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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Multidrug and Toxic Compound Extrusion-Type Transporters Implicated in Vacuolar Sequestration of Nicotine in Tobacco Roots^1,[C],[W]

Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630–0192, Japan (T.S., K.I., Y.S., H.T., T.H.); National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305–8602, Japan (Y.Y.); Research Institute for Sustainable Humanosphere, Kyoto University, Uji 611–0011, Japan (N.S., K.Y.); and RIKEN Plant Science Center, Yokohama, Kanagawa 230–0045, Japan (Y.G., K.T., K.M.)

Nicotine is a major alkaloid accumulating in the vacuole of tobacco (Nicotiana tabacum), but the transporters involved in the vacuolar sequestration are not known. We here report that tobacco genes (NtMATE1 and NtMATE2) encoding transporters of the multidrug and toxic compound extrusion (MATE) family are coordinately regulated with structural genes for nicotine biosynthesis in the root, with respect to spatial expression patterns, regulation by NIC regulatory loci, and induction by methyl jasmonate. Subcellular fractionation, immunogold electron microscopy, and expression of a green fluorescent protein fusion protein all suggested that these transporters are localized to the vacuolar membrane. Reduced expression of the transporters rendered tobacco plants more sensitive to the application of nicotine. In contrast, overexpression of NtMATE1 in cultured tobacco cells induced strong acidification of the cytoplasm after jasmonate elicitation or after the addition of nicotine under nonelicited conditions. Expression of NtMATE1 in yeast (Saccharomyces cerevisiae) cells compromised the accumulation of exogenously supplied nicotine into the yeast cells. The results imply that these MATE-type proteins transport tobacco alkaloids from the cytosol into the vacuole in exchange for protons in alkaloid-synthesizing root cells.

² These authors contributed equally to the article.

³ Present address: Faculty of Bioenvironmental Science, Kyoto Gakuen University, Kyoto 621–8555, Japan.

⁴ Present address: Faculty of Agriculture, Kyushu University, Fukuoka 812–8581, Japan.

The author responsible for the distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Takashi Hashimoto (hasimoto{at}bs.naist.jp).

^[C] Some figures in this article are displayed in color online but in black and white in the print edition.

^[W] The online version of this article contains Web-only data.

www.plantphysiol.org/cgi/doi/10.1104/pp.108.132811

^* Corresponding author; e-mail hasimoto{at}bs.naist.jp.

Received November 18, 2008; accepted December 16, 2008; published December 19, 2008.

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