Group translocation as a mechanism for sucrose transfer into vacuoles from sugarcane cells

Abstract

Isolated vacuoles from sugarcane cells took up uridine diphosphate glucose (UDP-Glc) from the surrounding medium at a rapid rate. After a 7-min incubation of vacuoles with UDP-[14C]Glc, sucrose and sucrose phosphate were identified in the vacuole extract. UDP-Glc in the incubation medium was converted to hexose phosphates, sucrose, and glucose, with very little UDP-Glc remaining. Fructose 6-phosphate was not required for UDP-Glc uptake nor was [(14)C]fructose 6-phosphate taken up even in the presence of UDP-Glc. Glucose 6-phosphate and glucose 1-phosphate also were not taken up into vacuoles. UDP-Glc uptake showed saturation kinetics with a K(m) of 0.7 mM and a V(max) of 11.1 nmol/min per 10(6) vacuoles. The optimum pH for UDP-Glc uptake was between 6.5 and 7.0. Uptake of UDP-Glc could be inhibited by p-chloromercuriphenylsulfonic acid, UDP, and GDP, and to a lesser extent by carbonyl cyanide m-chlorophenylhydrazone. The UDP-Glc binding site was specific for UDP-Glc; adenosine diphosphate glucose was not taken up, and guanosine diphosphate glucose did not compete with UDP-Glc for the binding site. The results suggest that sucrose transfer into vacuoles from sugarcane is via a group translocation mechanism, probably involving five tonoplast-bound enzymes.