Processing of V-ATPase subunit B of Mesembryanthemum crystallinum L. is mediated in vitro by a protease and/or reactive oxygen species.

Soluble proteins were isolated from leaves of the common ice plant Mesembryanthemum crystallinum L. in the CAM state of photosynthesis and tested for protease activity using amino acid-beta-naphthylamide (NA)-derivatives in a search for proteolytic activity responsible for cleavage of the V-ATPase subunit B. This cleavage is suggested to occur at the peptide bond between Met192 and Glu193. At neutral pH Met-NA was one of seven derivatives which were cleaved by proteases present in this fraction. Enzymes exhibiting proteolytic activity were separated from other soluble proteins by Superose 12-size exclusion FPLC. Incubation of partially purified protease with tonoplast-enriched membrane vesicle fractions isolated from M. crystallinum in the C3-state of photosynthesis led to a decrease in subunit B (55 kDa) protein amount and to the formation of the polypeptide Di (32 kDa), which has been previously suggested to represent a fragment of subunit B. Cleavage of subunit B and the appearance of Di also occurred during incubation of tonoplast vesicles in the presence of reactive oxygen species. In addition to Di, the polypeptide Ei (28 kDa) appeared after incubation with protease and/or reactive oxygen species. Taken into account that Di and Ei cross-reacted with an affinity purified antiserum directed against subunit B, Di as well as Ei might represent fragments of subunit B. These results open new perspectives with respect to the regulation of V-ATPase modification and turnover.