EXPRESSION OF HUMAN DOPAMINE-BETA-HYDROXYLASE IN MAMMALIAN-CELLS INFECTED BY RECOMBINANT VACCINIA VIRUS - MECHANISMS FOR MEMBRANE ATTACHMENT

Dopamine beta-hydroxylase (DBH) is found in neurosecretory vesicles in both membrane-bound and soluble forms, We expressed various human DBH cDNAs in two mammalian cell lines, using the vaccinia virus expressio n system. The expression of a full-length DBH cDNA (DBH-f) reproduced the native DBH electrophoretic pattern and led to the synthesis of an active enzyme composed of two subunits of 77 and 73 kDa. In contrast, a truncated cDNA lacking the first ATG (DBH-t) generated a single band of 73 kDa. Analysis of mutated recombinant clones demonstrates that t he two polypeptides do not result from the use of an alternative trans lation initiator codon. These results, combined with deglycosylation e xperiments, allow us to attribute the double band pattern to an option al cleavage of the signal peptide. When the NH2-terminal extremity is shortened, cleavage becomes obligatory, underlining the role of the fi rst 14 amino acids in the regulation of the cleavage of the signal pep tide, Subcellular analysis of recombinant DBH-t and DBH-f proteins ind icates that DBH is anchored to the membrane by two distinct mechanisms ; one of them is due to the non-removal of the signal peptide, whereas the second one is independent of the presence of the signal sequence. Moreover, quantification of the fractionation experiments suggests th at the two modes of membrane attachment are additive.