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Originally published In Press as doi:10.1074/jbc.M101088200 on June 19, 2001

J. Biol. Chem., Vol. 276, Issue 35, 33265-33272, August 31, 2001
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Dopamine beta -Monooxygenase Signal/Anchor Sequence Alters Trafficking of Peptidylglycine alpha -Hydroxylating Monooxygenase*

Ana Maria OyarceDagger , Tami C. Steveson§, Lixian Jin, and Betty A. Eipper§

From the Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2105

Dopamine beta -monooxygenase (DBM) and peptidylglycine alpha -hydroxylating monooxygenase (PHM) are essential for the biosynthesis of catecholamines and amidated peptides, respectively. The enzymes share a conserved catalytic core. We studied the role of the DBM signal sequence by appending it to soluble PHM (PHMs) and expressing the DBMsignal/PHMs chimera in AtT-20 and Chinese hamster ovary cells. PHMs produced as part of DBMsignal/PHMs was active. In vitro translated and cellular DBMsignal/PHMs had similar masses, indicating that the DBM signal was not removed. DBMsignal/PHMs was membrane-associated and had the properties of an intrinsic membrane protein. After in vitro translation in the presence of microsomal membranes, trypsin treatment removed 2 kDa from DBMsignal/PHMs while PHMs was entirely protected. In addition, a Cys residue in DBMsignal/PHMs was accessible to Cys-directed biotinylation. Thus the chimera adopts the topology of a type II membrane protein. Pulse-chase experiments indicate that DBMsignal/PHMs turns over rapidly after exiting the trans-Golgi network. Although PHMs is efficiently localized to secretory granules, DBMsignal/PHMs is largely localized to the endoplasmic reticulum in AtT-20 cells. On the basis of stimulated secretion, the small amount of PHMs generated is stored in secretory granules. In contrast, the expression of DBMsignal/PHMs in PC12 cells yields protein that is localized to secretory granules.

* This work was supported by National Institutes of Health Grants DA-11269 (to A. M. O) and DA-00266 (to B. A. E).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed at the current address: Dept. of Pharmacology and Therapeutics BHSB 209, Medical College of Ohio, Toledo, OH 43614-5804. Tel.: 419-383-5308; Fax: 419-383-2871; E-mail: aoyarce@mco.edu.

§ Current address: Dept. of Neuroscience, University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06030-3401.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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