AN ASPARTIC ENDOPEPTIDASE IS INVOLVED IN THE BREAKDOWN OF PROPEPTIDESOF STORAGE PROTEINS IN PROTEIN-STORAGE VACUOLES OF PLANTS

To understand the mechanism of the maturation of various proteins in p rotein-storage vacuoles, we purified a 48-kDa aspartic endopeptidase c omposed of 32-kDa and 16-kDa subunits from castor bean. Immunocytochem ical and cell fractionation analyses of the endosperm of maturing cast or bean seed showed that the aspartic endopeptidase was localized in t he matrix of the protein-storage vacuoles, where a variety of seed sto rage proteins were also present, The amount of the aspartic endopeptid ase increased at the mid-maturation stage of the seeds before accumula tion of the storage proteins. To determine how the aspartic endopeptid ase is responsible for maturation of seed proteins in concert with the vacuolar processing enzyme, we prepared S-35 labeled proproteins of s eed proteins from the endoplasmic reticulum fraction of pulse-labeled maturing endosperm and used the authentic proproteins as substrates fo r in vitro processing experiments. The purified aspartic endopeptidase was unable to convert any of three endosperm proproteins, pro2S album in, proglobulin, and proricin, into their mature sizes, while the puri fied vacuolar processing enzyme could convert all three proproteins. W e further examined the activity of aspartic endopeptidase on the cleav age of an internal propeptide of Arabidopsis pro2S albumin, which is k nown to be removed post-translationally. The aspartic endopeptidase cl eaved the propeptide at three sites under acidic conditions. These res ults suggest that aspartic endopeptidase cannot directly convert pro2S albumin into the mature form, but it may play a role in trimming the C-terminal propeptides from the subunits that are produced by the acti on of the vacuolar processing enzyme.