ABSTRACT
This chapter focuses on the functional study of the specific transporters. Detecting and quantifying their transport activity poses several challenges. First, like any organellar transport protein, the intracellular localization limits their tractability. Second, they usually have low substrate affinity, in the millimolar range. Finally, the translocation of molecule or ion through transporters is very slow as it requires structural transitions that alternately expose the substrate binding site to either side of the membrane. The mutant protein delivery approach, first applied to the lysosomal cystine transporter, cystinosin, replaces the poorly tractable lysosomal export by a classical cellular influx. Lowering the extracellular pH mimics the lysosomal lumen acidity and generally activates the misrouted transporter. An alternate approach, the counter-transport assay, consists in artificially loading lysosomes using an unlabelled ester and measuring an exchange reaction between the unlabelled, internal amino acid and the radioactive, externally added amino acid.
