Fig 1.
Basigin-MCT1 fusion constructs, expression in yeast, and transport functionality.
(A) Shown are fusion constructs of basigin (black/gray; PDB #6LZ0) with MCT1 (orange; PDB #6LZ0). Disulfide bridges are shown as orange spheres, and Glu218 of the basigin transmembrane helix is highlighted in red. (B) Western blot showing expression of MCT1 alone (58 kDa), and fused with BSGΔIg (WT, 67 kDa) or the E27R point mutant. Proteins were detected using a Penta-His antibody. (C) Live-cell confocal microscopy of fusion proteins via C-terminal GFP and soluble GFP (—). (D, E) Uptake of 14C-labeled l-lactate into jen1Δ ady2Δ yeast at pH 6.8 and a 1 mM inward gradient. Shown are curves for non-expressing cells (background, D, ○), cells expressing MCT1 alone (D, ●), and in fusion with BSGΔIg (E, ■), or BSGΔIg E27R (E, □). The data are normalized to 1 mg of cells and the background of non-expressing cells was subtracted. Error bars indicate ± S.E.M. from three biological replicates.
Table 1.
Kinetic parameters ± S.E.M. of MCT1/basigin fusion variants (nD: Not determined).
Fig 2.
Increased uptake of l-lactate by MCT1 fusions with basigin variants carrying the extracellular Ig-I domain.
(A) Western blot showing expression of MCT1 alone (58 kDa) and fused with BSGΔIg (67 kDa), BSG Ig-I (77 kDa), BSG Ig-I/C2 (85 kDa), and BSG var2 (87 kDa) using a Penta-His antibody. (B) Visualization of the constructs at the plasma membrane via C-terminal GFP and live-cell confocal microscopy. (C–E) Uptake of 14C-labeled l-lactate via MCT1 fusion constructs with basigin variants at pH 6.8 and a 1 mM inward gradient. Shown are BSG Ig-I (C, ◆), BSG Ig-I/C2 (D, ▼), BSG var2 (D, ▲), and the BSG Ig-I point mutants C23S (E, ⟠) and C82S (E, ◊). The background of non-expressing cells was subtracted, and the data were normalized to 1 mg of cells; error bars represent ± S.E.M. of three biological replicates. (F) Western blot showing expression of MCT1 fused to BSG Ig-I C23S and C82S. The arrow head indicates the expected molecular weight of 77 kDa (Penta-His antibody).
Fig 3.
Effect of the transmembrane proton gradient and altered substrate concentrations on transport via MCT1 in the presence and absence of the basigin Ig-I domain.
(A-C) Effect of adding 1 mM l-lactate at an external buffer pH of 6.8 to yeast cells without monocarboxylate transporters (A, 〇), or expressing MCT1 (B, ●) or MCT1 fused with BSG Ig-I (C, ◆). (D) Uptake of 14C-labeled l-lactate via MCT1 alone (●) and fused wit BSG Ig-I (◆) at an external pH 5.8 and a 1 mM inward l-lactate gradient. (E) Uptake capacities of cells expressing MCT1 alone (orange) and fused with BSG Ig-I (black) at pH 6.8 (based on data from Figs 1D and 2C) and pH 5.8 (data from 3D). (F) Michaelis-Menten kinetics for MCT1 alone and fused with BSG Ig-I. Km values were determined at pH 6.8 and are given in mM. In all cases, the background of non-expressing cells was subtracted; error bars indicate ± S.E.M. of three biological replicates.
Fig 4.
Shifted transmembrane l-lactate distribution and promotion of cell growth by the presence of the BSG Ig-I domain.
(A) Conversion of scales from the amount of intracellular substrate per cell mass to the molar concentration (nmol mg–1 to mM) by use of the freely diffusible aquaporin substrate glycerol. Uptake of 14C-labeled glycerol of AQP7 or AQP9 expressing cells was monitored for 15 min at pH 6.8 and 1 mM (●) and 5 mM (〇) inward gradients. The error bars denote ± S.E.M. from four biological replicates. (B) Intracellular l-lactate accumulation generated by expression of MCT1 alone (orange) and fusions with basigin variants (black); error bars denote ± S.E.M. from three biological replicates. (C) Relation of the internal/external l-lactate (ceq_in, ceq_ex) and proton concentrations ([H+]eq_in, [H+]eq_ex) at equilibrium. The tested basigin-MCT1 fusion constructs yielded different ratios (at 1 mM l-lactate, pH 6.8 conditions) indicating an equilibrative (K = 1) or accumulating effect (apparent K > 1) on intracellular l-lactate with respect to the overall buffer concentrations. (D) Growth of cells expressing MCT1 and basigin fusion variants on agar media containing 2% sodium l-lactate (left) or 2% glucose (growth control; right). Cell suspensions (5 μl) were spotted in ten-fold serial dilutions from a starting OD600 of 1.
Fig 5.
Two oppositely charged surface patches in the BSG Ig-I domain.
(A) Poisson-Boltzmann electrostatic potential of the extracellular domain of BSG var2 (PDB #3B5H) reveals two oppositely charged surface patches in the BSG Ig-I domain; the red/blue scale covers the range of –3 kT e– to +3 kT e–. (B–D) Effect of replacing charged residues in the BSG Ig-I domain by neutral ones on the electrostatic potential (left) and the l-lactate transport of fused MCT1 (right). The five Glu residues of the negative patch (pos. 114, 118, 120, 168, 172) were changed to Gln (B, ●), the five Lys/Arg residues of the positive patch (pos. 108, 111, 127, 201, 203) were mutated to Ala altogether (C, ●) or in two pairs of two (D, Lys108,111Ala: ●, Arg201,203Ala: 〇). The electrostatic surface in D shows wild-type BSG for better orientation. The gray shading indicates the corridor between the uptake capacities of MCT1 fused with wild-type BSG Ig-I (upper border, from Fig 2C) and MCT1 alone (lower border, from Fig 1D).
Fig 6.
Bivalent harvesting function of the BSG Ig-I domain for protons and l-lactate anions.
(A) In the absence of the extracellular BSG Ig-I domain, the environmental concentrations of l-lactate and protons determine transport. (B) The presence of the Ig-I domain creates a microenvironment and increases the local concentration of the substrate and protons at the MCT entry site driving accumulation in the cell.