TY - JOUR
T1 - Transmembrane segment 11 appears to line the purine permeation pathway of the Plasmodium falciparum Equilibrative Nucleoside Transporter 1 (PfENT1)
AU - Riegelhaupt, Paul M.
AU - Frame, I. J.
AU - Akabas, Myles H.
PY - 2010/5/28
Y1 - 2010/5/28
N2 - Purine transport is essential for malaria parasites to grow because they lack the enzymes necessary for de novo purine biosynthesis. The Plasmodium falciparum Equilibrative Nucleoside Transporter 1 (PfENT1) is a member of the equilibrative nucleoside transporter (ENT) gene family. PfENT1 is a primary purine transport pathway across the P. falciparum plasma membrane because PfENT1 knock-out parasites are not viable at physiologic extracellular purine concentrations. Topology predictions and experimental data indicate that ENT family members have eleven transmembrane (TM) segments although their tertiary structure is unknown. In the current work, we showed that a naturally occurring polymorphism, F394L, in TM11 affects transport substrate Kℳ. Weinvestigated the structure and function of the TM11 segment using the substituted cysteine accessibility method. We showed that mutation to Cys of two highly conserved glycine residues in a GXXXG motif significantly reduces PfENT1 protein expression levels. We speculate that the conserved TM11 GXXXG glycines may be critical for folding and/or assembly. Small, cysteine-specific methane-thiosulfonate (MTS) reagents reacted with four TM11 Cys substitution mutants, L393C, I397C, T400C, and Y403C. Larger MTS reagents do not react with the more cytoplasmic positions. Hypoxanthine, a transported substrate, protected L393C, I397C, and T400C from covalent modification by the MTS reagents. Plotted on an α-helical wheel, Leu-393, Ile-397, and Thr-400 lie on one face of the helix in a 60o arc suggesting that TM11 is largely α helical. We infer that they line a water-accessible surface, possibly the purine permeation pathway. These results advance our understanding of the ENT structure.
AB - Purine transport is essential for malaria parasites to grow because they lack the enzymes necessary for de novo purine biosynthesis. The Plasmodium falciparum Equilibrative Nucleoside Transporter 1 (PfENT1) is a member of the equilibrative nucleoside transporter (ENT) gene family. PfENT1 is a primary purine transport pathway across the P. falciparum plasma membrane because PfENT1 knock-out parasites are not viable at physiologic extracellular purine concentrations. Topology predictions and experimental data indicate that ENT family members have eleven transmembrane (TM) segments although their tertiary structure is unknown. In the current work, we showed that a naturally occurring polymorphism, F394L, in TM11 affects transport substrate Kℳ. Weinvestigated the structure and function of the TM11 segment using the substituted cysteine accessibility method. We showed that mutation to Cys of two highly conserved glycine residues in a GXXXG motif significantly reduces PfENT1 protein expression levels. We speculate that the conserved TM11 GXXXG glycines may be critical for folding and/or assembly. Small, cysteine-specific methane-thiosulfonate (MTS) reagents reacted with four TM11 Cys substitution mutants, L393C, I397C, T400C, and Y403C. Larger MTS reagents do not react with the more cytoplasmic positions. Hypoxanthine, a transported substrate, protected L393C, I397C, and T400C from covalent modification by the MTS reagents. Plotted on an α-helical wheel, Leu-393, Ile-397, and Thr-400 lie on one face of the helix in a 60o arc suggesting that TM11 is largely α helical. We infer that they line a water-accessible surface, possibly the purine permeation pathway. These results advance our understanding of the ENT structure.
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U2 - 10.1074/jbc.M110.115758
DO - 10.1074/jbc.M110.115758
M3 - Article
C2 - 20335165
AN - SCOPUS:77952752414
SN - 0021-9258
VL - 285
SP - 17001
EP - 17010
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 22
ER -