Phosphate uptake across the tonoplast of intact vacuoles isolated from suspension-cultured cells of Catharanthus roseus (L.) G. Don

Transport of inorganic orthophosphate (Pi) across the tonoplast membrane was studied using intact vacuoles isolated from suspension-cultured cells of Catharanthus roseus. Orthophosphate uptake was strongly stimulated in the presence of Mg-ATP and Mg-pyrophosphate and inhibited by bafilomycin and concanamycin which are potent inhibitors of the vacuolar H+-ATPase. These results indicated that the buildup of an electrochemical gradient by the H+ pumps tvas essential for the uptake of Pi. Potassium thiocyanate, which dissipates the membrane potential across the tonoplast, strongly inhibited the Mg-ATP-stimulated uptake of Pi, while only a weak inhibition was observed in the presence of NH4Cl, which dissipates the pH gl-adient. These results indicate that, as observed for other anions like malate or chloride, the electrical component is the driving force of Pi uptake, whereas the Delta pH plays only a minor role. Possible competitive inhibitors of Pi, MoO42-, VO43- and CrO42- were tested. Among them, CrO42- strongly inhibited Pi uptake into the vacuoles. Various inhibitors of anion transport were also tested. Only 4,4-diisothiocyanostilbene-2,2'-disulfonic acid strongly inhibited Pi uptake into the vacuoles. The function of the vacuolar Pi transporters for cytoplasmic Pi homeostasis is discussed.