The role of the prosequence of Rhizopus oryzae lipase (ROL) with a preprose
quence was analyzed by an expression system using Saccharomyces cerevisiae.
When the mature portion of ROL (mROL) fused to the pre-alpha -factor leade
r sequence was expressed, secretion of active mROL was not observed. Howeve
r, when mROL was synthesized together with the prosequence in trans (indivi
dually and coincidentally), secretion of active mROL was observed. The resu
lts indicate that the prosequence of ROL helped correct folding of mROL and
its subsequent secretion from the yeast cells, and that physical linkage (
cis) of the prosequence to the mature region was not prerequisite. From the
expression of the ROL mutants with deletions at the N-terminal end of the
prosequence together with mROL in trans, the residues from 20 to 37 in the
prosequence were essential for the secretion, and those from 38 to 57 were
essential for the formation of the active ROL and might play a role as an i
ntramolecular chaperone. The results using the fragment of the prosequence
confirmed that these residues (20-57) were significant for in vivo folding
and secretion of active mROL.