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Analytical and Fluorimetric Methods for the Characterization of the Transmembrane Transport of Specialized Metabolites in Plants

  • Inês Carqueijeiro
  • Viviana Martins
  • Henrique Noronha
  • Hernâni Gerós
  • Mariana SottomayorEmail author
Protocol
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Part of the Methods in Molecular Biology book series (MIMB, volume 1405)

Abstract

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 words

ABC transporters ACMA Alkaloids BY-2 cells Catharanthus roseus H+-antiport MATE transporters Tobacco Tonoplast vesicles Transmembrane transport Transmembrane pH gradient Uptake assay 

Notes

Acknowledgements

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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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Inês Carqueijeiro
    • 1
    • 2
    • 3
  • Viviana Martins
    • 1
    • 2
    • 4
    • 5
  • Henrique Noronha
    • 4
    • 5
  • Hernâni Gerós
    • 4
    • 5
  • Mariana Sottomayor
    • 1
    • 2
    • 3
    Email author
  1. 1.Instituto de Investigação e Inovação em SaúdeUniversidade do PortoPortoPortugal
  2. 2.IBMC – Instituto de Biologia Molecular e CelularUniversidade do PortoPortoPortugal
  3. 3.Departamento de Biologia, Faculdade de CiênciasUniversidade do PortoPortoPortugal
  4. 4.Centro de Investigação e de Tecnologias Agro-Ambientais e Biológicas (CITAB-UM)BragaPortugal
  5. 5.Grupo de Investigação em Biologia Vegetal Aplicada e Inovação Agroalimentar, Departamento de BiologiaUniversidade do MinhoBragaPortugal

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