Purification and reconstitution of polyspecific H + /organic cation antiporter human MATE1

Tatsuya Kawasaki, Takuya Matsumoto, Yuma Iwai, Mamiyo Kawakami, Narinobu Juge, Hiroshi Omote, Tomohiro Nabekura, Yoshinori Moriyama

Research output: Contribution to journalArticle

Abstract

Human MATE1 (multidrug and toxin extrusion 1, hMATE1) is a H + /organic cation (OC) exchanger responsible for the final step of toxic organic cation excretion in the kidney and liver. To investigate the mechanism of transport, we have established an in vitro assay procedure that includes its expression in insect cells, solubilization with octyl glucoside, purification, and reconstitution into liposomes. The resultant proteoliposomes containing hMATE1 as the sole protein component took up radiolabeled tetraethylammonium (TEA) in a ∆pH-dependent and electroneutral fashion. Furthermore, lipid-detergent micelle containing hMATE1 showed ∆pH-dependent TEA binding similar to transport. Mutated hMATE1 with replacement E273Q completely lacked these TEA binding and transport. In the case of divalent substrates, transport was electrogenic. These observations indicate that the stoichiometry of OC/H + exchange is independent of substrate charge. Purification and reconstitution of hMATE1 is considered to be suitable for understanding the detailed molecular mechanisms of hMATE1. The results suggest that Glu273 of hMATE1 plays essential roles in substrate binding and transport.

Original languageEnglish
Pages (from-to)2456-2464
Number of pages9
JournalBiochimica et Biophysica Acta - Biomembranes
Volume1860
Issue number11
DOIs
Publication statusPublished - Nov 2018
Externally publishedYes

Keywords

  • Multidrug and toxic compound extrusion
  • Organic cation transporter
  • Purification
  • Reconstitution

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Cell Biology

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