Molecular cloning and structure-function analysis of the plant proton-sucrose symporter.

摘要

A proton-sucrose symporter was cloned as part of a systematic investigation of sugar transport in plants. A clone contained the full-length cDNA of the symporter was identified by RT-PCR and subsequent screening of a sugar beet cDNA library. The putatively encoded protein shares 84% amino acid identity with the spinach homologue. It has a calculated molecular weight of 55 kD and is predicted to be a membrane protein with twelve transmembrane domains. The beet symporter is present as a single to low copy gene in the beet genome. The gene is expressed in all vegetative tissues except roots. In leaves, expression follows a sink-to-source transition, similar to that observed in photosynthesis and consistent with its putative role in phloem loading. The protein is also expressed in the storage hypocotyl, suggesting a possible involvement in sucrose uptake into storage cells. The carrier's transport function was demonstrated in Xenopus oocytes, and the activity was shown to be pH-dependent.; To study the structure-function relationships of the symporter, the Arabidopsis AtSUC1 was functionally expressed in yeast. Based on the symporter's sensitivity to DEPC, the conserved His-65 of AtSUC1 was investigated by site-directed mutagenesis. Results of yeast growth rates on sucrose-limited media and transport analysis showed mutation at this residue resulted in drastic changes in activity. Specifically, basic amino acid substitutions enhanced transport activity, suggesting His-65 is involved in the transport reaction. In support of that conclusion, His-65 was found to be the DEPC-sensitive and substrate-protectable residue of the carrier.; 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.