Bafilomycin Inhibits Vacuolar pH Regulation in a Fresh Water Charophyte, Chora corallina

Abstract Bafilomycin A 1 , known as an inhibitor of vacuolar type H + ‐ATPase, was used to study involvement of the vacuolar ATP‐dependent H + ‐pump in the vacuolar pH regulation in a fresh water charophyte, Chara corallina . When bafilomycin A 1 (100 nM) was externally given to intact cells, the vacuolar pH (about 5) was not affected. Internodal cells were then pretreated with 100 nM bafilomycin for 1 − 2 h and the vacuolar sap was replaced with a weakly buffered solution of pH 7.4. The readjustment of the modified vacuolar pH in bafilomycin‐treated cells was significantly retarded compared with that in untreated cells. Next, bafilomycin A 1 was directly introduced into the vacuole by vacuolar perfusion with the artificial cell sap of pH 7.4. At 100 nM bafilomycin A 1 , the decrease in the vacuolar pH was significantly inhibited. When cell sap was replaced with the artificial cell sap containing no buffer (pH 5.2 − 5.5), the vacuolar pH increased in the presence of vacuolar bafilomycin, suggesting that the PP 1 ‐ dependent H + pumping alone was not sufficient for the pH regulation of Chara vacuoles. Intracellular bafilomycin A 1 had no effect on the plasma membrane potential of tonoplast‐free cells, which is evidence that it does not affect the electrogenic H + ‐pump in the plasma membrane. Bafilomycin A 1 inhibited the ATP‐dependent H + transport of tonoplast vesicles but not the PP 1 ‐dependent H + transport. The ATPase activity of tonoplast vesicles was also inhibited by bafilomycin A 1 .