Demonstration of Cl– Requirement for Inhibition of Vacuolar Acidification by Cycloprodigiosin in Situ

Applying vacuole-perfusion and plasma membrane permeabilization techniques to internodal cells of Chara, we analyzed the requirement of Cl- for the action of cycloprodigiosin (cPrG) to inhibit vacuole acidification in situ. By combining the two techniques, the Cl- concentration on both sides of the tonoplast could be controlled. In permeabilized cell fragments lacking Cl- in the vacuole, the inhibitory effect of cPrG on vacuole acidification was cancelled. On the other hand, Cl- in the cytoplasm was not needed for the cPrG action. These results supported the function of cPrG as a H+/Cl- symporter. Requirement of Cl- for the cPrG action was also demonstrated in vacuole-perfused living cells. This is the first report on the mechanism of cPrG action in situ.