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 andantibodies were raised against the subunits of 55 and 33 kDa.Antibodies against mung bean H⁺-pyro-phosphatase (V-PPase) andradish VM23, which is a tonoplast intrinsic protein (TIP) anda water channel, cross-reacted with the vacuolar membrane proteinsof pear fruit. To clarify the roles of these proteins in developmentof pear fruit, we determined their levels relative to the totalamount of protein by immunoblot analysis. The levels of subunitsof the V-ATPase increased with fruit development. By contrast,the level of V-PPase was particularly high at the cell-divisionstage and remained almost the same at other stages. The changesin the activities of V-ATPase and V-PPase corresponded to thosein their protein levels. The ratio of V-PPase activity to V-ATPaseactivity indicated that V-PPase is a major H⁺-pump of the vacuolarmembranes of young fruit and that the contribution of V-ATPaseincreases with fruit development, finally, V-ATPase becomesthe major H⁺-pump during the later stages of fruit development.The level of a protein analogous to VM23 (VM23P) was especiallyhigh during the active cell-expansion stage in young fruit,and VM23P might, therefore, play an important role in the rapidexpansion of cells as a vacuolar water channel. Our resultsshow that the levels of V-ATPase, V-PPase and VM23P change differentlyand reflect the roles of the respective proteins in the developmentof pear fruit. ³Research Fellow of the Japan Society for the Promotion of Science ⁴Present address: Faculty of Agriculture, Tohoku University,1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, 981 Japan