Human proton coupled folic acid transporter is a monodisperse oligomer in the lauryl maltose neopentyl glycol solubilized state

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  • Nanda G. Aduri
  • Heidi A. Ernst, Københavns Universitet
  • ,
  • Bala K. Prabhala, Københavns Universitet
  • ,
  • Shweta Bhatt, Københavns Universitet
  • ,
  • Thomas Boesen
  • Michael Gajhede, Københavns Universitet
  • ,
  • Osman Mirza, Københavns Universitet

The human proton coupled folic acid transporter PCFT is the major import route for dietary folates. Mutations in the gene encoding PCFT cause hereditary folic acid malabsorption, which manifests itself by compromised folate absorption from the intestine and also in impaired folate transport into the central nervous system. Since its recent discovery, PCFT has been the subject of numerous biochemical studies aiming at understanding its structure and mechanism. One major focus has been its oligomeric state, with some reports supporting oligomers and others a monomer. Here, we report the overexpression and purification of recombinant PCFT. Following detergent screening, n-Dodecyl beta-D-maltoside (DDM) and lauryl maltose neopentyl glycol (LMNG) were chosen for further work as they exhibited the most optimal solubilization. We found that purified detergent solubilized PCFT was able to bind folic acid, thus indicating a functionally active protein. Size exclusion chromatography showed that PCFT in DDM was polydisperse; the LMNG preparation was clearly monodisperse but with shorter retention time than the major DDM peak. To assess the oligomeric state negative stain electron microscopy was performed which showed a particle with the size of a PCFT dimer. (C) 2017 Elsevier Inc. All rights reserved.

Original languageEnglish
JournalBiochemical and Biophysical Research Communications
Volume495
Issue2
Pages (from-to)1738-1743
Number of pages6
ISSN0006-291X
DOIs
Publication statusPublished - 8 Jan 2018

    Research areas

  • Negative stain EM, Oligomeric state, PCFT, Size exclusion chromatography, Substrate binding assay, FOLATE TRANSPORTER, IMPACT, MEMBRANE, MALDI-TOFMS, CRYO-EM, EM STRUCTURE DETERMINATION, IDENTIFICATION, Proton-Coupled Folate Transporter/chemistry, Humans, Protein Multimerization, Spodoptera, Recombinant Proteins/chemistry, Sf9 Cells, Protein Structure, Quaternary, Glucosides, Solubility, Folic Acid/metabolism, Models, Molecular, Detergents, Microscopy, Electron, Animals, Ligands, Glycols

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