Monitoring the function of membrane transport proteins in detergent-solubilized form

Abstract

Transport proteins constitute approximately 10% of most proteomes and play vital roles in the translocation of solutes across membranes of all organisms. Their (dys)function is implicated in many disorders, making them frequent targets for pharmacotherapy. The identification of substrates for members of this large protein family, still replete with many orphans of unknown function, has proven difficult, in part because high-throughput screening is greatly complicated by endogenous transporters present in many expression systems. In addition, direct structural studies require that transporters be extracted from the membrane with detergent, thereby precluding transport measurements because of the lack of a vectorial environment and necessitating reconstitution into proteoliposomes for activity measurements. Here, we describe a direct scintillation proximity-based radioligand-binding assay for determining transport protein function in crude cell extracts and in purified form. This rapid and universally applicable assay with advantages over cell-based platforms will greatly facilitate the identification of substrates for many orphan transporters and allows monitoring the function of transport proteins in a nonmembranous environment.

Fig. 1.

Monitoring the function of membrane transport proteins outside of the membrane. Schematic representation of the analyses of membrane transport proteins in different states. See Introduction for details.

Fig. 2.

Detection of transporter function in nonmembranous environment. (a) Optimization of the expression host. The activity of Tyt1 solubilized from membrane vesicles of L. lactis NZ9000 or E. coli CD41(DE3) was assayed for binding of 1 μM l-[³H]tyrosine by means of scintillation proximity (filled bars; n = 3). Membrane vesicles of L. lactis NZ9000/pNZ8048 or E. coli CD41(DE3)/pQE60 served as control (open bars; n = 3). Binding of (b) 0.1 μM l-[³H]leucine to crude membranes extracts of E. coli C41(DE3) containing LeuT_Aa, (c) 1 μM l-[³H]tyrosine to membrane extracts of L. lactis NZ9000 harboring MhsT_Bh, or (d) 1 μM l-[³H]proline to solubilized membranes of E. coli WG170 harboring PutP was detected outside of a membranous environment (n = 3). (b–d) Binding was assayed in the presence of 150 mM NaCl (NaCl), 150 mM NaCl plus 150 mM imidazole (Imidazole), or in the absence of NaCl. The results of the latter two conditions were not significantly different from those observed with sample originating from control vesicles (data not shown). Data were expressed as percentage of the highest signal without background correction. Specific ³H-substrate binding activity (after background correction) was (in pmol/μg solubilized membrane protein) 1.04 ± 0.09 [³H]leucine for LeuT_Aa (b), 0.074 ± 0.004 [³H]tyrosine for MhsT_Bh (c), and 0.033 ± 0.002 [³H]proline for PutP (d). All scintillation proximity binding assays shown in this report were performed in the T = 0 assay format (simultaneous incubation of protein sample, radiotracer, and SPA beads; see manufacturer's instructions for details). Performing the radiotracer binding step with the respective vesicles before solubilization (i.e., delayed SPA format) led to similar levels of binding (data not shown).

Fig. 3.

Tyrosine binding to Tyt1 is strictly Na⁺- and pH-dependent. (a) Binding of l-[³H]tyrosine to solubilized Tyt1 was performed in assay buffer composed of 50 mM Tris/Mes, pH 7.5/20% glycerol/2 mM TCEP/0.05% N-dodecyl-β-d-maltopyranoside/150 mM of the indicated salts (at the indicated ratios) (n = 6). (b) The effect of protons on the binding activity was performed by varying the pH of the assay buffer in the presence of 150 mM NaCl (n = 6). (c) Tyrosine binding to solubilized Tyt1 is not chloride-dependent as determined by the replacement of NaCl with other sodium salts. (d) Tyt1 exhibits an apparent 2 Na⁺:1 tyrosine binding stoichiometry. The apparent Na⁺-activation constant of l-tyrosine binding (KDNa⁺) was determined to be 91.5 ± 4 mM with a Hill coefficient of 1.94 ± 0.28. Binding of 1 μM l-[³H]tyrosine was performed by varying the NaCl concentration from 0–400 mM (equimolar replacement with Tris/Mes; n = 3).

