Human proton-coupled folate transporter (PCFT) membrane topology indicating residues evaluated with the substituted cysteine accessibility method (SCAM) in the current or previous reports. There are six extracellular (EL), and five intracellular (IL) predicted loops. The seven native cysteine residues are highlighted in orange. The residues studied include those in blue predicted to reside in a cytoplasmic loop and those in green predicted to reside in an extracellular loop, with experimental confirmation based on several reports (5, 9, 41). Residues in red are shown, in this report, to be posttranslationally modified when replaced with cysteine. The rectangle encloses the 1st intracellular loop.
MTSEA-biotin labeling of the substituted cysteine residues in human PCFT. A: MTSEA-biotin labeling of wild-type PCFT (PCFT-WT) and the S110C-CL (cysteine-less) mutant along with the negative control (PCFT-CL) and residues predicted to be extracellular (G207C-CL and E292C-CL) as positive, extracellular, controls. B: MTSEA-biotin labeling of residues previously shown to be extracellularly inaccessible and residues in the 1st intracellular loop. “Pull down” indicates proteins separated by streptavidin beads; proteins in crude membranes are indicated in the lower bands and serve as controls for PCFT expression. The films were subjected to low, medium, and high exposure times. The arrows indicate that G207C-CL transient transfectants were treated with 10 mM dl-dithiothreitol (DTT) for 10 min at room temperature before MTSEA-biotin labeling. The data are representative of at least three independent experiments.
The effects of DTT treatment as well as remodification by methanethiosulfonate reagents on transport function of the L290C-DSL and G207C-CL mutants. Transient transfectants were incubated with DTT to remove the modification from the substituted cysteine. After being washed, cells were treated with MTSEA-biotin (0.5 mM), sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES; 3 mM), or [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET; 3 mM) for 30 min at room temperature. The references set at 100% are the transport activities of the mutants after treatment with 10 mM DTT. Data are means ± SE from three independent measurements.
Specificity of the weak labeling, and labeling after membrane permeabilization by MTSEA-biotin, of the substituted cysteine residues within the 1st intracellular loop. A: impact of treatment with membrane-impermeant MTSES on MTSEA-biotin labeling. Transient transfectants were treated with 3 mM MTSES, or buffer alone, for 30 min at room temperature before exposure to MTSEA-biotin. B: MTSEA-biotin labeling was performed after transient transfectants were permeabilized with digitonin, (50 µg/ml), for 5 min at room temperature. The vertical line indicates the repositioned lanes. Each panel is a representative image from two separate experiments.
Efficiency of reducing reagents in removing the posttranslational modification of the L290C-DSL mutant. A: effect of reducing reagents on restoration of the transport activity of the L290C-DSL mutant with PCFT-DSL (DSL indicates the template in which serine is substituted for Cys66 and Cys298 to eliminate the disulfide bond that would be broken with DTT, making the molecule accessible), as a negative control. Transient transfectants were incubated with 10 mM reducing agents for 10 min at room temperature before [3H]methotrexate influx was measured. The reference is the activity of a mutant treated with 10 mM DTT. B: activation of the L290C-DSL was assessed at lower concentrations (1 and 0.1 mM) of reducing agents. The reference is the activity after 10 mM DTT treatment. The dotted line indicates the activity of L290C-DSL without treatment with a reducing agent. C: the impact of the inclusion of reducing agents in the growth medium on transport activity. Freshly made reducing agents were diluted in growth medium and added to cells immediately after transfection. Since activity was determined 2 days after transfection, the incubation period with reducing agents was also 2 days with one change of medium after 1 day. The cells were treated either with 10 mM DTT or with buffer alone for 10 min before measurement of PCFT function with [3H]methotrexate. The control in this experiment was the transport activities of transfectants neither treated with a reducing reagent in the growth medium nor with DTT afterwards. For all panels, data are means ± SE from three separate measurements.
SDS-PAGE analysis of dimer formation of the PCFT mutants posttranslationally modified. A: PCFT expressed on the plasma membrane in transient transfectants was labeled with lysine-targeted EZ-Link Sulfo-NHS-LC-Biotin and pulled down with streptavidin beads. DTT-free sample buffer was used to mix the crude membrane fractions at room temperature and to strip proteins from the beads at 100°C for 5 min. The protein samples were then mixed with equal volumes of either DTT-free sample buffer (DTT−) or DTT-containing sample buffer (DTT+) for at least 30 min at room temperature before being loaded on the gel. B: transient transfectants were treated successively with DTT, N-ethylmaleimide (NEM), and EZ-Link Sulfo-NHS-LC-Biotin before biotinylated PCFT was pulled down with streptavidin beads. DTT-containing or DTT-free sample buffer were used to process the crude membranes and the beads. Each panel is a representative image from at least two separate experiments.
Glutathionylation of the substituted cysteine residues in PCFT-mutants. Panel A: Glutathionylation of PCFT mutants that were found to be posttranslationally modified. After transient transfection, cells were grown in RPMI-1640 medium containing 22 µM BioGEE for 24 h before cells were processed for pull-down assay with streptavidin beads. The last lane of this panel serves as a quantitative control in which PCFT expressed on the plasma membrane (P.M.) was labeled with EZ-Link Sulfo-NHS-LC-Biotin. B: the effects of DTT on the removal of the glutathionylation modification from PCFT mutants. Transient transfectants were labeled with BioGEE for 24 h and then treated with DTT (DTT+) or buffer alone (DTT−) before pull-down assays were performed with streptavidin beads. C: the effects of addition of GSSG on biotinylation of PCFT by BioGEE in PCFT mutants. After transfection, cells were grown in medium containing 22 µM BioGEE in the presence or absence of 2 mM GSSG for 24 h. D: glutathionylation assays in a wider spectrum of PCFT mutants and PCFT-WT. For all panels, DTT-free sample buffer was used to process all samples and the images shown are representative of two independent analyses.