Palmitoylated Peptides from the Cysteine-rich Domain of SNAP-23 Cause Membrane Fusion Depending on Peptide Length, Position of Cysteines, and Extent of Palmitoylation

Abstract

Synaptosome-associated proteins SNAP-23/25, members of a family of proteins essential for exocytosis, have a highly conserved central cysteine-rich domain that plays an important role in membrane targeting. More than one cysteine in this domain is modified by palmitic acid through a thioester linkage. In an effort to address the biological significance of acylation of this domain, we have generated synthetic peptides corresponding to the cysteine-rich region of SNAP-23 and covalently modified the cysteines with palmitic acid. The interaction of acylated and nonacylated peptides with lipid vesicles and natural membranes has been investigated. Our results indicate that palmitoylation is essential for membrane association. The palmitoylated peptides were able to fuse both model and natural membranes. The extent of fusion depended on the length of the peptides and the number and positions of covalently linked palmitic acids. Peptide-mediated fusion was suppressed by lysolipid and involved both outer and inner leaflets of the lipid bilayer, which is characteristic of natural membrane fusion. Our results suggest an important role for the cysteine-rich palmitoylated domain of SNAP-23 in promoting membrane fusion in cells.The mixing of specific membrane compartments by the process of fusion is a highly regulated event in eukaryotic cells. The fusion process is exquisitely controlled in a manner that does not disturb the structural and functional identity of organelles (1, 2). Several proteins that are involved at different steps in the transport process, from the formation of a transport vesicle until its fusion with the target membrane, have been identified (3-7). The SNARE 1 proteins play a crucial role in the process of intracellular membrane fusion. For fusion to occur, transport vesicles with distinct v-SNAREs have to pair with cognate t-SNAREs at the appropriate target membrane. Syntaxin and SNAP-23/25 are members of the t-SNARE family of proteins, whereas synaptobrevin/vesicle-associated membrane protein are v-SNARE proteins (3-7).Syntaxin, SNAP-25, and vesicle-associated membrane proteins mediate fusion through the formation of helical bundles that span opposing membranes. Hydrophobic C-terminal domains anchor syntaxin and vesicle-associated membrane proteins to the lipid bilayer of the plasma and vesicle membranes, respectively. The crystal structure of the SNARE complex has identified the regions of SNAP-25 that are involved in binary and ternary interactions (8, 9). SNAP-25 and its non-neuronal homolog, syndet/SNAP-23 (10, 11) possess cysteine residues clustered in a relatively unstructured segment between two helical domains. The cysteine residues in this segment are palmitoylated (12)(13)(14). Mutants of syndet and SNAP-25 lacking cysteines, and therefore not palmitoylated, are localized predominantly in the cytoplasm of transfected cells (15,16). Based on mutagenesis of SNAP-25, a minimal plasma membranetargeting domain composed of residues 85-120 has been identified. This segment has the ability to target...

Identification of Soluble N-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor Exocytotic Machinery in Human Plasma Cells: SNAP-23 Is Essential for Antibody Secretion

“…The other modification is the palmitoylation of cysteine residues clustered in the segment in between the two helical domains forming the core complex. Recent reports suggest that these palmitic residues allow the insertion of SNAP-23 (and SNAP-25) into membranes, a phenomenon that is essential for membrane fusion (55,56). Moreover, early palmitoylation of SNAP-25 seems to be inherent to an intact secretory pathway (57).…”

SNAP-23 and syntaxin-2 localize to the extracellular surface of the platelet plasma membrane

“…59 24 This domain directs membrane targeting of SNAP-25 and is itself fusogenic. 41 Whether protein transduction domains or a palmitoylated membrane binding domain endow syntaxin-2 or SNAP-23, respectively, with the ability to traverse a lipid bilayer has not been evaluated.…”

“…24 This domain directs membrane targeting of SNAP-25 and is itself fusogenic. 41 Whether protein transduction domains or a palmitoylated membrane binding domain endow syntaxin-2 or SNAP-23, respectively, with the ability to traverse a lipid bilayer has not been evaluated.A third possibility is that a population of SNAP-23 and syntaxin-2 is sorted to an extracytoplasmic compartment during protein trafficking in megakaryocytes. Syntaxin isoforms are type IV integral membrane proteins that are targeted to membranes following translation rather than cotranslationally.…”

“…SNAP-23 binds membranes via a central membrane binding domain that contains 5 palmitoyl moieties. 41,42 Acyl-protein thioesterase 1 (APT1) specifically removes palmitate from palmitoylated proteins. 32,43,44 Incubation with APT1 did not activate platelets, as evidenced by lack of P selectin surface expression (data not shown), or compromise membrane integrity, as evidenced by the fact that platelets incubated with APT1 remained impermeant to sulforhodamine ( Figure 7A).…”

Dynamic cycling of t-SNARE acylation regulates platelet exocytosis

“…Instead, both proteins contain cysteine-rich regions: human syntaxin-11 has 8 cysteines with 6 at its C-terminus, and SNAP-23 contains 6 cysteines with 5 in a central domain. These cysteine-rich regions have been shown, in some cells, to be important for membrane association and are potential sites for S-acylation (16)(17)(18)(19)(20) (27,28).…”