The SNAP-25 Linker as an Adaptation Toward Fast Exocytosis

Abstract

The assembly of four soluble N-ethylmaleimide-sensitive factor attachment protein receptor domains into a complex is essential for membrane fusion. In most cases, the four SNARE-domains are encoded by separate membrane-targeted proteins. However, in the exocytotic pathway, two SNARE-domains are present in one protein, connected by a flexible linker. The significance of this arrangement is unknown. We characterized the role of the linker in SNAP-25, a neuronal SNARE, by using overexpression techniques in synaptosomal-associated protein of 25 kDa (SNAP-25) null mouse chromaffin cells and fast electrophysiological techniques. We confirm that the palmitoylated linker-cysteines are important for membrane association. A SNAP-25 mutant without cysteines supported exocytosis, but the fusion rate was slowed down and the fusion pore duration prolonged. Using chimeric proteins between SNAP-25 and its ubiquitous homologue SNAP-23, we show that the cysteine-containing part of the linkers is interchangeable. However, a stretch of 10 hydrophobic and charged amino acids in the C-terminal half of the SNAP-25 linker is required for fast exocytosis and in its absence the calcium dependence of exocytosis is shifted toward higher concentrations. The SNAP-25 linker therefore might have evolved as an adaptation toward calcium triggering and a high rate of execution of the fusion process, those features that distinguish exocytosis from other membrane fusion pathways. INTRODUCTIONThe soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are central to vesicular trafficking (Fasshauer, 2003;Jahn and Scheller, 2006;Rizo et al., 2006) and characterized by a stretch of 60 -70 amino acids, known as the SNARE motif (Terrian and White, 1997;Weimbs et al., 1997). The assembly of four SNARE domains into a coiled-coil bundle is a general mechanism in fusion of intracellular membrane compartments. The bundle is held together by layers of hydrophobic interaction, with the exception of the middle layer (layer "0"), which is formed by an arginine and three glutamines (Sutton et al., 1998). The SNARE domains can be subdivided into four homologous groups, named after their contribution to the zero layer: R, Qa, Qb, and Qc (Fasshauer et al., 1998b). SNARE assembly requires the participation of one domain from each group, resulting in a certain degree of specificity Scales et al., 2000a).In most cases, the four SNARE-domains are encoded by separate membrane-targeted proteins, but the SNAREs driving the fusion of vesicles with the plasma membrane (exocytosis) are special in that three proteins provide the four domains (Weimbs et al., 1998;Fukuda et al., 2000). One of the SNAREs in this pathway, exemplified by the best-known isoform synaptosomal-associated protein of 25 kDa (SNAP-25), seems to have been created by fusion of the Qb and Qc SNAREs. This arrangement necessitates a flexible linker, which runs back along the complex from the C-terminal end of the first (Qb) SNARE-domain and connects to the Nterminal end of the second SN...

Distinct Initial SNARE Configurations Underlying the Diversity of Exocytosis

“…The sustained phase is greater with SNAP23 than with SNAP25 (436,607), consistent with the idea that SNAP23 is involved in the unitary-SNARE configuration.…”

“…Interestingly, the kinetics of exocytosis are markedly altered by the linkers between the SNARE motif and the TMDs of VAMP (66,136,217,316,406) and syntaxin (668), and by the linker between the two SNARE motives in SNAP25 (69,436). Thus the linker regions outside the SNARE motif play an important role in the kinetics of membrane fusion mediated by SNAREs, in line with the idea that SNARE assembly can be induced up to the transmembrane regions (613).…”

“…Exocytosis of LDCVs containing a brain-derived neurotrophic factor (BDNF) is full fusion in dendrites and kiss-and-run fusion in axons (397). SNAREs, and many SNARE-interacting proteins, including Munc18a (173,292), synaptotagmins (579,742), Doc2 (178), SNAP25 (163,436), VAMP2 (66,316), syntaxin (227), SNARE complex (226), and complexin (9,17), affect fusion pore properties (see sect. V).…”

“…These states were interconvertible in a Ca 2ϩ -independent manner, with a time constant of 4 s (675). The fast trans-SNARE state was selectively eliminated by BoNTA (706), COOHterminal mutation of SNAP25 (608), and deletion of synaptotagmin1 (674) and was slowed by a R233Q mutation in synaptotagmin1 (605), replacement of SNAP25a with SNAP25b (607), and replacement of the SNAP25b COOHterminal linker with SNAP23 (436). The amplitude of the fast component, but not the slow component, of the capacitance increases was regulated by PKA-mediated phosphorylation of SNAP25 (437).…”

SNAP25 expression in mammalian retinal horizontal cells

“…Of particular interest is that exon 5b incorporates a palmitoylation site, leading to the plasma membrane association of the protein (Hess et al, 1992; Veit et al, 1996; Lane and Liu, 1997). These palmitoylated cysteines are important for vesicular release, as exocytosis is slowed when these sites are mutated (Nagy et al, 2008). …”