Figure 9.

Sidedness of the transport activity of BasC in PLs. (A and B) Effect of cysteine-modifying reagents MTSES (ES) and MTSEA (EA) on L-serine transport activity. Time course (0–60 s) influx of 10 µM [³H]L-serine (0.5 µCi/data point) in empty (empty circles) or filled with 4 mM L-alanine (filled circles) BasC-A20C-GFP-PLs (A) and time course (0–60 s) efflux of 10 µM [³H]L-serine (0.5 µCi/data point) filled BasC-A20C-GFP-PLs against medium without amino acid (empty circles) or with 4 mM alanine and nontreated (filled circles), treated with 1 mM MTSES (ES) for 15 min (magenta circles), or treated with 5 mM MTSEA (EA) for 30 min (orange circles; B). 10 µM [³H]L-serine influx and efflux occurs via right-side-out– and inside-out–oriented BasC in PLs, respectively. MTSES only reaches Cys 20 (C20) in right-side-out–oriented BasC. MTSEA reaches C20 independently of the orientation of BasC insertion in PLs. Influx was determined by the incorporation of [³H]L-serine into PLs, whereas efflux was determined by the decay of [³H]L-serine remaining in PLs. MTSES or MTSEA treatment abolished L-serine influx, while only treatment with MTSEA, but not MTSES, completely inhibited L-serine efflux. Data (mean ± SEM) correspond to quadruplicates of a representative experiment. Two additional independent experiments gave similar results. (C and D) Kinetics of [³H]L-serine/4 mM L-alanine exchange in BasC-A20C-GFP-PLs treated (magenta circles) and nontreated (black circles) with 1 mM MTSES for 15 min. (D) Eadie–Hofstee plot of the kinetics shown in C. Nontreated PLs (black circles) showed a complex kinetics with low (line) and high K_m components. MTSES treatment abolished the low K_m (i.e., apparent high-affinity) component. Data (mean ± SEM) correspond to quadruplicates of a representative experiment. Another independent experiment gave similar results.