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Am J Physiol Cell Physiol. 2012 Sep 15;303(6):C673-81. doi: 10.1152/ajpcell.00123.2012. Epub 2012 Jul 11.

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

Author information

1
Depts. of Medicine and Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461, USA. rongbao.zhao@einstein.yu.edu

Abstract

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.

PMID:
22785121
PMCID:
PMC3468349
DOI:
10.1152/ajpcell.00123.2012
[Indexed for MEDLINE]
Free PMC Article

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