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 expression 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 the two polypeptides do not result from the use of an alternative translation initiator codon. These results, combined with deglycosylation experiments, allow us to attribute the double band pattern to an optional cleavage of the signal peptide. When the NH2-terminal extremity is shortened, cleavage becomes obligatory, underlining the role of the first 14 amino acids in the regulation of the cleavage of the signal peptide. Subcellular analysis of recombinant DBH-t and DBH-f proteins indicates 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 that the two modes of membrane attachment are additive.
Houhou
L
L
Laboratoire de Génétique Moléculaire de la Neurotransmission et des Processus Neurodégénératifs, CNRS, Gif-sur-Yvette, France.
Lamouroux
A
A
Biguet
N F
NF
Mallet
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D002451:Cell Compartmentation; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics; D004299:Dopamine beta-Hydroxylase / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D010442:Peptide Chain Initiation, Translational; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014616:Vaccinia virus / Q000235:genetics
D018076:DNA, Complementary; D008565:Membrane Proteins; D011994:Recombinant Proteins; D004299:Dopamine beta-Hydroxylase