Application of vanadate-induced nucleotide trapping to plant cells for detection of ABC proteins.
The vanadate-induced nucleotide trapping technique, which has been conventionally used to characterize mammalian ATP-binding cassette (ABC) proteins, was applied to berberine-producing plant cell cultures, Thalictrum minus and Coptis japonica. One membrane protein at ca. 180 kDa was photoaffinity-labeled with 8-azido-[alpha-(32)P]ATP in the T. minus cells in the presence of vanadate, which was specifically induced by the addition of benzyladenine in a similar manner as the induction of berberine biosynthesis in these cell cultures, whereas three bands were observed in the C. japonica cells in the size region between 120 and 150 kDa corresponding to full-sized ABC protein. The benzyladenine-induced band in T. minus showed properties similar to those of human MDR1, including the recognition of berberine, which suggests that the ABC protein detected in T. minus takes this endogenous alkaloid as a putative substrate for transport. This is the first application of this technique to plant cells.
Terasaka
Kazuyoshi
K
Laboratory of Molecular and Cellular Biology of Totipotency, Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kitashirakawa, Kyoto, 606-8502 Japan.
Shitan
Nobukazu
N
Sato
Fumihiko
F
Maniwa
Fumio
F
Ueda
Kazumitsu
K
Yazaki
Kazufumi
K
D018528:ATP-Binding Cassette Transporters / Q000235:genetics* / Q000378:metabolism; D001599:Berberine / Q000378:metabolism; D002478:Cells, Cultured; D039323:Coptis / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D019920:Photoaffinity Labels; D031949:Thalictrum / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology*
D018528:ATP-Binding Cassette Transporters; D010761:Phosphorus Radioisotopes; D019920:Photoaffinity Labels; D001599:Berberine; D014638:Vanadates