Residues in the eighth transmembrane domain of the proton-coupled folate transporter (SLC46A1) play an important role in defining the aqueous translocation pathway and in folate substrate binding

Abstract

The proton-coupled folate transporter (PCFT-SLC46A1) is required for intestinal folate absorption and folate transport across the choroid plexus. This report addresses the structure/function of the 8th transmembrane helix. Based upon biotinylation of cysteine-substituted residues by MTSEA-biotin, 14 contiguous exofacial residues to Leu316 were accessible to the extracellular compartment of the 23 residues in this helix (Leu303–Leu325). Pemetrexed blocked biotinylation of six Cys-substituted residues deep within the helix implicating an important role for this region in folate binding. Accessibility decreased at 4°C vs RT. The influx Kt, Ki and Vmax were markedly increased for the P314C mutant, similar to what was observed for Y315A and Y315P mutants. However, the Kt, alone, was increased for the P314Y mutant. To correlate these observations with PCFT structural changes during the transport cycle, homology models were built for PCFT based upon the recently reported structures of bovine and rodent GLUT5 fructose transporters in the inward-open and outward- open conformations, respectively. The models predict substantial structural alterations in the exofacial region of the eighth transmembrane helix as it cycles between its conformational states that can account for the extended and contiguous aqueous accessibility of this region of the helix. Further, a helix break in one of the two conformations can account for the critical roles Pro314 and Tyr315, located in this region, play in PCFT function. The data indicates that the 8th TMD of PCFT plays an important role in defining the aqueous channel and the folate binding pocket.

A proton-coupled folate transporter mutation causing hereditary folate malabsorption locks the protein in an inward-open conformation

“…Residues that are involved in proton binding, protoncoupling, extracellular gating, folate substrate binding and carrier translocation rates have been identified (2,14,15). Homology models of PCFT in the inward-open (16,17), and more recently in both the inward-and outward-open (14,18,19), conformations, have been developed, the latter based upon the structures of the mammalian Glut 5 fructose transporter (20). These models have been useful in understanding the structural ramifications of functional studies in the absence of a crystal structure of the PCFT protein.…”

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

“…5,6,17 Influx measurements were made over 1 min at 37°C, as has been previously described. 18,19 Most experiments were performed at the optimal pH for this transporter, 5.5, in MBS buffer [20 mM 2-(N-morpholino)ethanesulfonic acid, 140 mM NaCl, 5 mM KCL, 2 mM MgCl 2 , and 5 mM dextrose]. To assess pH-dependence, we adjusted (i) the MBS buffer to pH 5.5, 6.0, and 6.5 and (ii) the HBS buffer [20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 140 mM NaCl, 5 mM KCL, 2 mM MgCl 2 , and 5 mM dextrose] to pH 7.0 and 7.4.…”

“…20 On the basis of these structures, we built a homology model for PCFT in these 2 conformations. 18 In Figure 7, the 11th transmembrane helix in the inward open conformation, along with the Asn411 residue, are highlighted in blue. The position of the helix in the outward open conformation, and the Asn411 residue, are highlighted in green.…”

“…The cells were then processed for protein and radioactivity, as has been previously described. 18,19 Analysis of PCFT protein expression at the cell surface Cell surface expression was determined by biotinylation of accessible PCFT lysine residues with EZ-Link sulfo-NHS-LC, as has been previously described in detail. 10,11 Briefly, washed cells were treated with 1 mg/mL of EZ-Link sulfo-NHS-LC for 30 min at room temperature (RT).…”

“…Homology models of PCFT were developed, as has been described in detail, 18 on the basis of the recently reported structures of the bovine (inward open) and rat (outward open) Glut5 fructose transporters as templates (Protein Data Bank codes 4YB9 and 4YBQ, respectively). 20 These Glut5 transporters share 88% sequence identity with each other and 13% sequence identity to PCFT, similar to the level of identity of the glycerol-3-phosphate proton symporter previously used for homology modeling of PCFT.…”