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.
Highlights
The SNAP1 receptor (SNARE) hypothesis [1] has been crucial to our understanding of the molecular machinery responsible for exocytosis
Exocytotic Kinetics Regulated by SNARE Domains forms of SNAP-25 in chromaffin cells is a valuable tool when studying its role in neurosecretion [14, 15]
Similar results (Fig. 1B) were observed using a construct lacking the last nine C-terminal residues of this SNARE (GFP-SNAP-25 ⌬9) and equivalent to the proteolyzed form produced by the action of botulinum toxin A [28, 29]. These results indicated that the amino acid L203, which is absent in this construct as well as in the SNAP-25 polypeptide cleaved by botulinum neurotoxin A, is critical for exocytosis
Summary
Generation of Constructs of GFP Coupled to SNAP-25 or Synaptobrevin—To produce an in-frame fusion of SNAP-25 to the C terminus of GFP, the cDNA corresponding to the SNAP-25a isoform [18] was cloned into the expression vector pEGFP-C3 (CLONTECH, Palo Alto, CA) as previously described [14]. The amplified DNA carried an internal ClaI site, close to the 5Ј-end, and a BamHI site at the 3Ј-end These enzymes were used to substitute the original SNAP-25 sequence by the modified one in the GFP-SNAP-25 construct. The strategy for generating the synaptobrevin mutants (A81D, A82D and L84E) was based in the presence of an SpeI site at the 5Ј-end of the region containing the nucleotides to be mutated and the previously mentioned BamHI site at the 3Ј-end. Primary cultures of chromaffin cells were infected with a Herpes Simplex virus (HSV-1) amplicon containing the constructs described above. For this purpose they were transferred from the pEGFP-C3 or pEGFP-N1 vectors to the pHSVpUC vector [20]. Complex dissociation was stopped by cooling the samples to 4 °C before complexes were analyzed by SDS-PAGE
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