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

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Abstract
Isolated vacuoles from sugarcane cells took up UDP 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 [14C]fructose 6-phosphate taken up even in the presence of UDP-Glc. G-6-P and glucose 1-phosphate also were not taken up into vacuoles. UDP-Glc uptake showed saturation kinetics with a Km of 0.7 mM and a Vmax of 11.1 nmol/min per 106 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; ADP glucose was not taken up, and GDP glucose did not compete with UDP-Glc for the binding site. Apparently, sucrose transfer into vacuoles from sugarcane is via a group translocation mechanism, probably involving 5 tonoplast-bound enzymes.