Carboxypeptidase E, a Prohormone Sorting Receptor, Is Anchored to Secretory Granules via a C-Terminal Transmembrane Insertion

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 CP...

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Published in:Biochemistry (Easton) 2002-01, Vol.41 (1), p.52-60
Main Authors: Dhanvantari, Savita, Arnaoutova, Irina, Snell, Chris R, Steinbach, Peter J, Hammond, Kelli, Caputo, Gregory A, London, Erwin, Loh, Y. Peng
Format: Article
Language:English
Subjects:
Acrylamide - chemistry
Animals
Biological Transport
Carboxypeptidase H
Carboxypeptidases - metabolism
Cell Membrane - metabolism
Circular Dichroism
Cytoplasmic Granules - metabolism
Endopeptidases - chemistry
Fluorescence
Immunomagnetic Separation
Lipid Bilayers
Lipid Metabolism
Lipids - analysis
Mice
Models, Molecular
Peptide Fragments
Pro-Opiomelanocortin - chemistry
Protein Conformation
Signal Transduction
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Summary: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 α-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 α-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.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi015698n