Dopamine β-Monooxygenase Signal/Anchor Sequence Alters Trafficking of Peptidylglycine α-Hydroxylating Monooxygenase

Ana Maria Oyarce,Lixian Jin,Tami C Steveson,Betty A Eipper

doi:10.1074/jbc.m101088200

Abstract

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.

Highlights

Catecholamine synthesis from tyrosine involves several cytosolic enzymes and one secretory granule enzyme, dopamine ␤-monooxygenase (DBM)1 [1]
We studied the role of the DBM signal sequence by appending it to soluble peptidylglycine ␣-hydroxylating monooxygenase (PHM) (PHMs) and expressing the DBMsignal/PHMs chimera in AtT-20 and Chinese hamster ovary cells
The NH2-terminal sequences of rat, mouse, human, and bovine DBM vary in length, but each contains a stretch of 20 hydrophobic amino acids, which is longer than the hydrophobic domains typically found in cleaved signal sequences [13, 14] (Fig. 1A)

Summary

Introduction

Catecholamine synthesis from tyrosine involves several cytosolic enzymes and one secretory granule enzyme, dopamine ␤-monooxygenase (DBM)1 [1]. Sequence analysis of rat DBM produced by in vitro transcription/translation in the presence of microsomal membranes indicated that the signal sequence was not removed [12]. These studies suggest that DBM is a type II integral membrane protein. Analysis of the genomic sequence encoding human DBM [15, 16] identified an in-frame upstream Met codon that is likely to represent the actual translational start site (Fig. 1A) [17, 18] With this modification, the similarity of human to mouse and rat DBM is increased greatly.

Methods

Results

Conclusion