Dopamine beta-monooxygenase signal/anchor sequence alters trafficking of peptidylglycine alpha-hydroxylating monooxygenase

Dopamine beta -monooxygenase (DBM) and peptidylglycine a-hydroxylating mono oxygenase (PHM) are essential for the biosynthesis of catecholamines and am idated 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. I n vitro translated and cellular DBMsignal/PHMs had similar masses, indicati ng that the DBM signal was not removed. DBMsignaI/PHMs was membrane-associa ted and had the properties of an intrinsic membrane protein. After in vitro translation in the presence of microsomal membranes, trypsin treatment rem oved 2 kDa from DBMsignal/PHMs while PHMs was entirely protected. In additi on, a Cys residue in DBMsignal/PHMs was accessible to Cys-directed biotinyl ation. Thus the chimera adopts the topology of a type Il membrane protein. Pulse-chase experiments indicate that DBMsignal/PHMs turns over rapidly aft er exiting the trans-Golgi network. Although PHMs is efficiently localized to secretory granules, DBMsignal/PHMs is largely localized to the endoplasm ic reticulum in AtT-20 cells. On the basis of stimulated secretion, the sma ll amount of PHMs generated is stored in secretory granules. In contrast, t he expression of DBMsignal/PHMs in PC12 cells yields protein that is locali zed to secretory granules.