm DTT for 5 min. Transport of l-[³H]serine was subsequently measured using a range of external concentrations (0.01, 0.1, 0.5, 1, 5, and 10 mm) for 1 min (linear conditions at all l-serine concentrations) into proteoliposomes containing 4 mm l-serine or l-arginine. The rate of l-[³H]serine transport into proteoliposomes containing l-arginine was linearly related to the concentration of l-serine at all concentrations tested, as expected for simple diffusion (data not shown). l-Serine/l-serine exchange was calculated as in Fig. 1 and is expressed as picomoles of l-serine/μg of protein in 1 min. The apparent K_M value for l-serine uptake into the proteoliposomes was 0.7 ± 0.1 mm (r = 0.98) and the V_max was 43 ± 2 pmol/μg of protein in 1 min. Data (mean ± S.E.) correspond to a representative experiment run in triplicate. When not visible, error bars are smaller than the symbols. prot, protein.

FIGURE 6.

SteT K295C mutants showed coupled exchange of l-serine and l-arginine. a, time course of 10 μm l-[³H]serine transport into proteoliposomes harboring the single-Cys SteT mutant K295C and containing 4 mm unlabeled l-serine (black squares) or l-arginine (gray squares). Diffusion of 10 μm l-[³H]serine into liposomes containing no protein is indicated by the dotted line. This was calculated using a diffusion coefficient of 2.5 × 10⁻⁶ min⁻¹ and a liposome volume of 62 nl/μg protein, as measured previously (30). Data (mean ± S.E.) correspond to a representative experiment with three replicates. Error bars when not visible are smaller than the symbols. b, transport of l-serine into proteoliposomes harboring K295C mutants. Proteoliposomes containing wild-type (SteT), its Cys-less variant, the single-Cys SteT mutant K295C, and the K295C mutant generated in a wild-type background were used. Transport of 10 μm l-[³H]serine into proteoliposomes containing 4 mm l-serine (black bars), l-arginine (gray bars), or no amino acid (white bars) was measured over a period of 10 min, except for the two K295C mutants, for which transport was measured for 30 s (during the linear portion of the uptake time course). Only for the two K295C mutants transport was greater in proteoliposomes containing l-arginine than in those containing no amino acids (at least p ≤ 0.01) (inverted triangles). Transport is expressed as picomoles/μg of protein in 10 min. Data (mean ± S.E.) correspond to a representative experiment with three replicates. prot, protein.

FIGURE 7.

Amino acid cis-inhibition pattern of wild-type (open bars) and K295C (closed bars) SteT transport activity. Proteoliposomes harboring wild-type SteT or its K295C mutant were used, as indicated. Transport of 10 μm l-[³H]serine was measured for 10 min (SteT) and 30 s (K295C) (linear conditions) in proteoliposomes containing or lacking 4 mm l-serine. Transport was measured in the absence or presence of the indicated amino acids at a concentration of 5 mm in the external medium (2 mm in the case of l-tyrosine). l-Serine/l-serine exchange was calculated by subtracting transport in proteoliposomes containing no amino acid from that in those containing l-serine. Protein-mediated transport corrected for diffusion in this manner is expressed as the percentage of l-serine/l-serine exchange in the absence of cis-inhibitors (3.5 ± 0.8 pmol/μg protein in 10 min for wild-type SteT and 5.0 ± 0.2 pmol/μg protein in 30 s for the K295C mutant of SteT). Common amino acids and d-stereoisomers are indicated with the three-letter code. Homoser, l-homoserine; P-l-Ser, phospho-l-serine; OH-Pro, hydroxyproline. Data (mean ± S.E.) correspond to a representative experiment with three replicates.

FIGURE 8.

Exchange of l-serine and different amino acids by wild-type SteT and its K295C mutant. Transport of 10 μm l-[³H]serine was measured in wild-type (a) and K295C (b) SteT proteoliposomes containing no amino acids or the indicated amino acids at a concentration of 4 mm concentration. Transport was measured over periods during which uptake was a linear function of time, namely 10 min (wild-type) or 30 s, 2 or 5 min, depending on the substrate (K295C). l-Serine exchange was calculated by subtracting transport in proteoliposomes containing no amino acid from that in those containing the assayed amino acid. Exchange is expressed as picomoles of l-serine/μg of protein in 10 min. Data (mean ± S.E.) correspond to representative experiments with three replicates. A second experiment gave similar results.

FIGURE 9.

Putative substrate binding pocket of wild-type and K295C SteT. Upper view of the putative substrate binding pocket of wild-type SteT (a) and its K295C mutant (b), seen from the periplasmic space. This structural model is based on the open-to-out structure conformation of AdiC (22). The TM8 residues Cys-291 and Lys-295 are located at the surface of the bottom of the substrate binding pocket. In contrast, the TM8 residue Gly-294 (spheres) is not accessible to the solvent. Mutation K295C enlarges the substrate binding pocket in ∼90 ų and residue Tyr-102, in TM3, became accessible at the bottom of the cavity.