SUC1 AND SUC2 - 2 SUCROSE TRANSPORTERS FROM ARABIDOPSIS-THALIANA - EXPRESSION AND CHARACTERIZATION IN BAKERS-YEAST AND IDENTIFICATION OF THE HISTIDINE-TAGGED PROTEIN

An important, most likely essential step for the long distance transpo rt of sucrose in higher plants is the energy-dependent, uncoupler-sens itive loading into phloem cells via a sucrose-H+ symporter. This paper describes functional expression in Saccharomyces cerevisiae of two cD NAs encoding energy-dependent sucrose transporters from the plasma mem brane of Arabidopsis thaliana, SUC1 and SUC2. Yeast cells transformed with vectors allowing expression of either SUC1 or SUC2 under the cont rol of the promoter of the yeast plasma membrane ATPase gene (PMA1) tr ansport sucrose, and to a lesser extent also maltose, across their pla sma membranes in an energy-dependent manner. The K-M-values for sucros e transport are 0.50 mM and 0.77 mM, respectively, and transport by bo th proteins is strongly inhibited by uncouplers such as carbonyl cyani de m-chloro-phenylhydrazone (CCCP) and dinitrophenol (DNP), or SH-grou p inhibitors. The VMAX but not the K-M-values of sucrose transport dep end on the energy status of transgenic yeast cells. The two proteins e xhibit different patterns of pH dependence with SUC1 being much more a ctive at neutral and slightly acidic pH values than SUC2. The proteins share 78% identical amino acids, their apparent molecular weights are 54.9 kDa and 54.5 kDA, respectively, and both proteins contain 12 put ative transmembrane helices. A modified SUC1-His6 cDNA encoding a hist idine tag at the SUC1 C-terminus was also expressed in S. cerevisiae. The tagged protein is fully active and is shown to migrate at an appar ent molecular weight of 45 kDa on 10% SDS-polyacrylamide gels.