Substitutions that lock and unlock the proton-coupled folate transporter (PCFT-SLC46A1) in an inward-open conformation

Srinivas Aluri,Rongbao Zhao,Kai Lin,Daniel Sanghoon Shin,Andras Fiser,I David Goldman

doi:10.1074/jbc.ra118.005533

Abstract

The proton-coupled folate transporter (PCFT) mediates intestinal absorption of folates and their transport from blood to cerebrospinal fluid across the choroid plexus. Substitutions at Asp-109 in the first intracellular loop between the first and second transmembrane domains (TMDs) abolish PCFT function, but protein expression and trafficking to the cell membrane are retained. Here, we used site-directed mutagenesis, the substituted-cysteine accessibility method, functional analyses, and homology modeling to determine whether the D109A substitution locks PCFT in one of its conformational states. Cys-substituted residues lining the PCFT aqueous translocation pathway and accessible in WT PCFT to the membrane-impermeable cysteine-biotinylation reagent, MTSEA-biotin, lost accessibility when introduced into the D109A scaffold. Substitutions at Gly-305 located exofacially within the eighth TMD, particularly with bulky residues, when introduced into the D109A scaffold largely restored function and MTSEA-biotin accessibility to Cys-substituted residues within the pathway. Likewise, Ser-196 substitution in the fifth TMD, predicted by homology modeling to be in proximity to Gly-305, also partially restored function found in solute transporters, is critical to oscillation of the carrier among its conformational states. Substitutions at Asp-109 and Gly-112 lock PCFT in an inward-open conformation, resulting in the loss of function. However, the integrity of the locked protein is preserved, indicated by the restoration of function after insertion of a second "unlocking" mutation. and accessibility. Similarly, the inactivating G112K substitution within the first intracellular loop was partially reactivated by introducing the G305L substitution. These data indicate that the first intracellular loop, with a sequence identical to "motif A" (GXXXDXXGR(R/K)).

Highlights

The proton-coupled folate transporter (PCFT) mediates intestinal absorption of folates and their transport from blood to cerebrospinal fluid across the choroid plexus
This laboratory previously reported that substitution of Asp-109 with a variety of other residues resulted in a complete loss of function but did not alter the level of PCFT protein expressed or that trafficked to the cell membrane [14]
The data in this paper indicate that the integrity of this loop is critical to the oscillation of the carrier among its conformational states and that mutations at the Asp-109 residue abolish the mobility of the protein completely while preserving expression of the protein and its trafficking to the cell membrane

Summary

To whom correspondence should be addressed

Blood to cerebrospinal fluid across the choroid plexus. Mutations in the PCFT gene that result in loss of function of the protein lead to the autosomal recessive disorder, hereditary folate malabsorption (HFM), in which there is impaired or complete loss of function of both processes [1,2,3]. Since the discovery of PCFT [2], there have been extensive studies employing site-directed mutagenesis that have defined the topology of this transporter and identified residues and regions that play a critical role in its function [1]. Among these are residues critical to proton coupling [8], proton binding [9], and helices that contribute to a gate at the exofacial boundary of the carrier [10]. Second mutations in remote exofacial regions of the protein were identified that, when inserted in the D109A scaffold, restored both transport activity and accessibility of Cys-substituted residues within the aqueous translocation pathway

Results

Discussion

Experimental procedures