Dopamine beta-monooxygenase signal/anchor sequence alters trafficking of peptidylglycine alpha-hydroxylating monooxygenase.
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.
Oyarce
A M
AM
Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2105, USA. aoyarce@mco.edu
Steveson
T C
TC
Jin
L
L
Eipper
B A
BA
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002417:Cattle; D002460:Cell Line; D006224:Cricetinae; D004299:Dopamine beta-Hydroxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D051379:Mice; D008861:Microsomes / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D021382:Protein Sorting Signals; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D014162:Transfection
D009097:Multienzyme Complexes; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D006899:Mixed Function Oxygenases; D004299:Dopamine beta-Hydroxylase; C035189:peptidylglycine monooxygenase