Molecular Cloning and Structure-Function Analysis of the Plant Proton-Sucrose Symporter

Abstract

To explore other functionally-important domains, the AtSUC1 cDNA was randomly mutagenized and transformed yeast were screened on sucrose- or maltose-limited media for growth. Three point mutations were identified---H65Q, G334S, and L461P. Significantly, they all displayed enhanced sucrose transport rate. H65Q and G334S also increased maltose transport activity. In the predicted "6-loop-6" topology of the symporter, these residues all face extracellularly, suggesting their involvement in substrate binding. To investigate this hypothesis, the symporter-expressing COS-1 cells were analyzed by epitope-tagging and immunofluorescence. The results showed that both N- and C-termini, as well as the central loop, are located on the cytoplasmic side of the membrane.