Modifications in the C Terminus of the Synaptosome-associated Protein of 25 kDa (SNAP-25) and in the Complementary Region of Synaptobrevin Affect the Final Steps of Exocytosis

Abstract

Fusion proteins made of green fluorescent protein coupled to SNAP-25 or synaptobrevin were overexpressed in bovine chromaffin cells in order to study the role of critical protein domains in exocytosis. Point mutations in the C-terminal domain of SNAP-25 (K201E and L203E) produced a marked inhibition of secretion, whereas single (Q174K, Q53K) and double mutants (Q174K/Q53K) of amino acids from the so-called zero layer only produced a moderate alteration in secretion. The importance of the SNAP-25 C-terminal domain in exocytosis was also confirmed by the similar effect on secretion of mutations in analogous residues of synaptobrevin (A82D, L84E). The effects on the initial rate and magnitude of secretion correlated with the alteration of single vesicle fusion kinetics since the amperometric spikes from cells expressing SNAP-25 L203E and K201E and synaptobrevin A82D and L84E mutants had lower amplitudes and larger half-width values than the ones from controls, suggesting slower neurotransmitter release kinetics than that found in cells expressing the wild-type proteins or zero layer mutants of SNAP-25. We conclude that a small domain of the SNAP-25 C terminus and its counterpart in synaptobrevin play an essential role in the final membrane fusion step of exocytosis.The SNAP 1 (soluble NSF attachment protein) receptor (SNARE) hypothesis (1) has been crucial to our understanding of the molecular machinery responsible for exocytosis. The molecular events taking place during the exocytotic fusion of cellular and vesicular membranes should provide fundamental information about the mechanism of membrane fusion common to different membrane trafficking processes (2). The assembly of a ternary complex formed by the plasma membrane proteins syntaxin (3) and SNAP-25 (4) and the vesicle-associated protein synaptobrevin (5) is considered to be one of the molecular events driving vesicle priming, involving maturation steps needed to promote the apposition and final fusion of membranes (6). A heptad repeat structural motif typical of coiledcoils forming proteins is present and could be the basis for the formation of the core complex. This was confirmed by the observed increase in ␣-helix content following assembly of the complex (7,8). In recent years, precise structural studies on the nature of the complex (9, 10) have shown that single fragments of 60 -70 residues of syntaxin and synaptobrevin together with two segments of SNAP-25 form helices in a four-stranded coil. Although most of the interactions between these helices are hydrophobic, there is a polar layer embedded in the middle of this rod-shaped structure formed by three glutamines and one arginine (zero layer), which is believed to be critical for SNARE complex formation. In addition, functional studies using specific neurotoxins (11), antibodies against critical domains (12), peptides imitating regions of SNAREs (13), and overexpression of altered forms of these proteins (14, 15) have revealed important details about the participation of SNAREs in the exocytotic proce...

Arg206 of SNAP-25 is essential for neuroexocytosis at the Drosophila melanogaster neuromuscular junction

“…This does explain the dominantnegative nature of the mutation introduced here with respect to neuroexocytosis, and fits well with the specific action of BoNT/A and BoNT/C which cleave SNAP-25 just before and after Arg198, respectively, causing a dominant-negative effect with the characteristic long duration of action of these two neurotoxins (Eleopra et al, 1997;Eleopra et al, 1998;Meunier et al, 2003). The present model also explains the remarkable findings that the replacements of Lys201 and Leu203 with a negatively charged glutamate residue do not affect SNARE assembly but inhibits exocytosis in chromaffin cells (Criado et al, 1999;Gil et al, 2002) as these changes are disturbing the protein-protein contacts between adjacent SNARE complexes.…”

The distribution of mitochondria and endoplasmic reticulum in relation with secretory sites in chromaffin cells

“…Chromaffin cells were isolated from bovine adrenal glands by collagenase digestion, and they were further separated from the debris and erythrocytes by centrifugation on Percoll gradients as described previously (Gutierrez et al, 1988;Gil et al, 2002). The cells were maintained as monolayer cultures in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 10 mM cytosine arabinoside, 10 mM 5-fluoro-29-deoxyuridine, 50 U/ml penicillin and 50 mg/ml streptomycin.…”

Complexin II plays a positive role in Ca 2+ -triggered exocytosis by facilitating vesicle priming

“…Because CPXs bind to the assembled SNARE core complex (7), and SNARE proteins are known to play a role in the behavior of the fusion pore (27)(28)(29)(30)-the aqueous pore that initially connects the vesicle lumen and extracellular space during exocytosis-we tested whether the kinetics of fusion pore dilation is altered in the CPX II Ϫ/Ϫ chromaffin cells. We used amperometry, an electrochemical technique that directly reports the kinetics and amount of catecholamine release from individual vesicles.…”