CLEAVAGE-SECRETION OF ANGIOTENSIN I-CONVERTING ENZYME IN YEAST

Angiotensin I-converting enzyme (ACE) is a type I transmembrane protei n composed of two domains (N and C domains) which undergoes a post-tra nslational proteolytic cleavage in mammalian cells to release the solu ble ectodomain. The protease involved in ACE cleavage-secretion (ACE-s ecretase) is not well characterised and eludes isolation: the presence of a yeast homologue, thus more amenable to genetic manipulation, wou ld facilitate its identification. We have expressed a secreted form of the ACE C domain; lacking the C-terminal membrane anchor (C domain(De lta COOH)), and the membrane-anchored C domain (C domain) in the yeast Pichia pastoris by fusion to prepro-alpha-factor. Immunofluorescent l abelling localises the ACE C domain to the periphery of yeast cells bu t not C domain(Delta COOH), however, expression of both C domain and C domain(Delta COOH) produced soluble enzymes in the culture medium. Im munocharacterisation of the two soluble forms of the C domain indicate s a proteolytic cleavage of the membrane-bound C domain to produce the soluble counterpart. Thus ACE undergoes a proteolytic cleavage in yea st. (C) 1997 Elsevier Science Ireland Ltd.