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Hereditary folate malabsorption due to a mutation in the external gate of the proton-coupled folate transporter SLC46A1

Srinivas Aluri, Rongbao Zhao, Charlotte Lubout, Susanna M. I. Goorden, Andras Fiser and I. David Goldman

Article Figures & Data

Figures

  • Figure 1.

    Expression and function of the N411K PCFT mutant. (A) Influx of 0.5 μM [3H] MTX was assessed over 1 min at 37°C in RFC-/PCFT-null HeLa R1-11 cells transfected with wild-type PCFT, the N411K mutant, or vector control. Data are indicated as percentage of PCFT-WT activity and are the average of 3 independent experiments ± SEM. (B) Expression at the cell surface is depicted in the upper row. PCFT expression in the crude membrane preparation is depicted in the lower row. The western blot is representative of 3 independent experiments.

  • Figure 2.

    Structural requirement at the Asn411 residue. (A) Expression at the cell surface (upper row), and in the crude membrane fraction (middle row), of the Asn411 mutants. β-Actin was the internal control (lower row). The blot is representative of 3 independent experiments. (B) Influx of 0.5 and 50 μM [3H] MTX was measured in HeLa R1-11 cells transfected with a variety of substitutions at the Asn411 residue. Data are the averages ± SEM of 3 independent experiments. *P < .01; **P < .0001, in comparison with PCFT-WT activity.

  • Figure 3.

    Effects of sulfhydryl modifications of the N411C PCFT mutant. Cells harboring the N411C PCFT mutation were treated with 3 mM MTSES, 3 mM MTSET, or 0.5 mM MTSEA-biotin for 30 min at pH 7.4 and RT followed by assessment of [3H]MTX influx. Data are percentages of untreated cells ± SEM, from the average of 3 independent experiments.

  • Figure 4.

    pH-dependence of [3H]MTX influx. Activities for the PCFT-WT and N411K PCFT mutant are normalized to the rate at pH 5.5, indicated as 100%. Data are the means ± SEM from 3 independent experiments.

  • Figure 5.

    Substrate specificity of transport mediated by the N411K PCFT mutant. Influx of tritiated PCFT substrates was assessed at a concentration far below the influx Kt for PCFT-WT, 0.5 µM (A) and at least an order of magnitude higher than the influx Kt for PCFT-WT, 50 µM (5 µM for pemetrexed) (B). Transport at the high concentration reflects the influx Vmax. Since a change in Vmax should result in a comparable proportional change in influx, irrespective of the substrate concentration, a discrepancy in the percentage change between the low and high substrate concentrations reflects an additional change in the influx Kt. The data shown are percentages of influx activity in relation to PCFT-WT ± SEM from 3 independent experiments. *Comparing the differences between the percentage reduction in transport of MTX (P < .01), pemetrexed (not significant), and folic acid (P <. 01) at the low vs high concentrations. **Comparing the difference between 5-formylTHF and 5-methylTHF influx at the high concentration (P < .05). ***Comparing influx of MTX (P < .005) and folic acid (P < .005) with transport of pemetrexed at the low substrate concentration.

  • Figure 6.

    Pemetrexed influx kinetics. Influx of 0.05 to 5.0 µM [3H]pemetrexed was assessed in cells transfected with either PCFT-WT or the N411K mutant. Data are best fit to the Michaelis-Menten equation V = Vmax[S]/(Kt + [S]), where [S] is extracellular substrate concentration, Vmax is the maximum transport rate, and Kt is the concentration at which influx is one half of maximum. Data shown are the means ± SEM from 3 independent experiments.

  • Figure 7.

    Homology modeling of PCFT. Homology models of PCFT based on the reported structures of the bovine and rat fructose transporters, Glut5, in the inward open and outward open conformations, respectively.18,20 The 11th TMD with the Asn411 residue is highlighted. The predicted structure in the inward open configuration is indicated in blue. The predicted structure in the outward open conformation is indicated in green. (Left) A planar view of the protein. (Right) A view from the extracellular compartment into the aqueous translocation pathway. The Asn411 residue protrudes into the aqueous pathway and shifts further into the pathway when the protein is in the inward open conformation.