Targeting of frog prodermorphin to the regulated secretory pathway by fusion to proenkephalin.
We have investigated the sorting and processing of the amphibian precursor prepro-dermorphin in mammalian cells. Dermorphin, a D-alanine-containing peptide with potent opioid activity, has been isolated from the skin of the frog Phyllomedusa sauvagei. The maturation of this peptide from the precursor involves several posttranslational steps. Recombinant vaccinia viruses were used to infect AtT-20, PC12, and HeLa cells to study the sorting and processing of prepro-dermorphin. While this precursor was not processed in any of the examined cell lines, AtT-20 cells were able to process approximately 40% of a chimeric precursor consisting of the first 241 amino acids of prepro-enkephalin fused to a carboxy-terminal part of pro-dermorphin. By immunogold-EM, we could show that the chimeric protein, but not pro-dermorphin, was sorted to dense-core secretion granules. The processing products could be released upon stimulation by 8-Br-cAMP. We conclude that the pro-enkephalin part of the fusion protein contains the information for targeting to the regulated pathway of secretion, while this sorting information is missing in pro-dermorphin. This indicates that sorting mechanisms may differ between amphibian and mammalian cells.
Seethaler
G
G
Institute of Molecular Biology, Austrian Academy of Sciences, Salzburg.
Chaminade
M
M
Vlasak
R
R
Ericsson
M
M
Griffiths
G
G
Toffoletto
O
O
Rossier
J
J
Stunnenberg
H G
HG
Kreil
G
G
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001001:Anura; D002460:Cell Line; D002678:Chimera; D003594:Cytoplasmic Granules / Q000378:metabolism / Q000648:ultrastructure; D004745:Enkephalins / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D016253:Microscopy, Immunoelectron; D009842:Oligopeptides / Q000235:genetics*; D010957:Plasmids; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014162:Transfection; D014616:Vaccinia virus / Q000235:genetics
D004745:Enkephalins; D009842:Oligopeptides; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; C029992:proenkephalin; C070894:prodermorphin; D015124:8-Bromo Cyclic Adenosine Monophosphate