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-step reaction carried out in sequence by the monooxygenase and lyase domains 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 enzymatically active. To address this question, PAM-3, a soluble bifunctional protein, 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 retained in the ER based on immunocytochemistry, pulse-chase experiments, and maintained endoglycosidase H sensitivity. The effect of the KDEL sequence was specific since PAM-3 with an inactive ER retention/retrieval signal (PAM-3-KDEV) moved through the secretory pathway like wild type PAM-3. In AtT-20 cells, PAM-3-KDEL was not subjected to the COOH-terminal endoproteolytic cleavage that generates a 75-kDa PAM protein from PAM-3 and PAM-3-KDEV. PAM-3-KDEL protein exhibited both monooxygenase and lyase activities with specific activities similar to those of the wild type PAM-3 and PAM-3-KDEV proteins. Thus, although PAM catalyzes a reaction that occurs primarily in the secretory granules, newly synthesized PAM protein becomes enzymatically competent in the ER.
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Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D005455:Fluorescent Antibody Technique; D006801:Humans; D015151:Immunoblotting; D007668:Kidney; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism; D014162:Transfection
D017931:DNA Primers; D009097:Multienzyme Complexes; D009842:Oligopeptides; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C073762:lysyl-aspartyl-glutamyl-leucine; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase