Changes in H⁺-pumps and a tonoplast intrinsic protein of vacuolar membranes during the development of pear fruit

Vacuolar H⁺-ATPase (V-ATPase) was purified from pear fruit and antibodies were raised against the subunits of 55 and 33 kDa. Antibodies against mung bean H⁺-pyrophosphatase (V-PPase) and radish VM23, which is a tonoplast intrinsic protein (TIP) and a water channel, cross-reacted with the vacuolar membrane proteins of pear fruit. To clarify the roles of these proteins in development of pear fruit, we determined their levels relative to the total amount of protein by immunoblot analysis. The levels of subunits of the V-ATPase increased with fruit development. By contrast, the level of V-PPase was particularly high at the cell-division stage and remained almost the same at other stages. The changes in the activities of V-ATPase and V-PPase corresponded to those in their protein levels. The ratio of V-PPase activity to V-ATPase activity indicated that V-PPase is a major H⁺-pump of the vacuolar membranes of young fruit and that the contribution of V-ATPase increases with fruit development, finally, V-ATPase becomes the major H⁺-pump during the later stages of fruit development. The level of a protein analogous to VM23 (VM23P) was especially high during the active cell-expansion stage in young fruit, and VM23P might, therefore, play an important role in the rapid expansion of cells as a vacuolar water channel. Our results show that the levels of V-ATPase, V-PPase and VM23P change differently and reflect the roles of the respective proteins in the development of pear fruit.