THE HEXOSE TRANSPORTERS AT THE PLASMA-MEMBRANE AND THE TONOPLAST OF TRANSFORMED PLANT-CELLS - KINETIC CHARACTERIZATION OF 2 DISTINCT CARRIERS

The plasma membrane hexose transporter and the tonoplast hexose transporter from heterotrophically grown transformed Nicotiana tabacum cells have been studied in vitro using membrane vesicles for trans-zero transport studies. In highly purified phase-partitioned outside-out plasma membrane vesicles (PMV) the hexose transporter showed an apparent K(m) value of 230-mu-M (substrate: 3-O-methyl-D-glucose (3-OMG); pH(i) 7.2/pH(o) 7.2), which was reduced to 120-mu-M when a pH gradient was imposed (pH(o) 5.7/pH(i) 7.2). However, the V(max) value was not affected indicating that no stable pH gradient was formed. Uptake experiments with C-14-labelled acetate supported this interpretation. Transport was insensitive to N-ethylmaleimide (NEM; up to 1 mM concentration) and p-chloromercuribenzene sulfonate (PCMBS; up to 500-mu-M), whereas the tonoplast hexose transporter (in mixed inside/out and outside/out vesicles) was inhibited by NEM in a substrate-protectable manner, and PCMBS was also inhibitory. Kinetically two components with apparent K(m) values of 6 and 20 mM could be distinguished for the tonoplast hexose transporter. Substrate specificities of both transporters were similar except for D-galactose and D-fructose. The results indicate structural differences between the tonoplast and plasma membrane hexose transporters in plants.