Abstract
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 K(m). We investigated 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 methanethiosulfonate (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 alpha-helical wheel, Leu-393, Ile-397, and Thr-400 lie on one face of the helix in a 60 degrees arc suggesting that TM11 is largely alpha helical. We infer that they line a water-accessible surface, possibly the purine permeation pathway. These results advance our understanding of the ENT structure.
Highlights
NIGMS Medical Scientist Training Program Grant T32-GM007288
Hypoxanthine Protection Experiments—For the Cys mutants that reacted with an MTS reagent, we investigated whether coapplication of hypoxanthine, a PfENT1 transported substrate, with the MTS reagent would protect the Cys mutant from covalent modification
GXXXG motif are highlighted by the vertical stripes, and the 394 position of the polymorphism is indicated by the vertical arrow below the sequences
Summary
Identification of Naturally Occurring PfENT1 Polymorphisms— To search for polymorphisms within the PfENT1 gene, we PCR amplified PfENT1 from the genomic DNA of 11 separate parasite strains. Western Blot of Total and Membrane Protein—Three days post-injection, ϳ70 oocytes for each condition were transferred to a dish containing calcium-free buffer (115 mM NaCl, 2.5 mM KCl, 1.8 mM MgCl2, 10 mM HEPES, pH 8) and reacted with 0.5 mg/ml sulfo-NHS-LC biotin for 30 min at room temperature to biotinylate cell surface proteins. For all of the substrates tested, no significant difference was observed between DEPC-water injected or uninjected oocytes For this reason, these data sets were pooled to represent background uptake values. Oocytes were washed 3–5 times with room temperature E1 buffer to remove MTS reagent and transferred to a solution containing 100 nM [8-3H]adenosine (23 Ci/mmol) in E1 buffer, to measure PfENT1-mediated adenosine uptake as described above. A paired two tailed t test was used to compare the effects of dipyridamole
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