Substituted-cysteine accessibility and cross-linking identify an exofacial cleft in the 7th and 8th helices of the proton-coupled folate transporter (SLC46A1)

The proton-coupled folate transporter (PCFT-SLC46A1) is required for folate transport across the apical membrane of the small intestine and across the choroid plexus. This study focuses on the structure/function of the 7th transmembrane domain (TMD), and its relationship to the 8th TMD as assessed by the substituted cysteine accessibility method (SCAM) and dicysteine cross-linking. Nine exofacial residues (I278C; H281C–L288C) of 23 residues in the 7th TMD were accessible to 2-((biotinoyl)amino)ethyl methanethiosulfonate (MTSEA-biotin). Pemetrexed, a high-affinity substrate for PCFT, decreased or abolished biotinylation of seven of these residues consistent with their location in or near the folate binding pocket. Homology models of PCFT based on Glut5 fructose transporter structures in both inward- and outward- open conformations were constructed and predicted that two pairs of residues (T289-I304C and Q285-Q311C) from the 7th and 8th TMDs should be in sufficiently close proximity to form a disulfide bond when substituted with cysteines. The single Cys-substituted mutants were accessible to MTSEA-biotin and functional with and without pretreatment with dithiotreitol. However, the double mutants were either not accessible at all, or accessibility was markedly reduced and function markedly impaired. This occurred spontaneously without inclusion of an oxidizing agent. Dithiotreitol restored accessibility and function consistent with disulfide bond disruption. The data establish the proximity of exofacial regions of the 7th and 8th TMDs and their role in defining the aqueous translocation pathway and suggest that these helices may be a component of an exofacial cleft through which substrates enter the protein binding pocket in its outward-open conformation.