Multidrug and toxic compound extrusion-type transporters implicated in vacuolar sequestration of nicotine in tobacco roots.
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.
Shoji
Tsubasa
T
Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan.
Inai
Koji
K
Yazaki
Yoshiaki
Y
Sato
Yasutaka
Y
Takase
Hisabumi
H
Shitan
Nobukazu
N
Yazaki
Kazufumi
K
Goto
Yumi
Y
Toyooka
Kiminori
K
Matsuoka
Ken
K
Hashimoto
Takashi
T
D000470:Alkaloids / Q000378:metabolism / Q000633:toxicity; D003001:Cloning, Molecular; D003517:Cyclopentanes / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D018432:Drug Resistance, Multiple / Q000235:genetics*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009538:Nicotine / Q000378:metabolism* / Q000633:toxicity*; D027701:Organic Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D054883:Oxylipins / Q000378:metabolism; D018517:Plant Roots / Q000187:drug effects / Q000502:physiology*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014026:Nicotiana / Q000187:drug effects / Q000235:genetics* / Q000502:physiology*; D014162:Transfection; D014617:Vacuoles / Q000187:drug effects / Q000502:physiology*
D000470:Alkaloids; D003517:Cyclopentanes; D018076:DNA, Complementary; D018744:DNA, Plant; D027701:Organic Cation Transport Proteins; D054883:Oxylipins; D049452:Green Fluorescent Proteins; D009538:Nicotine; C011006:jasmonic acid