Fig. 4.

Accessibility and functional consequences of modifications of Cys-substituted residues at position 188, 190, 191, and 193 of human PCFT. A: [³H]MTX (0.5 μM) influx at pH 5.5 over 1 min. Influx mediated by the mutants is indicated as percentage of WT-PCFT activity. B: expression of the mutants in the crude membrane preparation and biotinylated on the plasma membrane. C, Top: labeling of the Cys-substituted PCFT residues with the membrane-impermeable 2-[(biotinoyl)amino]ethyl methanethiosulfonate (MTSEA biotin). Bottom: expression levels of the mutant PCFTs in the crude membrane preparation. D: impact of modification of the Cys-substituted residues with [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET) on PCFT function. Cysteine modification with MTSET (1 mg/ml) was carried out at room temperature for 30 min. After the modification, [³H]MTX influx (0.5 μM) was assessed at pH 5.5 over 1 min. Influx is expressed as pmol MTX/mg protein/min. For A and D, data are the means ± SE from three independent experiments. For B and C, The blots are representative of 2 independent experiments.