ADDITION OF AN ENDOPLASMIC-RETICULUM RETENTION RETRIEVAL SIGNAL DOES NOT BLOCK MATURATION OF ENZYMATICALLY ACTIVE PEPTIDYLGLYCINE ALPHA-AMIDATING MONOOXYGENASE

Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the COOH -terminal alpha-amidation of neural and endocrine peptides via a two-s tep reaction carried out in sequence by the monooxygenase and lyase do mains contained in this bifunctional protein. Peptide alpha-amidation is thought to take place primarily in the secretory granules in which mature bioactive peptides are stored, and it is not known where in the secretory compartment newly synthesized PAM protein becomes enzymatic ally active. To address this question, PAM-3, a soluble bifunctional p rotein, was modified by addition of the KDEL endoplasmic reticulum (ER ) retention/retrieval signal to its COOH terminus. PAM 3 KDEL protein stably expressed in hEK-293 cells or in AtT-20 cells was efficiently r etained in the ER based on immunocytochemistry, pulse-chase experiment s, and maintained endoglycosidase H sensitivity. The effect of the KDE L sequence was specific since PAM-3 with an inactive ER retention/retr ieval signal (PAM-3-KDEV) moved through the secretory pathway Like wil d type PAM-3. In AtT-20 cells, PAM-3-KDEL was not subjected to the COO H-terminal endoproteolytic cleavage that generates a 75 kDa PAM protei n from PAM-3 and PAM-3 KDEV. PAM-3-KDEL protein exhibited both monooxy genase and lyase activities with specific activities similar to those of the wild type PAM-3 and PAM-3-KDEV proteins. Thus, although PAM cat alyzes a reaction that occurs primarily in the secretory granules, new ly synthesized PAM protein becomes enzymatically competent in the ER.