Specific cellular proteins associate with angiotensin-converting enzyme and regulate its intracellular transport and cleavage-secretion

Angiotensin-converting enzyme (ACE) is an extensively glycosylated type I e ctoprotein anchored in the plasma membrane by a hydrophobic transmembrane d omain. In tissue culture as well as in vivo, the extracellular domain of AC E is released into the culture medium by a regulated proteolytic cleavage. To identify the cellular proteins that regulate ACE processing and cleavage -secretion, ACE-bound proteins were purified by affinity chromatography and characterized by microsequencing and Western blotting. One protein was ide ntified as ribophorin and another as immunoglobulin-binding protein (BiP), a chaperone. Metabolic labeling and immunoprecipitation of ACE confirmed it s interaction with BiP. Overexpression of BiP inhibited ACE secretion, an e ffect accentuated by the expression of an enzymatically inactive mutant BiP , This inhibition was caused by the retention of ACE precursors by BiP in t he endoplasmic reticulum, as revealed by immunoprecipitation and immunofluo rescence experiments. However, treatment with a phorbol ester, phorbol 12-m yristate 13-acetate, enhanced ACE secretion even from cells overexpressing BiP. Western blot analysis of ACE-associated proteins with antibodies to pr otein kinase C (PRC) revealed the presence of its specific isozymes. Treatm ent with phorbol 12-myristate 13-acetate caused marked reduction in ACE ass ociation of selective PKC species. Thus, our studies have identified PKC an d BiP as two proteins that directly interact with ACE and modulate its cell -surface expression and cleavage secretion.