The C-terminal Region of Carboxypeptidase E Involved in Membrane Binding Is Distinct from the Region Involved with Intracellular Routing

Abstract

Carboxypeptidase E (CPE) is involved in the biosynthesis of numerous peptide hormones and neurotransmitters. Previously, the C-terminal region of CPE has been shown to participate in the binding of the protein to membranes and to also contribute to the sorting of CPE into the regulated pathway. In this study, the role of the C-terminal region of CPE was further examined using several approaches. A series of CPE mutants with C-terminal deletions was expressed in the baculovirus system; constructs with a deletion of 14 or 23 residues were expressed at levels comparable to wild-type CPE. In contrast, deletion of 33 or more residues eliminated CPE activity, and the resulting protein was not secreted from the cells. Even though CPE mutants with a deletion of 14 or 23 residues were expressed normally, the resulting protein was mainly soluble, whereas approximately 55% of wild-type CPE was membrane associated. When expressed in AtT-20 cells, CPE with a deletion of 43 C-terminal amino acids was not secreted, whereas CPE with a deletion of 23 residues was secreted via the regulated pathway. Pulse-chase analysis revealed the protein with a deletion of 43 residues to be degraded in a non-acidic intracellular compartment. To investigate whether the C-terminal region of CPE can confer membrane binding and regulated pathway sorting to another protein, portions of the CPE C-terminal region were attached to the C terminus of albumin and the fusion proteins expressed in AtT-20 cells. Of the constructs examined, only the protein containing 51 amino acids of CPE was sorted to the regulated pathway, although with reduced efficiency compared to endogenous CPE. Although the C-terminal 14 amino acids of CPE are sufficient to target albumin to membranes, this fusion protein is not sorted into the regulated pathway. Taken together, these results indicate that the C-terminal 14 amino acids of CPE are important for membrane binding and that membrane binding and sorting require distinct signals.