The characterization of membrane transport of specialized metabolites is essential to understand their metabolic fluxes and to implement metabolic engineering strategies towards the production of increased levels of these valuable metabolites. Here, we describe a set of procedures to isolate tonoplast membranes, to check their purity and functionality, and to characterize their transport properties. Transport is assayed directly by HPLC analysis and quantification of the metabolites actively accumulated in the vesicles, and indirectly using the pH sensitive fluorescent probe ACMA (9-amino-6- chloro-2-methoxyacridine), when a proton antiport is involved.
Keywords: ABC transporters; ACMA; Alkaloids; BY-2 cells; Catharanthus roseus; H+-antiport; MATE transporters; Tobacco; Tonoplast vesicles; Transmembrane pH gradient; Transmembrane transport; Uptake assay.