Identification of a functionally critical GXXG motif and its relationship to the folate binding site of the proton-coupled folate transporter (PCFT-SLC46A1)

• Published in: American Journal of Physiology: Cell Physiology Vol. 303; no. 6; pp. C673 - C681
• Main Authors: Zhao, Rongbao, Shin, Daniel Sanghoon, Fiser, Andras, Goldman, I David
• Format: Journal Article
• Language: English
• Published: United States American Physiological Society 15.09.2012
• Subjects: Amino Acid Motifs - physiology; Amino Acid Sequence; Amino acids; Binding sites; Binding Sites - physiology; Conserved Sequence; Folic Acid - chemistry; Folic Acid - metabolism; Genes; Glycine - physiology; HeLa Cells; Humans; Isoleucine - genetics; Membranes; Molecular Sequence Data; Mutation; Protein Transport - physiology; Proton-Coupled Folate Transporter - chemistry; Proton-Coupled Folate Transporter - physiology; Protons; Signal Transduction - physiology; Vitamin B
• ISSN: 0363-6143; 1522-1563
• DOI: 10.1152/ajpcell.00123.2012

The proton-coupled folate transporter (PCFT) mediates intestinal folate absorption, and loss-of-function mutations in this gene result in the autosomal recessive disorder hereditary folate malabsorption. The current study, focused on a structure-functional analysis of this transporter, identified Gly-189 and Gly-192 (a GxxG motif) located in the fifth transmembrane domain as residues that could not be replaced with alanine without a loss of function. In contrast, function was preserved when Gly-56 and Gly-59 (the other conservative GXXG motif in human PCFT) were replaced with alanine. Similarly, Gly-93 and Gly-97, which constitute the only conserved GXXXG dimerization motif in human PCFT, tolerated alanine substitution. To explore the role of this region in folate binding, the residues around Gly-189 and Gly-192 were analyzed by the substituted cysteine accessibility method. Both I188C and M193C mutants were functional and were inhibited by membrane-impermeable sulfhydryl-reactive reagents; this could be prevented with PCFT substrate, but the protection was sustained at 0°C only for the I188C mutant, consistent with localization of Ile-188 in the PCFT folate binding pocket. The functional role of residues around Gly-189 and Gly-192 is consistent with a molecular structural model in which these two residues along with Ieu-188 are accessible to the PCFT aqueous translocation pathway.