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 Mesembry anthemum 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 s ubunit B. This cleavage is suggested to occur at the peptide bond between M et192 and Glu193. At neutral pH Met-NA was one of seven derivatives which w ere cleaved by proteases present in this fraction. Enzymes exhibiting prote olytic activity were separated from other soluble proteins by Superose 12-s ize exclusion FPLC. Incubation of partially purified protease with tonoplas t-enriched membrane vesicle fractions isolated from M. crystallinum in the C-3-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 sub unit B and the appearance of D-i also occurred during incubation of tonopla st vesicles in the presence of reactive oxygen species. In addition to D-i, the polypeptide E-i (28 kDa) appeared after incubation with protease and/o r reactive oxygen species. Taken into account that D-i and E-i cross-reacte d with an affinity purified antiserum directed against subunit B, D-i as we ll as E-i might represent fragments of subunit B. These results open new pe rspectives with respect to the regulation of V-ATPase modification and turn over.