Plants produce a large variety of alkaloids, which have diverse chemical structures and biological activities. Many of these alkaloids accumulate in vacuoles. Although some membrane proteins on tonoplasts have been identified as alkaloid uptake transporters, few have been characterized to date, and relatively little is known about the mechanisms underlying alkaloid transport and accumulation in plant cells. Berberine is a model alkaloid. Although all genes involved in berberine biosynthesis, as well as the master regulator, have been identified, the gene responsible for the final accumulation of berberine at tonoplasts has not been determined. This study showed that a multidrug and toxic compound extrusion protein 1 (CjMATE1) may act as a berberine transporter in cultured Coptis japonica cells. CjMATE1 was found to localize at tonoplasts in C. japonica cells and, in intact plants, to be expressed preferentially in rhizomes, the site of abundant berberine accumulation. Cellular transport analysis using a yeast expression system showed that CjMATE1 could transport berberine. Expression analysis showed that RNAi suppression of CjbHLH1, a master transcription factor of the berberine biosynthetic pathway, markedly reduced the expression of CjMATE1 in a manner similar to the suppression of berberine biosynthetic genes. These results strongly suggest that CjMATE1 is the transporter that mediates berberine accumulation in vacuoles. (C) 2017 Elsevier Ltd. All rights reserved.