Extracellular Rhizopus niveus aspartic proteinase-I (RNAP-I) was secreted effectively by Saccharomyces cerevisiae when RNAP-I with its preprosequence was synthesized in this organism (Horiuchi, H., Ashikari, T., Amachi, T., Yoshizumi, H., Takagi, M., and Yano, K. (1990) Agric. Biol. Chem. 54, 1771-1779). Certain deletions (DELTApro, DELTA1, DELTA2), and amino acid substitutions (MI) in the prosequence blocked secretion of RNAP-I, although the protease protection assay revealed that even DELTApro could be translocated across the membrane of the endoplasmic reticulum. When Apro or Ml was synthesized simultaneously with the wild-type preprosequence in S. cerevisiae, secretion of RNAP-I was recovered. Therefore, the physical linkage of the prosequence to the mature region is not a prerequisite for secretion of active RNAP-I. Purified RNAP-I with the prosequence once denatured in 6 m guanidine HCl could be renatured and activated to have its enzymatic activity by removing guanidine HCl in vitro, but RNAP-I without the prosequence could not. Furthermore, the wild-type prosequence helped the recovery of the activity of the denatured RNAP-I in trans, but the prosequence of M1 with which secretion of RNAP-I was not observed in vivo, did not. From these results we concluded that the prosequence of RNAP-I supports correct folding of RNA-P-I in the endoplasmic reticulum lumen and its subsequent secretion in S. cerevisiae. The functional role of the prosequence of an aspartic proteinase was elucidated.
