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.
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.
Gil
Anabel
A
Department of Biochemistry and Molecular Biology and the Instituto de Neurociencias, Universidad Miguel Hernández-Consejo Superior de Investigaciones Cientificas, 03550 San Juan, Alicante, Spain.
Gutiérrez
Luis M
LM
Carrasco-Serrano
Carmen
C
Alonso
M Teresa
MT
Viniegra
Salvador
S
Criado
Manuel
M
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000378:metabolism; D005089:Exocytosis; D049452:Green Fluorescent Proteins; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D050825:Synaptosomal-Associated Protein 25; D013696:Temperature; D013997:Time Factors
D008164:Luminescent Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins; D050825:Synaptosomal-Associated Protein 25; D049452:Green Fluorescent Proteins