Photosynthetic control of the plasma membrane H⁺-ATPase in Vallisneria leaves. II. Presence of putative isogenes and a protein equipped with a C-terminal autoinhibitory domain

Abstract.

In vitro treatment with trypsin of plasma membrane (PM) vesicles isolated from the leaves of Vallisneria gigantea Graebner, an aquatic monocot, produced a marked decrease in the K _m for ATP and an increase in the V _max of H⁺-transporting activity. Concomitantly, the removal of 8 kDa of the C-terminal domain from the 94-kDa PM H⁺-ATPase was confirmed by immunoblotting using different kinds of polyclonal antibody. Three partial clones of putative PM H⁺-ATPase genes (Vga1, 2, and 3) were isolated from leaves by reverse transcription polymerase chain reaction. Northern blotting analysis revealed that the expression level of Vga3 was high and that of the other two genes was much lower. The H⁺-transporting activity of PM vesicles was substantially suppressed in the presence of inorganic phosphate (Pi), which has been supposed to be a noncompetitive inhibitor of the PM H⁺-ATPase, coincident with an increase in the K _m for ATP and a decrease in the V _max. After treatment of the isolated PM vesicles with trypsin, the inhibitory effect of Pi was no longer evident. This result indicates that Pi inhibited the activity through the C-terminal autoinhibitory domain of the PM H⁺-ATPase. Furthermore, Pi increased the K _m for ATP of the H⁺-transporting activity in the PM vesicles isolated from both dark-adapted and red-light-irradiated leaves. The results suggest that regulation of the K _m for ATP through the operation of photosynthesis is independent of regulation through the cytoplasmic level of Pi.