Molecular Cloning, Immunochemical Localization to the Vacuole, and Expression in Transgenic Yeast and Tobacco of a Putative Sugar Transporter from Sugar Beet

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Abstract
Several plant genes have been cloned that encode members of the sugar transporter subgroup of the major facilitator superfamily of transporters. Here we report the cloning, expression, and membrane localization of one of these porters found in sugar beet (Beta vulgaris L.). This clone, cDNA-1, codes for a protein with 490 amino acids and an estimated molecular mass of 54 kD. The predicted membrane topology and sequence homology suggest that cDNA-1 is a member of the sugar transporter family. RNA gel blot analysis revealed that this putative sugar transporter is expressed in all vegetative tissues and expression increases with development in leaves. DNA gel blot analysis indicated that multiple gene copies exist for this putative sugar transporter in the sugar beet genome. Antibodies directed against small peptides representing the N- and C-terminal domains of the cDNA1 protein identified a 40-kD polypeptide in microsomes isolated from cDNA-1-transformed yeast (Saccharomyces cerevisiae). Moreover, the same protein was identified in sugar beet and transgenic tobacco (Nicotaina tobacum L.) membrane fractions. Detailed analysis of the transporter's distribution across linear sucrose gradients and flotation centrifugations showed that it co-migrates with tonoplast membrane markers. We conclude that this carrier is located on the tonoplast membrane and that it may mediate sugar partitioning between the vacuole and cytoplasmic compartments.