A point mutation in the juxtamembrane stalk of human angiotensin I-converting enzyme invokes the action of a distinct secretase

Angiotensin I-converting enzyme (ACE) is one of a number of integral membra ne proteins that is proteolytically shed from the cell surface by a zinc me tallosecretase, Mutagenesis of Asn(631) to Gin in the juxtamembrane stalk r egion of ACE resulted in more efficient secretion of the mutant protein (AC E(NQ)) as determined by pulse-chase analysis. In contrast to the wild-type ACE, the cleavage of ACE(NQ) was not blocked by the metallosecretase inhibi tor batimastat but by the serine protease inhibitor, 1,3-dichloroisocoumari n. Incubation of the cells at 15 degreesC revealed that ACE(NQ) was cleaved in the endoplasmic reticulum, and mass spectrometric analysis of the secre ted form of the protein indicated that it had been cleaved at the Asn(635)- Ser(636) bond, three residues N-terminal to the normal secretase cleavage s ite at Arg(638)-Ser(639). These data clearly show that a point mutation in the juxtamembrane region of an integral membrane protein can invoke the act ion of a mechanistically and spatially distinct secretase, In light of this observation, previous data on the effect of mutations in the juxtamembrane stalk of shed proteins being accommodated by a single secretase having a r elaxed specificity need to be re-evaluated.