Functional roles of aspartate residues of the proton-coupled folate transporter (PCFT-SLC46A1); a D156Y mutation causing hereditary folate malabsorption

Abstract

The proton-coupled folate transporter (PCFT; SLC46A1) mediates folate transport into enterocytes in the proximal small intestine; pcft loss-of-function mutations are the basis for hereditary folate malabsorption. The current study explored the roles of Asp residues in PCFT function. A novel, homozygous, loss-of-function mutation, D156Y, was identified in a child of Pakistani origin with hereditary folate malabsorption. Of the 6 other conserved Asp residues, only one, D109, is shown to be required for function. D156Y, along with a variety of other substitutions at this site (Trp, Phe, Val, Asn, or Lys), lacked function due to instability of the PCFT protein. Substantial function was preserved with Glu, Gly, and, to a lesser extent, with Ser, Thr, and Ala substitutions. This correlated with PCFT bio-tinylated at the cell surface. In contrast, all D109 mutants, including D109E, lacked function irrespective of pH (4.5, 5.5, and 7.4) or substrate concentration (0.5-100 μM), despite surface expression comparable to wild-type PCFT. Hence, D156 plays a critical role in PCFT protein stability, and D109, located in the first intracellular loop between the second and third transmembrane domains, is absolutely required for PCFT function.

Figure 1

Identification of a novel D156Y PCFT mutation in a subject with HFM and its expression as well as transport function. (A) A chromatogram showing a homozygous mutation in the PCFT gene in a subject with HFM. (B) [³H]-MTX (0.5μM) influx over 1 minute at pH 5.5 and 37°C in Hela-R1-11 cells transiently transfected with wild-type PCFT, D156Y-PCFT, or the vector (mock). (C) Western blot analysis of cells transiently transfected with wild-type PCFT, the D156Y mutant, or vector: crude cell membrane fraction (left); biotinylated protein at the cell surface (right). Vertical lines have been inserted to indicate repositioned gel lanes.

Figure 2

A topologic model for human PCFT. All 7 Asp residues are indicated. D54 is semiconserved; D72, D109, D156, D170, D286, and D331 are fully conserved among species (monkey, horse, rat, mouse, dog, cow, opossum, xenopus, and zebra fish). D156 is mutated in the subject with HFM.

Figure 3

Functional assessment of PCFT aspartate mutants. [³H]-MTX influx was assessed at pH 5.5 and 37°C over 1 minute at a concentration of 0.5μM in Hela-R1-11 transfectants. (A) Initial screening of Asp mutants. (B) Studies focused on D109, D156, and D331 residues replaced with Glu, Asp, or Ala. Data are the mean ± SEM from 3 independent experiments.

Figure 4

Expression and function of D156 PCFT mutants. (A) [³H]-MTX influx was performed at 0.5μM and pH 5.5 over 1 minute. Data are the mean ± SEM from 3 independent experiments performed on different days. (B) Western blot assay of the crude cell-membrane fraction (top panel) and biotinylated protein at the cell surface (middle panel). β-actin was the loading control (bottom panel). These blots are representative of 3 experiments and were performed on 2 gels at the same time.

Figure 5

MTX influx kinetics mediated by D156 PCFT mutants. (A) [³H]-MTX influx kinetics mediated by D156G and D156S PCFT, compared with wild-type PCFT. Influx was assessed at pH 5.5 at 37°C over 1 minute with [³H]-MTX concentrations of 0.1, 0.2, 0.5, 1, 2.5, 5, and 10μM. Data are the mean ± SEM from 3 independent experiments. (B) Western blot of biotinylated protein obtained from cells transfected with wild-type PCFT and the D156G and D156S mutants. Wild-type PCFT was diluted to more accurately quantify, by comparison, levels of mutant PCFTs. Ten μL of the biotinylated wild-type PCFT protein fraction was added to 10, 20, 25, and 30 μL of 2× SDS-loading buffer, described as 1:2, 1:3, 1:3.5, and 1:4 dilutions, respectively. The data are representative of 2 independent experiments. A vertical line has been inserted to indicate a repositioned gel lane.

Figure 6

Functional properties of D109 PCFT mutants. (A) [³H]-MTX influx (0.5μM) over 1 minute at pH 5.5. Data are the mean ± SEM from 3 independent experiments. (B) Western blot assay of the crude cell-membrane fraction (top panel) and biotinylated protein (middle panel) of D109 PCFT mutants. β-actin was the loading control (bottom panel). The graph shown is a representative of 2 independent experiments. The vertical line between the D109S and Mock lanes indicates repositioned gel lanes.

Figure 7

Impact of MTX concentration and pH on the activity of PCFT D109 mutants. (A) [³H]-MTX influx (10μM) over 1 minute at pH 5.5. (B) [³H]-MTX influx (100μM) at pH 5.5. (C) [³H]-MTX influx (0.5μM) at pH 4.5. (D) [³H]-MTX influx (100μM) at pH 4.5.