Sorting during transport to the surface of PC12 cells: divergence of synaptic vesicle and secretory granule proteins.
PC12 cells, a cell line derived from a rat pheochromocytoma, have both regulated and constitutive secretory pathways. Regulated secretion occurs via large dense core granules, which are related to chromaffin granules and are abundant in these cells. In addition, PC12 cells also contain small electron-lucent vesicles, whose numbers increase in response to nerve growth factor and which may be related to cholinergic synaptic vesicles. These could characterize a second regulated secretory pathway. We have investigated the trafficking of protein markers for both these organelles. We have purified and characterized the large dense core granules from these cells using sequential velocity and equilibrium gradients. We demonstrate the copurification of the major PC12 soluble regulated secretory protein (secretogranin II) with this organelle. As a marker for the synaptic vesicle-like organelles in this system, we have used the integral membrane glycoprotein p38 or synaptophysin. We show that the p38-enriched fraction of PC12 cells comigrates with rat brain synaptic vesicles on an equilibrium gradient. We also demonstrate that p38 purifies away from the dense core granules; less than 5% of this protein is found in our dense granule fraction. Finally we show that p38 does not pass through the dense granule fraction in pulse-chase experiments. These results rule out the possibility of p38 reaching the small clear vesicles via mature dense granules and imply that these cells may have two independently derived regulated pathways.
Cutler
D F
DF
Department of Biochemistry, Wolfson Laboratories, Imperial College, London, England.
Cramer
L P
LP
D000310:Adrenal Gland Neoplasms; D000818:Animals; D000906:Antibodies; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003594:Cytoplasmic Granules / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000235:genetics* / Q000302:isolation & purification; D010673:Pheochromocytoma; D011499:Protein Processing, Post-Translational; D013572:Synaptic Vesicles / Q000378:metabolism*
D000906:Antibodies; D009363:Neoplasm Proteins