EXPRESSION AND ACTIVATION OF THE VACUOLAR PROCESSING ENZYME IN SACCHAROMYCES-CEREVISIAE

Vacuolar processing enzymes (VPEs) are cysteine proteinases responsibl e for maturation of various vacuolar proteins in plants. A larger prec ursor to VPE synthesized on rough endoplasmic reticulum is converted t o an active enzyme in the vacuoles. In this study, a precursor to cast or bean VPE was expressed in a pep4 strain of the yeast Saccharomyces cerevisiae to examine the mechanism of activation of VPE. Two VPE prot eins of 59 and 46 kDa were detected in the vacuoles of the transforman t. They were glycosylated in the yeast cells, although VPE is not glyc osylated in plant cells in spite of the presence of two N-linked glyco sylation sites. During the growth of the transformant, the level of th e 59 kDa VPE increased slightly until a rapid decrease occurred after 9 h. By contrast, the 46 kDa VPE appeared simultaneously with the disa ppearance of the 59 kDa VPE. Vacuolar processing activity increased wi th the accumulation of the 46 kDa VPE, but not of the 59 kDa VPE. The specific activity of the 46 kDa VPE was at a similar revel to that of VPE in plant cells. The 46 kDa VPE instead of proteinase A mediated th e conversion of procarboxypeptidase Y to the mature form. This indicat es that proteinase A responsible for maturation of yeast vacuolar prot eins can be replaced functionally by plant VPE. These findings suggest that an inactive VPE precursor synthesized on the endoplasmic reticul um is transported to the vacuoles in the yeast cells and then processe d to make an active VPE by serf-catalytic proteolysis within the vacuo les.