FUNCTIONAL RECONSTITUTION OF THE SOLUBILIZED ARABIDOPSIS-THALIANA STP1 MONOSACCHARIDE-H-CEREVISIAE( SYMPORTER IN LIPID VESICLES AND PURIFICATION OF THE HISTIDINE TAGGED PROTEIN FROM TRANSGENIC SACCHAROMYCES)

Complete DNA sequences encoding the Arabidopsis thaliana STP1 monosacc haride/H+ symporter or a histidine-tagged STP1-His6 protein were expre ssed in baker's yeast Saccharomyces cerevisiae. Both wild-type STP1 an d the recombinant his-tagged protein were located in the plasma membra nes of transformed yeast cells. The C-terminal modification caused no loss of transport activity compared with the wild-type protein. Anti-S TP1-antibodies were used to confirm the identity of the protein in yea st and to compare the apparent molecular weights of STP1 proteins in m embrane extracts from yeast or Arabidopsis thaliana. Purified yeast pl asma membranes were fused with proteoliposomes consisting of Escherich ia coli lipids and beef heart cytochrome-e oxidase. Addition of ascorb ate/TMPD/cytochrome-c to these fused vesicles caused an immediate form ation of membrane potential (inside negative; monitored with [H-3]tetr aphenylphosphonium cations) and a simultaneous, uncoupler-sensitive in flux of D-glucose into the energized vesicles. STP1-His6 protein is fu nctionally active after solubilization with octyl-PD-glucoside, which was shown by insertion of the protein into proteoliposomes by detergen t dilution and determination of the resulting transport capacity. Dete rgent extracts from either total membranes or plasma membranes of tran sgenic yeast cells were used for one-step purification of the STP1-His 6 protein on Ni2+-NTA columns. The identity of the purified protein wa s checked by immunoblotting and N-terminal sequencing.