A novel loss-of-function mutation in the proton-coupled folate transporter from a patient with hereditary folate malabsorption reveals that Arg 113 is crucial for function.
Hereditary folate malabsorption (HFM) patients harbor inactivating mutations including R113S in the proton-coupled folate transporter (PCFT), an intestinal folate transporter with optimal activity at acidic pH. Here we identified and characterized a novel R113C mutation residing in the highly conserved first intracellular loop of PCFT. Stable transfectants overexpressing a Myc-tagged wild-type (WT) and mutant R113C PCFT displayed similar transporter targeting to the plasma membrane. However, whereas WT PCFT transfectants showed a 22-fold increase in [(3)H]folic acid influx at pH 5.5, R113C or mock transfectants showed no increase. Moreover, WT PCFT transfectants displayed a 50% folic acid growth requirement concentration of 7 nM, whereas mock and R113C transfectants revealed 24- to 27-fold higher values. Consistently, upon fluorescein-methotrexate labeling, WT PCFT transfectants displayed a 50% methotrexate displacement concentration of 50 nM, whereas mock and R113C transfectants exhibited 12- to 14-fold higher values. Based on the crystal structure of the homologous Escherichia coli glycerol-3-phosphate transporter, we propose that the cationic R113 residue of PCFT is embedded in a hydrophobic pocket formed by several transmembrane helices that may be part of a folate translocation pore. These findings establish a novel loss of function mutation in HFM residing in an intracellular loop of PCFT crucial for folate transport.
Lasry
Inbal
I
The Fred Wyszkowski Cancer Research Laboratory, Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel.
Berman
Bluma
B
Straussberg
Rachel
R
Sofer
Yael
Y
Bessler
Hanna
H
Sharkia
Mohamad
M
Glaser
Fabian
F
Jansen
Gerrit
G
Drori
Stavit
S
Assaraf
Yehuda G
YG
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D016466:CHO Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002648:Child; D003241:Consanguinity; D006224:Cricetinae; D003412:Cricetulus; D017931:DNA Primers / Q000235:genetics; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000378:metabolism*; D006720:Homozygote; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008286:Malabsorption Syndromes / Q000235:genetics* / Q000378:metabolism*; D008727:Methotrexate / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D002352:Carrier Proteins; D017931:DNA Primers; D058973:Folate Receptors, GPI-Anchored; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D005492:Folic Acid; D001120:Arginine; D008727:Methotrexate