Cleavage-secretion of angiotensin I-converting enzyme in yeast.
Angiotensin I-converting enzyme (ACE) is a type I transmembrane protein composed of two domains (N and C domains) which undergoes a post-translational proteolytic cleavage in mammalian cells to release the soluble ectodomain. The protease involved in ACE cleavage-secretion (ACE-secretase) is not well characterised and eludes isolation: the presence of a yeast homologue, thus more amenable to genetic manipulation, would facilitate its identification. We have expressed a secreted form of the ACE C domain, lacking the C-terminal membrane anchor (C domain(deltaCOOH)), and the membrane-anchored C domain (C domain) in the yeast Pichia pastoris by fusion to prepro-alpha-factor. Immunofluorescent labelling localises the ACE C domain to the periphery of yeast cells but not C domain(deltaCOOH), however, expression of both C domain and C domain(deltaCOOH) produced soluble enzymes in the culture medium. Immunocharacterisation of the two soluble forms of the C domain indicates a proteolytic cleavage of the membrane-bound C domain to produce the soluble counterpart. Thus ACE undergoes a proteolytic cleavage in yeast.
Williams
T A
TA
INSERM unité 36, Collège de France, Paris. williams@infobiogen.fr
Gouttaya
M
M
Tougard
C
C
Michaud
A
A
Chauvet
M T
MT
Corvol
P
P
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D000072235:Mating Factor; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010452:Peptide Biosynthesis; D010455:Peptides; D007703:Peptidyl-Dipeptidase A / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D010843:Pichia; D011480:Protease Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D013379:Substrate Specificity
D010455:Peptides; D011480:Protease Inhibitors; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000072235:Mating Factor; D010450:Endopeptidases; D007703:Peptidyl-Dipeptidase A; C077675:angiotensin converting enzyme secretase