CELL-SURFACE LOCALIZATION OF PROTEOLYSIS OF HUMAN ENDOTHELIAL ANGIOTENSIN I-CONVERTING ENZYME - EFFECT OF THE AMINO-TERMINAL DOMAIN IN THE SOLUBILIZATION PROCESS

Angiotensin-converting enzyme (ACE) belongs to the type I class of ect oproteins and is solubilized by Chinese hamster ovary cells transfecte d with the full-length human ACE cDNA. ACE release in Chinese hamster ovary cells involves a proteolytic cleavage occurring in the carboxyl- terminal region, between Arg-1137 and Leu-1138, The subcellular locali zation of ACE proteolysis was established by pulse chase experiments, cell surface immunolabeling, and biotinylation of radiolabeled mature proteins, The proteolysis of ACE takes place primarily at the plasma m embrane, The solubilization of ACE is less than 2% within 1 h, is incr eased 2.4-fold by phorbol esters, but is not influenced by ionophores. An ACE mutant lacking the transmembrane domain and the cytosolic part (ACE(Delta COOH)), is secreted at a faster rate without a carboxyl-te rminal cleavage, and phorbol esters or ionophores have no effect on it s rate of production in the medium, Therefore, the proteolysis of ACE is dependent on the presence of the membrane anchor and suggests that the secretase(s) involved is also membrane-associated, An ACE mutant l acking the aminoterminal domain (ACE(CF)) is secreted 10 fold faster c ompared with wild-type ACE, The solubilization of ACE(CF) occurs at th e plasma membrane and is stimulated 2.7-fold by phorbol esters, and th e cleavage site is localized between Arg-1227 and Val-1228. The amino- terminal domain of ACE slows down the proteolysis and seems to act as a ''conformational inhibitor'' of the proteolytic process, possibly vi a interactions with the ''stalk'' of ACE and the secretase(s) itself.