title: Carboxypeptidase E, a prohormone sorting receptor, is anchored to secretory granules via a C-terminal transmembrane insertion.
abstract: Carboxypeptidase E (CPE) is a sorting receptor that directs the prohormone pro-opiomelanocortin (POMC) to the regulated secretory pathway, and is also a prohormone processing enzyme in neuro/endocrine cells. It has been suggested that the 25 C-terminal amino acids are necessary for the binding of CPE to secretory granule membranes, but its orientation in the membrane is not known. In this study, we examined the structure and orientation of the membrane-binding domain at the C-terminus of CPE. In vitro experiments using model membranes demonstrated that the last 22 amino acids of CPE (CP peptide) insert in a shallow orientation into lipid bilayers at low pH. Circular dichroism analysis indicated that the CP peptide adopts a partial alpha-helical configuration at low pH, and helix content increases when it is bound to lipid. Protease protection experiments, immunolabeling, and immunoisolation of intact secretory granules with a C-terminal antibody revealed a cytoplasmic domain in CPE, consistent with a transmembrane orientation of this protein. We conclude that the membrane-binding domain of CPE must adopt an alpha-helical configuration to bind to lipids, and that CPE may require another integral membrane "chaperone" protein to insert through the lipid bilayer in a transmembrane fashion.
authors.affiliation: Section on Cellular Neurobiology, Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-4480, USA.
authors.forename: Savita
authors.initials: S
authors.lastname: Dhanvantari
authors.forename: Irina
authors.initials: I
authors.lastname: Arnaoutova
authors.forename: Chris R
authors.initials: CR
authors.lastname: Snell
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Steinbach
authors.forename: Kelli
authors.initials: K
authors.lastname: Hammond
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Caputo
authors.forename: Erwin
authors.initials: E
authors.lastname: London
authors.forename: Y Peng
authors.initials: YP
authors.lastname: Loh
mesh_terms: D020106:Acrylamide / Q000737:chemistry; D000818:Animals; D001692:Biological Transport; D043423:Carboxypeptidase H; D002268:Carboxypeptidases / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002942:Circular Dichroism; D003594:Cytoplasmic Granules / Q000378:metabolism; D010450:Endopeptidases / Q000737:chemistry; D005453:Fluorescence; D018189:Immunomagnetic Separation; D008051:Lipid Bilayers; D050356:Lipid Metabolism; D008055:Lipids / Q000032:analysis; D051379:Mice; D008958:Models, Molecular; D010446:Peptide Fragments; D011333:Pro-Opiomelanocortin / Q000737:chemistry; D011487:Protein Conformation; D015398:Signal Transduction
chemical_list: D008051:Lipid Bilayers; D008055:Lipids; D010446:Peptide Fragments; D020106:Acrylamide; D011333:Pro-Opiomelanocortin; D002268:Carboxypeptidases; D010450:Endopeptidases; D043423:Carboxypeptidase H