Analytical and Fluorimetric Methods for the Characterization of the Transmembrane Transport of Specialized Metabolites in Plants
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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.
Key wordsABC transporters ACMA Alkaloids BY-2 cells Catharanthus roseus H+-antiport MATE transporters Tobacco Tonoplast vesicles Transmembrane transport Transmembrane pH gradient Uptake assay
This work was supported by: (1) Fundo Europeu de Desenvolvimento Regional funds through the Operational Competitiveness Programme COMPETE and by National Funds through Fundação para a Ciência e a Tecnologia (FCT) under the projects FCOMP-01-0124-FEDER-037277 (PEst-C/SAU/LA0002/2013) and FCOMP-01-0124-FEDER-019664 (PTDC/BIA-BCM/119718/2010); (2) by the FCT scholarships co-supported by FCT and POPH-QREN (European Social Fund), SFRH/BD/41907/2007 (IC) and SFRH/BD/74257/2010 (HN); (3) by a Postdoctoral fellowship financed by national funds through FCT under the project Incentivo/SAU/LA0002/2014 (VM); (4) by a Scientific Mecenate Grant from Grupo Jerónimo Martins.
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