Figure 2.

5-HT transport activity and cell surface expression of EL1 cysteine mutants. (A) 5-HT transport activity. HeLa cells expressing each EL1 cysteine mutant were assayed for [³H]5-HT uptake for 10 min as described under ‘Materials and methods’. The activity of each mutant transporter is represented as a percentage of parental mutant X5C activity (0.16±0.03 pmol/mg per min). Each value represents the mean and SD of three experiments, each of which was performed in triplicates. (B) Binding activity of EL1 cysteine mutants. Membranes prepared from HeLa cells expressing each of the EL1 cysteine mutants and the control X5C were incubated with [¹²⁵I]β-CIT as described (Androutsellis-Theotokis & Rudnick 2002). The binding to each mutant transporter is represented as a percentage of the amount bound to parental mutant X5C (1.52±0.11 pmol/mg). The data represent means from three experiments. (C) Surface expression of EL1 cysteine mutants. HeLa cells expressing the EL1 mutants were treated with NHS-SS-biotin to label cell surface proteins. The cells were lysed in detergent, and surface proteins were extracted with streptavidin beads. SERT mutants were detected by immunoblotting with a monoclonal antibody to the FLAG tag attached to the SERT COOH-terminus. The 96-kDa form of SERT, which represents mature and fully glycosylated protein is shown here. (D) Quantitation of surface expression. The surface expression levels of EL1 mutants were measured by densitometry and are shown as a percentage of X5C. The data represent averaged results from three experiments.