Synthesis, degradation, and subcellular localization of synaptotagmin IV, a neuronal immediate early gene product

Synaptotagmin IV (Syt IV) is an immediate early gene induced by depolarizat ion in rat PC12 cells and in rat hippocampus. We prepared an antiserum to S yt IV protein. The 46-kDa Syt IV protein is nearly undetectable by western blotting in unstimulated PC12 cells. After depolarization, Syt IV increases rapidly, peaks at 4 h, and decays to near baseline levels by 12 h. Forskol in stimulation also leads to rapid Syt IV protein accumulation. The rate of Syt IV protein synthesis, determined by labeling with radioactive amino ac ids and immunoprecipitation, is low in unstimulated PC12 cells, but increas es over the first 3 h after forskolin stimulation and remains elevated for several hours. Syt IV protein is relatively labile; metabolically labeled S yt IV has a half-life of similar to 2 h in PC12 cells. Sucrose density grad ient fractionation and vesicle immunoisolation experiments suggest that Syt IV protein is present in both synaptic-like microvesicles and secretory gr anules. Vesicles immunoisolated from forskolin-treated PC12 cells with anti -Syt I antibody contain radioactively labeled Syt IV, demonstrating that Sy t I and Syt IV colocalize in common vesicles. These results suggest that Sy t IV protein, after its stimulation-induced synthesis, is rapidly transport ed to secretory vesicles where it may transiently modulate the exocytotic m achinery.