Activation of the Dunaliella acidophila plasma membrane H⁺-ATPase by trypsin cleavage of a fragment that contains a phosphorylation site

Trypsin treatment of purified H⁺-ATPase from plasma membranes of the extreme acidophilic alga Dunaliella acidophila enhances ATP hydrolysis and H⁺ pumping activities. The activation is associated with an alkaline pH shift, an increase in V_max, and a decrease in K_m(ATP). The activation is correlated with cleavage of the 100-kD ATPase polypeptide to a fragment of approximately 85 kD and the appearance of three minor hydrophobic fragments of 7 to 8 kD, which remain associated with the major 85-kD polypeptide. The N-terminal sequence of the small fragments has partial homology to residues 713 to 741 of Arabidopsis thaliana plasma membrane H⁺-ATPases. Incubation of cells with ³²P-labeled orthophosphate (³²Pi) results in incorporation of ³²P into the ATPase 100-kD polypeptide. Trypsin treatment of the ³²Pi-labeled ATPase leads to complete elimination of label from the approximately 85-kD polypeptide. Cleavage of the phosphorylated enzyme with endoproteinase Glu-C (V-8) yields a phosphorylated 12-kD fragment. Peptide mapping comparison between the 100-kD and the trypsinized 85-kD polypeptides shows that the 12-kD fragment is derived from the trypsin-cleaved part of the enzyme. The N-terminal sequence of the 12-kD fragment closely resembles a C-terminal stretch of an ATPase from another Dunaliella species. It is suggested that trypsin activation of the D. acidophila plasma membrane H⁺-ATPase results from elimination of an autoinhibitory domain at the C-terminal end of the enzyme that carries a vicinal phosphorylation site.