Identification of a functionally critical GXXG motif and its relationship to the folate binding site of the proton-coupled folate transporter (PCFT-SLC46A1).
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.
Depts. of Medicine and Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461, USA. rongbao.zhao@einstein.yu.edu
Rongbao
R
Zhao
Daniel Sanghoon
DS
Shin
Andras
A
Fiser
I David
ID
Goldman
D020816:Amino Acid Motifs / Q000502:physiology; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000502:physiology; D017124:Conserved Sequence; D005492:Folic Acid / Q000737:chemistry / Q000378:metabolism*; D005998:Glycine / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D007532:Isoleucine / Q000235:genetics; D008969:Molecular Sequence Data; D021381:Protein Transport / Q000502:physiology; D058979:Proton-Coupled Folate Transporter / Q000737:chemistry* / Q000502:physiology*; D011522:Protons; D015398:Signal Transduction / Q000502:physiology
D058979:Proton-Coupled Folate Transporter; D011522:Protons; C505058:SLC46A1 protein, human; D007532:Isoleucine; D005492:Folic Acid; D005998:Glycine