Fig. 4.

Determination of the substrate specificity of Tyt1 and MhsT. (a) Tyt1 binding is highly specific. The substrate specificity of solubilized Tyt1 was measured by a competition assay. The binding of 0.1 μM (final concentration) l-[³H]tyrosine was assayed in the presence or absence of 10 μM nonradioactive amino acid (or tyrosine analogue) as indicated (a–c, in the one-letter amino acid code used; the tyrosine analogues shown in a are: tyramine, d-tyrosine (D-Tyr), dopamine, α-methyl-l-tyrosine (a-Met-Tyr), 5-diiodo-l-tyrosine (Diiodo-Tyr), and 3-(3,4-dihydrophenyl)-l-alanine (DOPA). (b) MshT of B. halodurans is a multisubstrate transporter. Binding of 0.1 μM l-[³H]tyrosine was tested in the presence or absence of 10 μM nonradioactive amino acid as indicated (n = 3). l-[³H]tyrosine binding to MshT_Bh was inhibited ≥50% by 10 μM l-tyrosine, l-phenylalanine, l-tryptophan, l-valine, l-isoleucine, or l-threonine. (c) This specificity pattern was confirmed by assessing the binding activity of a 1 μM concentration of the indicated l-[³H]amino acids (n = 3) as well as by transport assays (data not shown; M.Q., Hideaki Yano, and J.A.J., manuscript in preparation). Data represent the background-corrected binding activity measured in the presence of 150 mM imidazole, which was not significantly different from that observed with sample originating from control vesicles.

Fig. 5.

Optimization of reconstitution conditions for Tyt1. (a) l-[³H]tyrosine binding to Tyt1 is saturable with a K_D^Tyr of 1.35 ± 0.17 μM (n = 3). Tyt1 function is maintained during the solubilization process with a molar binding ratio of ≈1 (0.96 ± 0.04 mol of tyrosine per mol of Tyt1; n = 3). (b) Effect of detergents on the binding of 1 μM l-[³H]tyrosine to purified and desalted Tyt1 (n = 3). Detergents used were: n-dodecyl-β-d-maltopyranoside (C12M), n-decyl-β-d-maltopyranoside (C10M), n-nonyl-β-d-maltopyranoside (C9M), n-nonyl-β-d-thiomaltopyranoside (C9TM), n-octyl-β-d-thiomaltopyranoside (C8TM), CYMAL-5 (CY5), CYMAL-4 (CY4), CYMAL-3 (CY3), Triton X-100 (TX100), n-nonyl-β-d-glucopyranoside (C9G), n-octyl-β-d-glucopyranoside (C8G), n-octyl-β-d-thioglucopyranoside (C8TG), n-dodecyl-N,N-dimethylamine-N-oxide (LDAO), and polyoxyethylene(8)dodecyl ether (C₁₂E₈). Data in a and b represent the background-corrected binding activity. Nonspecific background binding activity was determined in the presence of 150 mM imidazole and was not significantly different from that observed with sample originating from control vesicles. (c) Upon the functional reconstitution of Tyt1 in proteoliposomes tyrosine transport by Tyt1-containing proteoliposomes is saturable with a K_0.5^Tyr of 0.9 ± 0.19 μM and a V_max^Tyr of 470 ± 42 nmol × mg Tyt1⁻¹× min⁻¹ (n = 3), resulting in a catalytic turnover number (k_cat) of 0.4 ± 0.04 × s⁻¹. (d) Membrane-inserted Tyt1 exhibits an apparent K_0.5^Na+ of 0.72 ± 0.05 mM with a Hill coefficient of 1.8 ± 0.21 (n = 3).