The membrane proteins SiaQ and SiaM form an essential stoichiometric complex in the sialic acid tripartite ATP-independent periplasmic (TRAP) transporter SiaPQM (VC1777-1779) from Vibrio cholerae

Abstract

Tripartite ATP-independent periplasmic (TRAP) transporters are widespread in bacteria but poorly characterized. They contain three subunits, a small membrane protein, a large membrane protein, and a substrate-binding protein (SBP). Although the function of the SBP is well established, the membrane components have only been studied in detail for the sialic acid TRAP transporter SiaPQM from Haemophilus influenzae, where the membrane proteins are genetically fused. Herein, we report the first in vitro characterization of a truly tripartite TRAP transporter, the SiaPQM system (VC1777-1779) from the human pathogen Vibrio cholerae. The active reconstituted transporter catalyzes unidirectional Na(+)-dependent sialic acid uptake having similar biochemical features to the orthologous system in H. influenzae. However, using this tripartite transporter, we demonstrate the tight association of the small, SiaQ, and large, SiaM, membrane proteins that form a 1:1 complex. Using reconstituted proteoliposomes containing particular combinations of the three subunits, we demonstrate biochemically that all three subunits are likely to be essential to form a functional TRAP transporter.

FIGURE 1.

Topology models of HiSiaQM, VcSiaQ, and VcSiaM. Consensus topology models of HiSiaQM from H. influenzae (A) and VcSiaQ (left) and VcSiaM (right) from V. cholerae (B). To construct the consensus models, a multiple sequence alignment was performed on the integral membrane proteins from seven TRAP transporters previously predicted to be specific to Neu5Ac (15). Included in this alignment were three TRAP transporters whose integral membrane component was composed of two separate proteins and four fused membrane components. The boundaries of the TMHs for each protein were predicted using the algorithm TMHMM (36). The predicted transmembrane helix boundaries for each protein and the multiple sequence alignment were overlaid, allowing us to define the consensus boundaries of the transmembrane helices. The topology models were then constructed in CoralDRAW. The linker helix of HiSiaQM is indicated by a dashed box.

FIGURE 2.

Expression and purification of the components of VcSiaPQM. A, Coomassie-stained SDS-polyacrylamide gel of the purification of the VcSiaQM complex (His-tagged on the N terminus of VcSiaQ) using IMAC. Lane 1, molecular weight ladder; lane 2, total membrane vesicles; lane 3, column flow-through; lane 4, 40 mm imidazole washing step; lanes 5–7, 500 mm imidazole elution steps. B, Coomassie-stained SDS-polyacrylamide gel of N-terminally tagged VcSiaP purified in one IMAC step. Lane 1, molecular mass marker with the sizes of the proteins indicated in kDa; lane 2, IMAC-purified VcSiaP.

FIGURE 3.

Energetic characterization of VcSiaPQM. A, transport of 5 μm [¹⁴C]Neu5Ac into the lumen of VcSiaQM-containing proteoliposomes in the presence of 5 μm VcSiaP and the following electrochemical gradients: Δ_μNa+ΔΨ (closed circles), Δ_μNa alone (open circles), Δ_μNa+ΔpH (open squares), ΔpH alone (closed squares), and ΔΨ alone (closed triangles). B, cold chase assay with unlabeled Neu5Ac. Transport of 5 μm [¹⁴C]Neu5Ac into the lumen of proteoliposomes containing VcSiaQM (circles, also in the presence of 5 μm VcSiaP) with an applied Δ_μNa + ΔΨ or NanT (triangles) with an applied ΔpH. At 100 s (arrow), either 1 mm unlabeled Neu5Ac (closed symbols) or an equivalent volume of distilled H₂O (open symbols) was added to the reaction. C, solute counterflow activity of proteoliposomes prepared via extrusion containing lumenal buffer with 1 mm unlabeled Neu5A and either VcSiaQM with 5 μm VcSiaP (circles) or NanT (triangles).

FIGURE 4.

Requirements of different transporter subunits for transporter function. A, transport of 5 μm [¹⁴C]Neu5Ac into the lumen of proteoliposomes containing VcSiaQM (co-expressed, closed circle), VcSiaQ and SiaM (separately expressed, open circles), VcSiaQ alone (closed inverted triangles), and VcSiaM alone (open triangles). All transport assays were performed with Δ_μNa + ΔΨ applied and in the presence of 5 μm VcSiaP. B, silver-stained SDS-polyacrylamide gel of the proteoliposomes containing different combinations of reconstituted VcSiaQ and VcSiaM. Lane 1, molecular weight ladder; lane 2, VcSiaQ and VcSiaM co-expressed, co-purified, and reconstituted; lane 3, VcSiaQ and VcSiaM, separately expressed, purified, mixed, and then reconstituted; lane 4, VcSiaM reconstituted alone; lane 5, VcSiaQ reconstituted alone. The top and bottom arrows indicate the position of the bands for VcSiaM and VcSiaQ, respectively.

FIGURE 5.

Size-exclusion chromatography of VcSiaQM and HiSiaQM. A, SEC trace of IMAC-purified VcSiaQM (black line) and HiSiaQM (gray line) showing absorbance (A₂₈₀) versus retention time. B and C, Coomassie-stained SDS-polyacrylamide gels of the fractions taken across the SEC peaks of VcSiaQM and HiSiaQM, respectively.

FIGURE 6.

Molecular masses of HiSiaQM and VcSiaQM determined by AUC and SEC-MALLS. A, SEC-MALLS trace of VcSiaQM (black lines) and HiSiaQM (gray lines) showing the relative absorbance at A₂₈₀ trace normalized to 1 (thin lines) and the molar masses of the species present in the samples (thick lines). B, representative dataset from the sedimentation equilibrium experiment performed on HiSiaQM using a Beckman Optima XL/1 analytical ultracentrifuge at 20 °C. C, table summarizing the data obtained for the masses of VcSiaQM and HiSiaQM using AUC, SEC-MALLS, and the colorimetric maltoside assay.