Electrostatic anchoring precedes stable membrane attachment of SNAP25/SNAP23 to the plasma membrane

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.DOI: http://dx.doi.org/10.7554/eLife.19394.001

Membrane‐mediated disorder‐to‐order transition of SNAP25 flexible linker facilitates its interaction with syntaxin‐1 and SNARE‐complex assembly

“…Some experiments have shown SNAP25 binding membranes even when the 4 cysteines are unpalmitoylated (24,25). Recent research showed that positively charged residues near cysteines also play an important role in the membrane-binding (25). Consistent with the report, our data also indicate that positively charged residues near cysteine participate in membrane interaction (Fig.…”

“…[ 31 P] spectra of liposomes and 1 H-15 N HSQC of the SNAP25 loop region show that the SNAP25 membrane interaction is weakened by the amino acid changes, suggesting that the cysteines are crucial for its membrane-binding. Some experiments have shown SNAP25 binding membranes even when the 4 cysteines are unpalmitoylated (24,25). Recent research showed that positively charged residues near cysteines also play an important role in the membrane-binding (25).…”

“…Palmitoylation of the 4 cysteines increases the affinity for the presynaptic membrane by increasing its hydrophobicity (20,21,23). Further efforts showed, although substitution of the cysteines inhibits association, SNAP25 can bind membranes without palmitoylation (24,25). On the other hand, the loop region influences the assembly of the SNARE complex and helps regulate synaptic and neural plasticity (26,27).…”

Structural Basis for Substrate Recognition by the Ankyrin Repeat Domain of Human DHHC17 Palmitoyltransferase

“…It must be noted however that palmitoylation of the cysteines in Snap25b is a necessary but not sufficient event for proper targeting of Snap25b to the plasma membrane. Recent evidence has pointed to the importance of electrostatic interactions between charged residues in Snap25b and membrane lipids (Weber et al, 2017). In the same report, the authors propose anchoring of Snap25b preceding the palmitoylation by plasma membrane DHHCs.…”

The Linker Domain of SNAP25 Acts as a Flexible Molecular Spacer to Ensure Efficient S-Acylation

“…Our previous work suggested that the hydrophobic cysteine-rich domain of SNAP25 (residues 85-92) plays an important role in initial membrane interaction of SNAP25 with Golgi membranes prior to S-acylation [22]. In contrast, a study by the Lang group suggested a role for positively-charged amino acids in the (large) linker domain in mediating this initial membrane interaction [30]. However, it is worthwhile further reflecting on the proposed role of positively-charged amino acids in initial membrane interaction of SNAP25 [30].…”