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Vol. 17, Issue 2, 711-722, February 2006
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Department of Biochemistry, University of Wisconsin, Madison, WI 53706
Submitted May 3, 2005;
Accepted November 14, 2005
Monitoring Editor: Keith Mostov
Soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins mediate cellular membrane fusion events and provide a level of specificity to donor–acceptor membrane interactions. However, the trafficking pathways by which individual SNARE proteins are targeted to specific membrane compartments are not well understood. In neuroendocrine cells, synaptosome-associated protein of 25 kDa (SNAP25) is localized to the plasma membrane where it functions in regulated secretory vesicle exocytosis, but it is also found on intracellular membranes. We identified a dynamic recycling pathway for SNAP25 in PC12 cells through which plasma membrane SNAP25 recycles in 
3 h. Approximately 20% of the SNAP25 resides in a perinuclear recycling endosome–trans-Golgi network (TGN) compartment from which it recycles back to the plasma membrane. SNAP25 internalization occurs by constitutive, dynamin-independent endocytosis that is distinct from the dynamin-dependent endocytosis that retrieves secretory vesicle constituents after exocytosis. Endocytosis of SNAP25 is regulated by ADP-ribosylation factor (ARF)6 (through phosphatidylinositol bisphosphate synthesis) and is dependent upon F-actin. SNAP25 endosomes, which exclude the plasma membrane SNARE syntaxin 1A, merge with those derived from clathrin-dependent endocytosis containing endosomal syntaxin 13. Our results characterize a robust ARF6-dependent internalization mechanism that maintains an intracellular pool of SNAP25, which is compatible with possible intracellular roles for SNAP25 in neuroendocrine cells.
Abbreviations used: ARF, ADP-ribosylation factor; BFA, brefeldin A; CHX, cycloheximide; CTxB, cholera toxin B; FRAP, fluorescence recovery after photobleaching; GFP, green fluorescent protein; PBS, phosphate-buffered saline; PIP2, phosphatidylinositol(4,5)bisphosphate; PI(4)P 5-kinase I, phosphatidylinositol(4)phosphate 5-kinase type I; SNAP25, synaptosome-associated protein of 25 KDa; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; Tf, transferrin.
Address correspondence to: Thomas F.J. Martin (tfmartin{at}facstaff.wisc.edu).
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