Effect of forskolin on synaptotagmin IV protein trafficking in PC12 cells.
Synaptotagmin IV (Syt IV) was originally described as an immediate early gene product induced by forskolin or membrane depolarization in PC12 cells; however, nothing is known about the subcellular localization and transport of the newly translated Syt IV protein in PC12 cells. In this study, we investigated the transport mechanism of Syt IV protein induced by forskolin and found that forskolin treatment dramatically increases the Syt IV protein level (approximately 10-fold, to a level comparable to that of Syt IX) and promotes the transport of Syt IV protein from the Golgi to the cell periphery by a microtubule-dependent motor(s). The expression levels and subcellular localizations of two major Syt isoforms (I and IX) in PC12 cells, on the other hand, were unaffected by such treatment. Immunoelectron microscopic analysis showed that some Syt IV signals are clearly associated with dense-core vesicles in forskolin-treated PC12 cells, although the majority of the Syt IV molecules at the cell periphery were present on clear vesicular structures other than dense-core vesicles. An N-terminal antibody-uptake experiment indicated that Syt IV-containing vesicles in forskolin-treated PC12 cells undergo Ca(2+)-dependent exocytosis, because uptake of the anti-Syt IV-N antibody from the culture medium was slightly, but significantly, increased after forskolin treatment. Our results indicate that forskolin (or the increased cAMP level) is important for the transport of the Syt IV protein from the Golgi to the cell periphery, but not sufficient for the sorting of all Syt IV molecules to mature dense-core vesicles.
Fukuda
Mitsunori
M
Fukuda Initiative Research Unit, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama. mnfukuda@brain.riken.go.jp
Yamamoto
Akitsugu
A
D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology*; D003470:Culture Media / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005089:Exocytosis; D006056:Golgi Apparatus / Q000378:metabolism; D007150:Immunohistochemistry; D017774:Long-Term Potentiation; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry; D016253:Microscopy, Immunoelectron; D008870:Microtubules / Q000378:metabolism; D008954:Models, Biological; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D016716:PC12 Cells; D020033:Protein Isoforms; D021381:Protein Transport; D051381:Rats; D013347:Subcellular Fractions / Q000378:metabolism; D050857:Synaptotagmins; D021601:trans-Golgi Network / Q000737:chemistry
D002135:Calcium-Binding Proteins; D003470:Culture Media; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D020033:Protein Isoforms; C494294:Syt4 protein, rat; D050857:Synaptotagmins; D005576:Colforsin; D000242:Cyclic AMP; D002118:Calcium