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PLoS One
2013 Oct 08;810:e78301. doi: 10.1371/journal.pone.0078301.
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Delineating the extracellular water-accessible surface of the proton-coupled folate transporter.
Duddempudi PK
,
Goyal R
,
Date SS
,
Jansen M
.
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The proton-coupled folate transporter (PCFT) was recently identified as the major uptake route for dietary folates in humans. The three-dimensional structure of PCFT and its detailed interplay with function remain to be determined. We screened the water-accessible extracellular surface of HsPCFT using the substituted-cysteine accessibility method, to investigate the boundaries between the water-accessible surface and inaccessible buried protein segments. Single-cysteines, engineered individually at 40 positions in a functional cysteine-less HsPCFT background construct, were probed for plasma-membrane expression in Xenopus oocytes with a bilayer-impermeant primary-amine-reactive biotinylating agent (sulfosuccinimidyl 6-(biotinamido) hexanoate), and additionally for water-accessibility of the respective engineered cysteine with the sulfhydryl-selective biotinylating agent 2-((biotinoyl)amino)ethyl methanethiosulfonate. The ratio between Cys-selective over amine-selective labeling was further used to evaluate three-dimensional models of HsPCFT generated by homology / threading modeling. The closest homologues of HsPCFT with a known experimentally-determined three-dimensional structure are all members of one of the largest membrane protein super-families, the major facilitator superfamily (MFS). The low sequence identity--14% or less--between HsPCFT and these templates necessitates experiment-based evaluation and model refinement of homology/threading models. With the present set of single-cysteine accessibilities, the models based on GlpT and PepTSt are most promising for further refinement.
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24205192
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Figure 2. Transport activity of wild-type (WT) and Cys-less (CYS-) ct-V5-HsPCFT.Five days after the oocytes were injected with WT PCFT and PCFT CYS- mRNA, the uptake of 0.015 µM [3H]folic acid was measured at pH 5.5 over 10 minutes. Transport of folic acid through PCFT is proton-coupled and therefore facilitated by acidic pH. Therefore, uptake was studied at pH 5.5 [6]. Uninjected or water-injected oocytes (Uninj) were used as negative controls. Data represent the mean ± SEM from three different batches of oocytes. One-way ANOVA with Bonferroni’s multiple comparison test results shown. *** indicates p < 0.001; ns indicates not significant. Wild-type and Cys- are also not significantly different when analyzed with unpaired t test.
Figure 3. Accessibility of primary amines and Cys in HsPCFT wild-type, Cys-, and single-Cys constructs.(A) Fifteen residues were selected within and close to ECL1 and ECL2. (B) Six residues were investigated in each ECL3 and ECL4. (C) Seven residues each were probed in ECL5 and ECL6. NH2-biotinylation shows Western blot after plasma membrane fraction isolation following surface-NH2 biotinylation with sulfo-NHS-LC-biotin / avidin beads. Uninjected oocytes (uninj.) served as negative control, and PCFT V5 and PCFT CYS- injected oocytes served as positive control. SH-biotinylation shows Western blot after avidin isolation of MTSEA-biotin labeled Cys. Single-Cys engineered at positions indicated on top of the lanes. Equal amounts of protein samples were electrophoretically separated on 4-15 % precast gels, transferred to PVDF membranes, and probed with V5-HRP antibody (1/5,000 dilution in 5 % milk). The experiment was repeated on three different batches of oocytes and a representative Western blot is shown for each condition. Lanes in boxes are from separate experiments as additional positions were probed later.
Figure 4. Color-coded Cys-accessibility normalized to NH2-accessibility.(A) ECL1 and ECL2. (B) ECL3 and ECL4. (C) ECL5 and ECL6. (D) Additional positions from all ECLs. Results shown in individual panels represent positions investigated on the same Western blot.
Figure 5. Color-coded Cys-accessibility normalized to NH2-accessibility plotted on two-dimensional model.
Figure 6. Three-dimensional models showing color-coded Cys-accessibility normalized to NH2-accessibility.Gene names are given above the respective panels, and pdb # and algorithm inside the top left corner of the upper panel for each model. View normal to the membrane in top row and from extracellular side in bottom row. The figures were prepared using the PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC.
Figure 1. Initial modeling of PCFT.(A) A three-dimensional homology model of PCFT was built by the SWISS-MODEL server using an HHpred generated alignment with GlpT as input and the crystal structure of GlpT as a template. View parallel to the membrane showing all twelve transmembrane segments, color-coding as in panel C. (B) View perpendicular to the membrane looking into the predicted aqueous translocation pathway from the extracellular side. (C) Cartoon representation of the secondary structure of PCFT generated from the homology model by extrapolating information regarding the extent and orientation of transmembrane segments and loops. Gray-filled circles represent residues individually mutated to Cys. Black-filled circles identify endogenous Cys that were mutated to Ser. Numbering of extracellular loops (ECL) and intracellular loops (ICL) as indicated. Glycosylation sites indicated by “*”, and ECL surface Lys by “+”. Note, that the two Cys in ECL1 and ECL4 were also used as single-Cys mutants.
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