PnuT uses a facilitated diffusion mechanism for thiamine uptake

Abstract

Membrane transporters of the bacterial pyridine nucleotide uptake (Pnu) family mediate the uptake of various B-type vitamins. For example, the PnuT transporters have specificity for vitamin B1 (thiamine). It has been hypothesized that Pnu transporters are facilitators that allow passive transport of the vitamin substrate across the membrane. Metabolic trapping by phosphorylation would then lead to accumulation of the transported substrates in the cytoplasm. However, experimental evidence for such a transport mechanism is lacking. Here, to determine the mechanism of thiamine transport, we purify PnuT_Sw from Shewanella woodyi and reconstitute it in liposomes to determine substrate binding and transport properties. We show that the electrochemical gradient of thiamine solely determines the direction of transport, consistent with a facilitated diffusion mechanism. Further, PnuT_Sw can bind and transport thiamine as well as the thiamine analogues pyrithiamine and oxythiamine, but does not recognize the phosphorylated derivatives thiamine monophosphate and thiamine pyrophosphate as substrates, consistent with a metabolic trapping mechanism. Guided by the crystal structure of the homologous nicotinamide riboside transporter PnuC, we perform mutagenesis experiments, which reveal residues involved in substrate binding and gating. The facilitated diffusion mechanism of transport used by PnuT_Sw contrasts sharply with the active transport mechanisms used by other bacterial thiamine transporters.

Figure 1.

Structural properties of Pnu transporters. (a) Sequence alignment of TM1–3 and TM5–7 of PnuC (N. mucosa) and PnuT (S. woodyi). Conserved residues in the substrate binding site and the potential gate regions are colored yellow and cyan, respectively. (b) Substrate binding site of PnuC_Nm (Protein Data Bank ID 4QTN) viewed from the membrane plane. A single protomer of PnuC_Nm is shown, with the two structurally related three-helix domains TM1–3 and TM5–7 colored blue and cyan, respectively, and TM4 in light gray. The nonconserved N-terminal TM is not shown for clarity. The residues of the binding site and potential gates are colored as in panel (a), and shown in stick representation. Residue numbering according to the sequence of PnuT_Sw, the numbers in brackets are from PnuC_Nm. The substrate nicotinamide riboside of PnuC is shown in black stick representation. (c) Domain structure of PnuC_Nm. The protein is viewed from the periplasmic side along an axis perpendicular to the plane of the membrane. The two structurally related three-helix domains TM1–3 and TM5–7 are colored blue and cyan, respectively.

Figure 2.

Static light scattering (SEC-MALLS) analysis of PnuT_Sw. The elution profile of a size exclusion chromatography experiment is shown with a black line and calculated molecular mass of protein (25 kD), the detergent micelle (DDM), and protein–detergent complex in red, green, and blue, respectively.

Figure 3.

Substrate binding to PnuT_Sw. (a–c) Examples of isothermal titration calorimetry experiments showing (a) thiamine, (b) pyrithiamine, and (c) oxythiamine binding to PnuT_Sw.

Figure 4.

Thiamine transport by PnuT_Sw reconstituted in proteoliposomes. (a) Transport measured at different voltages across the liposomal membrane: −110 mV (black circles), −55 mV (open circles), −40 mV (black triangles), −22 mV (open triangles), and 0 mV (black squares). The gray dashed line shows the thiamine association to protein-free liposomes (membrane voltage −110 mV). In the latter case, the scale of the y-axis is pmol of thiamine per 250 µg lipids. (b) Transport measured at different external pH values: pH 5.5 (squares), pH 6.5 (circles), pH 7.5 (triangles), and pH 8.5 (inverted triangles). The internal pH was 7.5 in all cases. (c) Transport measured in the presence (open circles) of absence (black circles) of external Na⁺. In the latter case, sodium chloride was replaced by choline chloride. (d) Demonstration of bidirectionality of thiamine transport. The luminal and external pH were identical (pH 7.5). Closed circles: The membrane voltage was clamped at −110 mV using valinomycin at the onset of the experiment, and FCCP was added after six minutes. Open circles: Valinomycin was added only after 6 min. Inverted triangles: No valinomycin was present, and FCCP was added after 6 min. All error bars indicate the SD from three experiments.

Figure 5.

Determination of K_m and V_max of PnuT_Sw reconstituted in proteoliposomes for thiamine transport. The error bars indicate the SD calculated from three independent experiments. The 95% confidence interval is indicated.

Figure 6.

Competitive inhibition of thiamine transport by PnuT_Sw reconstituted in proteoliposomes by thiamine analogues. (a) [³H]thiamine uptake in the presence of 1 mM of thiamine analogues. TMP (open triangles), TPP (open circles), oxythiamine (black inverted triangles), and pyrithiamine (black squares). Positive control with no additions (black circles). (b) Counter flow of [³H]thiamine into proteoliposomes loaded with 1 mM thiamine (black inverted triangles), pyrithiamine (black circles), oxythiamine (open circles), TMP (open triangles), and TPP (black squares). All error bars indicate the SD from three experiments.

Figure 7.

Initial rates of thiamine transport by PnuT_Sw mutants reconstituted in proteoliposomes. The error is the SD from three experiments.