SNAP25, but Not Syntaxin 1A, Recycles via an ARF6-regulated Pathway in Neuroendocrine Cells

Abstract

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 approximately 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.

Differential palmitoylation regulates intracellular patterning of SNAP25

“…Clear colocalisation of eGFP-SNAP25b with both Rab11 and TGN38 was observed, whereas we did not detect any overlap with GM130 (Fig. 1B), in good agreement with previous work (Aikawa et al, 2006b). Analysis of covariance of the fluorescence signals provided a quantitative measure of the colocalisation of SNAP25b with both Rab11 and TGN38 (Fig.…”

“…Consistent with this, previous work has also highlighted an important role for SNAP23, a ubiquitous homologue of SNAP25, in endosomal recycling of internalised transferrin to the basolateral plasma membrane in Madin-Darby canine kidney (MDCK) cells (Leung et al, 1998). The endosomal function of SNAP25 is supported by an ARF6-dependent cycling pathway, operating between the plasma membrane and the RE and trans Golgi network (TGN) compartments, which ensures a sufficient pool of SNAP25 to support endosomal membrane fusion (Aikawa et al, 2006b); disruption of this pathway inhibits trafficking of cargo to REs (Aikawa et al, 2006a). Although this cycling pathway is central to the intracellular functions of SNAP25, it is not clear how entry into this pathway is achieved.…”

“…Following palmitoylation at the Golgi, SNAP25 is probably delivered to the plasma membrane by vesicular transport; however, it is not clear whether SNAP25 traffics directly to the plasma membrane from the Golgi or whether it transits via other compartments such as REs and the TGN. It is clear that SNAP25 levels at the RE and TGN compartments are not maintained by new protein synthesis, as cycloheximide had very little effect on this localisation (Aikawa et al, 2006b;Gonzalo and Linder, 1998). Instead, a constitutive cycling pathway replenishes RE and TGN compartments with SNAP25 derived from the plasma membrane (Aikawa et al, 2006b).…”

“…It is clear that SNAP25 levels at the RE and TGN compartments are not maintained by new protein synthesis, as cycloheximide had very little effect on this localisation (Aikawa et al, 2006b;Gonzalo and Linder, 1998). Instead, a constitutive cycling pathway replenishes RE and TGN compartments with SNAP25 derived from the plasma membrane (Aikawa et al, 2006b). The continued enrichment of the SNAP25(C88L) mutant at the RE and TGN compartments following cycloheximide treatment suggests that changes in plasma membrane to RE and TGN cycling contribute to the increased intracellular targeting of this mutant.…”

“…A previous study, in PC12 cells, described an intracellular pool of SNAP25 on REs and the TGN that cycles between these compartments and the plasma membrane (Aikawa et al, 2006b). This intracellular pool is readily observed for both endogenous SNAP25 and eGFP-tagged SNAP25b (Fig.…”