Controlled Proteolysis Mimics the Effect of Fusicoccin on the Plasma Membrane H+-ATPase

We analyzed the effects of controlled treatments with trypsin of plasma membrane (PM) isolated from radish (Raphanus sativus L.) seedlings on the activity of the PM H+-ATPase, and we compared them with those of fusicoccin (FC). Mild treatments of the PM with trypsin, which led to a decrease of the molecular mass of the peptide of about 10 kD, markedly increased the H+-ATPase activity. The effect strongly increased with the increase of pH of the assay medium from 6.1 to 7.5, so the pH optimum of the enzyme activity shifted from 6.8 in untreated PM to 7.1 in trypsin-treated PM. The proteolytic treatment activated only the portion of PM H+-ATPase activity that is stable to preincubation in assay medium in the absence of ATP and determined a strong increase of Vmax and a less marked decrease of the apparent Km for Mg-ATP. All of these effects were very similar to those determined by FC, which activated the PM H+-ATPase without promoting its proteolytic cleavage. FC did not further activate the H+-ATPase activity of trypsin-treated PM under conditions in which the FC receptor was protected from the attack of trypsin. Conversely, trypsin treatment had little effect on the PM H+-ATPase preactivated with FC. Moreover, the activity of the PM H+-ATPase preactivated with FC was not further activated by Iysolecithin. These results indicate that the modification of the PM H+-ATPase of higher plants triggered by the FC-receptor complex hinders the inhibitory interaction of the regulatory C-terminal domain with the active site.