Abstract
The proton-coupled folate transporter (PCFT, SLC46A1) is required for folate intestinal absorption and transport across the choroid plexus. Recent work has identified a F392V mutation causing hereditary folate malabsorption. However, the residue properties responsible for this loss of function remains unknown. Using site-directed mutagenesis, we observed complete loss of function with charged (Lys, Asp, and Glu) and polar (Thr, Ser, and Gln) Phe-392 substitutions and minimal function with some neutral substitutions; however, F392M retained full function. Using the substituted-cysteine accessibility method (with N-biotinyl aminoethyl methanethiosulfonate labeling), Phe-392 mutations causing loss of function, although preserving membrane expression and trafficking, also resulted in loss of accessibility of the substituted cysteine in P314C-PCFT located within the aqueous translocation pathway. F392V function and accessibility of the P314C cysteine were restored by insertion of a G305L (suppressor) mutation. A S196L mutation localized in proximity to Gly-305 by homology modeling was inactive. However, when inserted into the inactive F392V scaffold, function was restored (mutually compensatory mutations), as was accessibility of the P314C cysteine residue. Reduced function, documented with F392H PCFT, was due to a 15-fold decrease in methotrexate influx Vmax, accompanied by a decreased influx Kt (4.5-fold) and Ki (3-fold). The data indicate that Phe-392 is required for rapid oscillation of the carrier among its conformational states and suggest that this is achieved by dampening affinity of the protein for its folate substrates. F392V and other inactivating Phe-392 PCFT mutations lock the protein in its inward-open conformation. Reach (length) and hydrophobicity of Phe-392 appear to be features required for full activity.
Highlights
The proton-coupled folate transporter (PCFT), a member (SLC46A1) of the superfamily of solute transporters, harnesses the proton gradient as the energy source to achieve uphill transport of folates into cells [1, 2]
The current study demonstrates that Phe-392 is critical to sustaining oscillation of the PCFT protein among its conformational states and suggests that this is achieved by dampening the affinity of the protein for its folate substrates
Previous studies showed that (i) P314C is accessible to the membrane-impermeant MTSEA-biotin at room temperature, consistent with its accessibility to the extracellular milieu [18, 19]; (ii) P314C accessibility is lost when PCFT is locked in the inward-open conformation; and (iii) P314C is highly active; its Vmax is ;4- to 5-fold greater than that of PCFT-WT [18, 19, 24]
Summary
The proton-coupled folate transporter (PCFT, SLC46A1) is required for folate intestinal absorption and transport across the choroid plexus. The proton-coupled folate transporter (PCFT), a member (SLC46A1) of the superfamily of solute transporters, harnesses the proton gradient as the energy source to achieve uphill transport of folates into cells [1, 2] Both the affinity for folate substrates and the rate of oscillation of the carrier among its conformational states increase as the extracellular pH is decreased to ;5.5, the optimal pH for this transporter [1, 3]. Like residues in Motif A in the first intracellular loop [15], mutations at Phe-392 that result in a marked loss of function lock the protein in an inward-open conformation that can be substantially reversed by the introduction of second mutations within the protein
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