Cloning, Localization, and Expression Analysis of a New Tonoplast Monosaccharide Transporter from Vitis vinifera L
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Tonoplast sugar transporters are important for sugar partitioning, immobilization, and accumulation during fruit development and ripening. Here we report the cloning, localization, and functional analysis of one of these transporters in grape berries (Vitis vinifera L.). This clone, named VvTMT1, encodes a 742-aa protein with a calculated molecular mass of 80.2 kDa. Predicted membrane topology and phylogenetic analysis suggest that VvTMT1 belongs to the major facilitator superfamily of membrane carriers. Semiquantitative RT-PCR suggests that VvTMT1 is a sink-specific transporter, whose expression decreases with berry development. Heterologous expression of VvTMT1 in yeast can partially restore growth of the hxt-null strain in glucose and other monosaccharide media, indicating that VvTMT1 is a functional monosaccharide transporter. Induction of VvTMT1-GFP fusion protein expression in transgenic yeast revealed its tonoplast localization. The subcellular localization of VvTMT1 in plants was shown by immunogold labeling of grape berry mesocarp cells and VvTMT1-GFP transient expression in tobacco epidermis cells. Based on the above analyses of VvTMT1, this is the first report of a functional tonoplast-localized monosaccharide transporter in grapevine.
KeywordsGrape berry Immunogold electron microscopy Subcellular localization Tonoplast Tonoplast monosaccharide transporter Vacuole Vitis vinifera L
This work was financially supported by the National Natural Science Foundation of China (30471212 and 30500347). We are grateful to Prof. Dr. Doris Rentsch (University of Bern, Switzerland) for the gift of the pDR195 vector, to Prof. Dr. Eckhard Boles (University of Frankfurt, Germany) for providing the S. cerevisiae strain EBY.VW4000, and to Dr. Camille Vainstein for language proof reading.
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