TRANSPORT AND ACTIVATION OF THE VACUOLAR ASPARTIC PROTEINASE PHYTEPSIN IN BARLEY (HORDEUM-VULGARE L.)

The primary translation product of barley aspartic proteinase, phyteps in (EC 3.4.23.40), consists of a signal sequence, a propart, and matur e enzyme forms. Here, we describe post-translational processing and ac tivation of phytepsin during its transport to the vacuole in roots, as detected by using metabolic labeling and immunoprecipitation. After r emoval of the signal sequence, the glycosylated precursor of 53 kDa (P 53) was produced and further processed to polypeptides of 31 and 15 kD a (P31 + P15) and, subsequently, to polypeptides of 26 and 9 kDa (P26 + P9), 45 min and 24 h after synthesis, respectively. The processing o ccurred in a late-Golgi compartment or post-Golgi compartment, because brefeldin A inhibited the processing, and P53 acquired partial endogl ycosidase H resistance 30 min after synthesis, whereas P15 was complet ely resistant. The N-glycosylation inhibitor tunicamycin had no effect on transport, but the absence of glycans on P53 accelerated the prote olytic processing. Phytepsin was also expressed in baculovirus-infecte d insect cells. The recombinant prophytepsin underwent autoproteolytic activation in vitro and showed enzymatic properties similar to the en zyme purified from grains. However, a comparison of the in vitro/in vi vo processing sites revealed slight differences, indicating that addit ional proteases are needed for the completion of the maturation in viv o.