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Bacterial Multidrug Exporters pp 343–352Cite as

Reconstitution and Transport Analysis of Eukaryotic Transporters in the Post-Genomic Era

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1700))

Abstract

Measuring transport activity through reconstituted proteoliposomes is a key technique to resolve numerous problems found in the traditional methods. The system includes overexpression, purification, and reconstitution of transporters. Mixing of purified transporter with lipid and dilution below the critical micelle concentration result in rapid generation of proteoliposomes. Incubation of proteoliposomes in the presence of a driving force initiates substrate uptake. After starting the reaction, samples are passed through a gel filtration column to separate proteoliposomes from the reaction mixture. Here, we describe step-by-step procedures for such reconstitution assays.

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References

  1. Kasahara M, Hinkle PC (1977) Reconstitution and purification of the d-glucose transporter from human erythrocytes. J Biol Chem 252:7384–7390

    CAS  PubMed  Google Scholar 

  2. Newman MJ, Foster DL, Wilson TH et al (1981) Purification and reconstitution of functional lactose carrier from Escherichia coli. J Biol Chem 256:11804–11808

    CAS  PubMed  Google Scholar 

  3. Omote H, Miyaji T, Hiasa M et al (2016) Structure, function, and drug interactions of neurotransmitter transporters in the post-genome era. Annu Rev Pharmacol Toxicol 56:385–402

    Article  CAS  PubMed  Google Scholar 

  4. Juge N, Moriyama S, Miyaji T et al (2015) Plasmodium falciparum chloroquine resistance transporter is a H+-coupled polyspecific nutrient and drug exporter. Proc Natl Acad Sci U S A 112:3356–3361

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Miyaji T, Kuromori T, Takeuchi Y et al (2015) AtPHT4;4 is a chloroplast-localized ascorbate transporter in Arabidopsis. Nat Commun 6:5928

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Omote H, Miyaji T, Juge N et al (2011) Vesicular neurotransmitter transporter: bioenergetics and regulation of glutamate transport. Biochemistry 50:5558–5565

    Article  CAS  PubMed  Google Scholar 

  7. Omote H, Moriyama Y (2013) Vesicular neurotransmitter transporters: an approach for studying transporters with purified proteins. Physiology (Bethesda) 28:39–50

    Article  CAS  Google Scholar 

  8. Otsuka M, Matsumoto T, Morimoto R et al (2005) A human transporter protein that mediates the final excretion step for toxic organic cations. Proc Natl Acad Sci U S A 102:17923–17928

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Juge N, Yoshida Y, Yatsushiro S et al (2006) Vesicular glutamate transporter contains two independent transport machineries. J Biol Chem 281:39499–39506

    Article  CAS  PubMed  Google Scholar 

  10. Miyaji T, Echigo N, Hiasa M et al (2008) Identification of a vesicular aspartate transporter. Proc Natl Acad Sci U S A 105:11720–11724

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Sawada K, Echigo N, Juge N et al (2008) Identification of a vesicular nucleotide transporter. Proc Natl Acad Sci U S A 105:5683–5686

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Juge N, Gray JA, Omote H et al (2010) Metabolic control of vesicular glutamate transport and release. Neuron 68:99–112

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Kato Y, Omote H, Miyaji T (2013) Inhibitors of ATP release inhibit vesicular nucleotide transporter. Biol Pharm Bull 36:1688–1691

    Article  CAS  PubMed  Google Scholar 

  14. Miyaji T, Sawada K, Omote H et al (2011) Divalent cation transport by vesicular nucleotide transporter. J Biol Chem 286:42881–42887

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Leviatan S, Sawada K, Moriyama Y et al (2010) Combinatorial method for overexpression of membrane proteins in Escherichia coli. J Biol Chem 285:23548–23556

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Moriyama Y, Iwamoto A, Hanada H et al (1991) One-step purification of Escherichia coli H+-ATPase (F0F1) and its reconstitution into liposomes with neurotransmitter transporters. J Biol Chem 266:22141–22146

    CAS  PubMed  Google Scholar 

  17. Komatsu T, Hiasa M, Miyaji T et al (2011) Characterization of the human MATE2 proton-coupled polyspecific organic cation exporter. Int J Biochem Cell Biol 43:913–918

    Article  CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Department of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, 700-8530, Japan

    Hiroshi Omote & Yoshinori Moriyama

Authors
  1. Hiroshi Omote
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  2. Yoshinori Moriyama
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Corresponding author

Correspondence to Hiroshi Omote .

Editor information

Editors and Affiliations

  1. Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka, Japan

    Akihito Yamaguchi

  2. Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka, Japan

    Kunihiko Nishino

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Omote, H., Moriyama, Y. (2018). Reconstitution and Transport Analysis of Eukaryotic Transporters in the Post-Genomic Era. In: Yamaguchi, A., Nishino, K. (eds) Bacterial Multidrug Exporters. Methods in Molecular Biology, vol 1700. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7454-2_19

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  • DOI: https://doi.org/10.1007/978-1-4939-7454-2_19

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7452-8

  • Online ISBN: 978-1-4939-7454-2

  • eBook Packages: Springer Protocols

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