SUC1 and SUC2: two sucrose transporters from Arabidopsis thaliana ; expression and characterization in baker's yeast and identification of the histidine‐tagged protein

Abstract

An important, most likely essential step for the long distance transport of sucrose in higher plants is the energy‐dependent, uncoupler‐sensitive loading into phloem cells via a sucrose‐H + symporter. This paper describes functional expression in Saccharomyces cerevisiae of two cDNAs encoding energy‐dependent sucrose transporters from the plasma membrane of Arabidopsis thaliana, SUC1 and SUC2 . Yeast cells transformed with vectors allowing expression of either SUC1 or SUC2 under the control of the promoter of the yeast plasma membrane ATPase gene ( PMA1 ) transport sucrose, and to a lesser extent also maltose, across their plasma membranes in an energy‐dependent manner. The K M ‐values for sucrose transport are 0.50 mM and 0.77 mM, respectively, and transport by both proteins is strongly inhibited by uncouplers such as carbonyl cyanide m ‐chlorophenylhydrazone (CCCP) and dinitrophenol (DNP), or SH‐group inhibitors. The V MAX but not the K M ‐values of sucrose transport depend on the energy status of transgenic yeast cells. The two proteins exhibit different patterns of pH dependence with SUC1 being much more active 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 putative transmembrane helices. A modified SUC1‐His6 cDNA encoding a histidine 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 apparent molecular weight of 45 kDa on 10% SDS—polyacrylamide gels.