Abstract
The SNAREs SNAP25 and SNAP23 are proteins that are initially cytosolic after translation, but then become stably attached to the cell membrane through palmitoylation of cysteine residues. For palmitoylation to occur, membrane association is a prerequisite, but it is unclear which motif may increase the affinities of the proteins for the target membrane. In experiments with rat neuroendocrine cells, we find that a few basic amino acids in the cysteine-rich region of SNAP25 and SNAP23 are essential for plasma membrane targeting. Reconstitution of membrane-protein binding in a liposome assay shows that the mechanism involves protein electrostatics between basic amino acid residues and acidic lipids such as phosphoinositides that play a primary role in these interactions. Hence, we identify an electrostatic anchoring mechanism underlying initial plasma membrane contact by SNARE proteins, which subsequently become palmitoylated at the plasma membrane.
Highlights
Palmitoylation is a post-translational modification of a protein which causes its stable attachment to a cellular membrane
Stable attachment to membranes is achieved after palmitoylation of a cysteine cluster, which is most probably catalyzed by the plasma membrane resident palmitoyl acyltransferase DHHC2
We examined whether the extent of plasma membrane targeting correlates with the degree of palmitoylation for wt-SNAP25 (+3), SNAP25-5 and SNAP25+10, using SNAP25(Cto-G) as a negative control
Summary
Palmitoylation is a post-translational modification of a protein which causes its stable attachment to a cellular membrane. Examples of proteins that follow this paradigm are the homologous SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) proteins SNAP25 and SNAP23, which after translation are initially cytosolic proteins. In order to function in vesicle fusion, they relocate to the plasma membrane. SNAP23 is ubiquitously expressed, whereas the neuronal SNAP25 is highly abundant in the synapse and in the plasma membrane of neuroendocrine cells (Jahn and Fasshauer, 2012; Wilhelm et al, 2014; Knowles et al, 2010). Stable attachment to membranes is achieved after palmitoylation of a cysteine cluster, which is most probably catalyzed by the plasma membrane resident palmitoyl acyltransferase DHHC2. DHHC2 is characterized by the presence of a conserved DH(H/Y)C motif and can palmitoylate SNAP25 and SNAP23 (Greaves et al, 2010)
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