Expression of dopamine beta-hydroxylase in Drosophila Schneider 2 cells. Evidence for a mechanism of membrane binding other than uncleaved signal peptide.
To characterize the mechanism of membrane attachment of dopamine beta-hydroxylase, an expression system producing the processed form of this enzyme has been developed. We have replaced the endogenous signal peptide of bovine dopamine beta-hydroxylase with a heterologous signal peptide which is efficiently recognized and cleaved in Drosophila Schneider 2 cells. A cDNA encoding this chimeric recombinant bovine enzyme has been stably transfected into Schneider 2 cells. The inducible expression of active dopamine beta-hydroxylase in these cells has been verified by Western blotting and enzyme activity assays. N-terminal sequence analysis of purified recombinant enzyme demonstrates complete removal of the signal peptide. Subcellular analysis shows that the recombinant enzyme exists as both a soluble and a membrane-bound form in these cells. These data demonstrate that the endogenous signal peptide is not required for the formation of the membranous dopamine beta-hydroxylase and further that the enzyme can be bound to membranes via a mechanism other than uncleaved signal sequence.
Gibson
K R
KR
Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, D.C. 20007.
Vanek
P G
PG
Kaloss
W D
WD
Collier
G B
GB
Connaughton
J F
JF
Angelichio
M
M
Livi
G P
GP
Fleming
P J
PJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology*; D003001:Cloning, Molecular; D004299:Dopamine beta-Hydroxylase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004330:Drosophila; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D021382:Protein Sorting Signals / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013347:Subcellular Fractions / Q000201:enzymology; D014162:Transfection
D009838:Oligodeoxyribonucleotides; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D004299:Dopamine beta-Hydroxylase