Differential palmitoylation regulates intracellular patterning of SNAP25

Abstract

SummarySNAP25 regulates membrane fusion events at the plasma membrane and in the endosomal system, and a functional pool of the protein is delivered to recycling endosomes (REs) and the trans Golgi network (TGN) through an ARF6-dependent cycling pathway. SNAP25 is a peripheral membrane protein, and palmitoylation of a cluster of four cysteine residues mediates its stable association with the membrane. Here, we report that palmitoylation also determines the precise intracellular distribution of SNAP25, and that mutating single palmitoylation sites enhances the amount of SNAP25 at the RE and TGN. The farnesylated CAAX motif from Hras was ligated onto a SNAP25 mutant truncated immediately distal to the cysteine-rich domain. This construct displayed the same intracellular distribution as full-length SNAP25, and decreasing the number of cysteine residues in this construct increased its association with the RE and TGN, confirming the dominant role of the cysteine-rich domain in directing the intracellular distribution of SNAP25. Marked differences in the localisations of SNAP25-CAAX and Hras constructs, each with two palmitoylation sites, were observed, showing that subtle differences in palmitoylated sequences can have a major impact upon intracellular targeting. We propose that the cysteinerich domain of SNAP25 is designed to facilitate the dual function of this SNARE protein at the plasma membrane and endosomes, and that dynamic palmitoylation acts as a mechanism to regulate the precise intracellular patterning of SNAP25.

Endoplasmic Reticulum Localization of DHHC Palmitoyltransferases Mediated by Lysine-based Sorting Signals

“…This suggests that DHHC3 proteins localized to the ER retain full palmitoyltransferase activity against this substrate protein. In addition to the Golgi, we have previously suggested that dynamic palmitoylation of mature SNAP25 might also occur at recycling endosomes and/or the trans-Golgi network (TGN) (17,22). Thus, although we did not detect the dilysine mutants of DHHC3 at the Golgi by immunofluorescence (Fig.…”

“…HA-tagged versions of DHHC3, DHHC4, and DHHC6 were kindly provided by Dr. Masaki Fukata (National Institute of Physiological Sciences, Osaka, Japan) (7). Mcherry-Rab11 was as described previously (22). TGN38-GFP was a gift from Professor George Banting (University of Bristol) (23).…”

Protein palmitoylation and subcellular trafficking

“…There are excellent recent reviews dealing with palmitoylation and sorting of H-Ras, N-Ras, SNAP25, G-Protein Coupled Receptors as well as transmembrane proteins such as the co-receptors CD4 and CD8 and the adaptor LAT [70][71][72][73][74][75]. In addition to all these proteins, we will also describe herein other selected examples of proteins in which palmitoylation regulate intracellular sorting, localization and function as well.…”

Phosphatidylinositol 4-Kinase IIα Is Palmitoylated by Golgi-localized Palmitoyltransferases in Cholesterol-dependent Manner

“…Turnover-Palmitoylation is a potentially reversible post-translational modification (27)(28)(29)(30). We compared the rates of Myc-PI4KII␣ turnover at the protein and palmitate levels by pulse-chase labeling.…”