Abstract
The proton-coupled folate transporter (PCFT) SLC46A1 mediates uphill folate transport into enterocytes in proximal small intestine coupled to the inwardly directed proton gradient. Hereditary folate malabsorption is due to loss-of-function mutations in the PCFT gene. This study addresses the functional role of conserved charged amino acid residues within PCFT transmembrane domains with a detailed analysis of the PCFT E185 residue. D156A-, E185A-, E232A-, R148A-, and R376A-PCFT mutants lost function at pH 5.5, as assessed by transient transfection in folate transport-deficient HeLa cells. At pH 7.4, function was preserved only for E185A-PCFT. Loss of function for E185A-PCFT at pH 5.5 was due to an eightfold decrease in the [3H]methotrexate (MTX) influx V max; the MTX influx Kt was identical to that of wild-type (WT)-PCFT (1.5 μM). Consistent with the intrinsic functionality of E185A-PCFT, [3H]MTX influx at pH 5.5 or 7.4 was trans-stimulated in cells preloaded with nonlabeled MTX or 5-formyltetrahydrofolate. Replacement of E185 with Leu, Cys, His, or Gln resulted in a phenotype similar to E185A-PCFT. However, there was greater preservation of activity (∼38% of WT) for the similarly charged E185D-PCFT at pH 5.5. All E185 substitution mutants were biotin accessible at the plasma membrane at a level comparable to WT-PCFT. These observations suggest that the E185 residue plays an important role in the coupled flows of protons and folate mediated by PCFT. Coupling appears to have a profound effect on the maximum rate of transport, consistent with augmentation of a rate-limiting step in the PCFT transport cycle.
| Original language | English (US) |
|---|---|
| Journal | American Journal of Physiology - Cell Physiology |
| Volume | 297 |
| Issue number | 1 |
| DOIs | |
| State | Published - Jul 2009 |
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Keywords
- Folate transport
- Heme carrier protein-1
- Hereditary folate malabsorption
- Methotrexate
- Pemetrexed
- Proton-coupled folate transporter/heme carrier protein-1
- Proton-coupled transporters
ASJC Scopus subject areas
- Cell Biology
- Physiology
Cite this
Role of the glutamate 185 residue in proton translocation mediated by the proton-coupled folate transporter SLC46A1. / Unal, Ersin Selcuk; Zhao, Rongbao; Goldman, I. David.
In: American Journal of Physiology - Cell Physiology, Vol. 297, No. 1, 07.2009.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Role of the glutamate 185 residue in proton translocation mediated by the proton-coupled folate transporter SLC46A1
AU - Unal, Ersin Selcuk
AU - Zhao, Rongbao
AU - Goldman, I. David
PY - 2009/7
Y1 - 2009/7
N2 - The proton-coupled folate transporter (PCFT) SLC46A1 mediates uphill folate transport into enterocytes in proximal small intestine coupled to the inwardly directed proton gradient. Hereditary folate malabsorption is due to loss-of-function mutations in the PCFT gene. This study addresses the functional role of conserved charged amino acid residues within PCFT transmembrane domains with a detailed analysis of the PCFT E185 residue. D156A-, E185A-, E232A-, R148A-, and R376A-PCFT mutants lost function at pH 5.5, as assessed by transient transfection in folate transport-deficient HeLa cells. At pH 7.4, function was preserved only for E185A-PCFT. Loss of function for E185A-PCFT at pH 5.5 was due to an eightfold decrease in the [3H]methotrexate (MTX) influx V max; the MTX influx Kt was identical to that of wild-type (WT)-PCFT (1.5 μM). Consistent with the intrinsic functionality of E185A-PCFT, [3H]MTX influx at pH 5.5 or 7.4 was trans-stimulated in cells preloaded with nonlabeled MTX or 5-formyltetrahydrofolate. Replacement of E185 with Leu, Cys, His, or Gln resulted in a phenotype similar to E185A-PCFT. However, there was greater preservation of activity (∼38% of WT) for the similarly charged E185D-PCFT at pH 5.5. All E185 substitution mutants were biotin accessible at the plasma membrane at a level comparable to WT-PCFT. These observations suggest that the E185 residue plays an important role in the coupled flows of protons and folate mediated by PCFT. Coupling appears to have a profound effect on the maximum rate of transport, consistent with augmentation of a rate-limiting step in the PCFT transport cycle.
AB - The proton-coupled folate transporter (PCFT) SLC46A1 mediates uphill folate transport into enterocytes in proximal small intestine coupled to the inwardly directed proton gradient. Hereditary folate malabsorption is due to loss-of-function mutations in the PCFT gene. This study addresses the functional role of conserved charged amino acid residues within PCFT transmembrane domains with a detailed analysis of the PCFT E185 residue. D156A-, E185A-, E232A-, R148A-, and R376A-PCFT mutants lost function at pH 5.5, as assessed by transient transfection in folate transport-deficient HeLa cells. At pH 7.4, function was preserved only for E185A-PCFT. Loss of function for E185A-PCFT at pH 5.5 was due to an eightfold decrease in the [3H]methotrexate (MTX) influx V max; the MTX influx Kt was identical to that of wild-type (WT)-PCFT (1.5 μM). Consistent with the intrinsic functionality of E185A-PCFT, [3H]MTX influx at pH 5.5 or 7.4 was trans-stimulated in cells preloaded with nonlabeled MTX or 5-formyltetrahydrofolate. Replacement of E185 with Leu, Cys, His, or Gln resulted in a phenotype similar to E185A-PCFT. However, there was greater preservation of activity (∼38% of WT) for the similarly charged E185D-PCFT at pH 5.5. All E185 substitution mutants were biotin accessible at the plasma membrane at a level comparable to WT-PCFT. These observations suggest that the E185 residue plays an important role in the coupled flows of protons and folate mediated by PCFT. Coupling appears to have a profound effect on the maximum rate of transport, consistent with augmentation of a rate-limiting step in the PCFT transport cycle.
KW - Folate transport
KW - Heme carrier protein-1
KW - Hereditary folate malabsorption
KW - Methotrexate
KW - Pemetrexed
KW - Proton-coupled folate transporter/heme carrier protein-1
KW - Proton-coupled transporters
UR - http://www.scopus.com/inward/record.url?scp=67650073369&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=67650073369&partnerID=8YFLogxK
U2 - 10.1152/ajpcell.00096.2009
DO - 10.1152/ajpcell.00096.2009
M3 - Article
C2 - 19403800
AN - SCOPUS:67650073369
VL - 297
JO - American Journal of Physiology - Renal Fluid and Electrolyte Physiology
JF - American Journal of Physiology - Renal Fluid and Electrolyte Physiology
SN - 1931-857X
IS - 1
ER -