title: The pro region is not required for the expression or intracellular routeing of carboxypeptidase E.
abstract: Carboxypeptidase E (CPE) is initially synthesized as a larger precursor containing an additional 14-residue propeptide that is highly conserved between human and rat. Previous studies have established that the proenzyme is enzymically active and that deletion of the pro region does not affect the expression of the active enzyme. In the present study the function of the pro region was examined both by deleting this region from CPE and by attaching this region to the N-terminus of albumin. CPE lacking the pro region is sorted into the regulated secretory pathway in AtT-20 cells, based on confocal microscopy and examination of the stimulated secretion of the protein. Stimulation of AtT-20 cells with either forskolin or phorbol 12-myristate 13-acetate induces the secretion of wild-type CPE and of CPE lacking the pro region to similar extents, indicating a similar efficiency of sorting of the mutant. When the pro region of proalbumin is replaced with the pro region of CPE followed by expression in AtT-20 cells, the protein is not sorted into the regulated pathway, based on the lack of stimulated secretion. Confocal microscopy suggests that the proCPE/albumin protein is retained in the endoplasmic reticulum to a greater extent than is proalbumin. Pulse-chase analysis indicates that the pro region of CPE is not efficiently removed from the N-terminus of albumin, and the small amount of propeptide cleavage that does occur takes place soon before secretion of the protein. In contrast, confocal microscopy indicates that the majority of the propeptide is removed from CPE, and that this cleavage occurs in the trans-Golgi network or soon after sorting into the secretory vesicles. Taken together, these results suggest that the pro region of CPE is not required for the expression or intracellular routeing of this protein.
authors.affiliation: Department of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
authors.forename: L
authors.initials: L
authors.lastname: Song
authors.forename: L D
authors.initials: LD
authors.lastname: Fricker
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western; D043423:Carboxypeptidase H; D002268:Carboxypeptidases / Q000737:chemistry* / Q000378:metabolism; D004792:Enzyme Precursors / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D051379:Mice; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D011487:Protein Conformation; D051381:Rats; D014162:Transfection
chemical_list: D004792:Enzyme Precursors; D002268:Carboxypeptidases; D043423:Carboxypeptidase H