TY - JOUR
T1 - Purification and reconstitution of polyspecific H + /organic cation antiporter human MATE1
AU - Kawasaki, Tatsuya
AU - Matsumoto, Takuya
AU - Iwai, Yuma
AU - Kawakami, Mamiyo
AU - Juge, Narinobu
AU - Omote, Hiroshi
AU - Nabekura, Tomohiro
AU - Moriyama, Yoshinori
N1 - Funding Information:
This study was supported in part by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan to Y.M. ( JP18056014 ), the Smoking Research Foundation to Y.M. ( FP01002041 ), grant-in-aid for Challenging Exploratory Research to H.O. ( JP25650035 ), Precursory Research for Embryonic Science and Technology to N.J. ( JPMJPR14L4 ), and Research Support 2017 from Astellas Pharma Inc. to T.N. ( RS2017A001092 ).
Publisher Copyright:
© 2018 Elsevier B.V.
PY - 2018/11
Y1 - 2018/11
N2 - 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.
AB - 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.
KW - Multidrug and toxic compound extrusion
KW - Organic cation transporter
KW - Purification
KW - Reconstitution
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U2 - 10.1016/j.bbamem.2018.07.005
DO - 10.1016/j.bbamem.2018.07.005
M3 - Article
C2 - 30028956
AN - SCOPUS:85050557711
VL - 1860
SP - 2456
EP - 2464
JO - Biochimica et Biophysica Acta - Biomembranes
JF - Biochimica et Biophysica Acta - Biomembranes
SN - 0005-2736
IS - 11
ER -