Glycosylation and membrane insertion of newly synthesized rat dopamine beta-hydroxylase in a cell-free system without signal cleavage.
Dopamine beta-hydroxylase (DBH, EC 1.14.17.1) is present in both membrane-bound and soluble forms in neurosecretory vesicles. This study was designed to investigate the differences between membrane-bound and soluble DBH and how they may arise from translation of a single mRNA. Antisera to a peptide corresponding to the carboxyl terminus of rat DBH was found to specifically immunoprecipitate the 77- and 73-kDa subunits of newly synthesized DBH in rat brain. Thus, both soluble and membrane-bound forms contain the same carboxyl terminus. To investigate differences at the amino terminus, full-length rat DBH mRNA, translated in a cell-free system, produced a 66-kDa peptide. An additional higher molecular mass product was synthesized upon co-translational addition of microsomal membranes. This product was glycosylated since it bound to concanavalin A-Sepharose and reverted to the 66-kDa polypeptide after treatment with endoglycosidase H. This glycosylated product was resistant to protease digestion and fractionated with microsomal membranes on sucrose gradients, indicating that it is incorporated into the microsomal membranes. Amino-terminal sequencing of the glycosylated translation product indicated that the amino-terminal "signal" sequence was not cleaved. The results indicate that in the cell-free system newly synthesized DBH undergoes glycosylation and incorporation into microsomal membranes without cleavage of the NH2-terminal signal sequence.
Feng
Z
Z
Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla 10595.
Angeletti
R H
RH
Levin
B E
BE
Sabban
E L
EL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002474:Cell-Free System; D002499:Centrifugation, Density Gradient; D004247:DNA; D004285:Dogs; D004299:Dopamine beta-Hydroxylase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006031:Glycosylation; D007425:Intracellular Membranes / Q000201:enzymology*; D008297:Male; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D017386:Sequence Homology, Amino Acid
D021382:Protein Sorting Signals; D012333:RNA, Messenger; D004247:DNA; D004299:Dopamine beta-Hydroxylase