A single amino acid near the C terminus of the synaptosome-associated protein of 25 kDa (SNAP-25) is essential for exocytosis in chromaffin cells

Amperometry in chromaffin cells expressing green fluorescent protein (GFP) fused to synaptosome-associated protein of 25 kDa (SNAP-25) have been used to test the involvement of single amino acids in exocytotic function, overc oming some of the limitations of studies based on Botulinum neurotoxin clea vage, as this occurs at defined sites of the protein. Constructs containing either the whole SNAP-25 polypeptide or several deleted forms larking its C-terminal domain were heavily overexpressed in transfected cells. All GFP- fusions were located in both the cytoplasm and the plasma membrane. Althoug h a construct containing complete SNAP-25 sustained normal secretion, remov al of four or more amino acids of its C terminus greatly altered the overal l rate and extent of exocytosis, Further mutational analysis proved that Le u(203), the fourth residue from the C terminus, is critical for secretion. Kinetics of single granule fusions from cells expressing truncated forms sh owed slow onset and decay times when compared with control cells expressing full SNAP-25, Thus, these data provide direct evidence for the involvement of a specific residue of SNAP-25 in exocytosis and show that overexpressio n of GFP-SNAP contructs combined with single vesicle fusion measurements co nstitutes a powerful approach to dissect the structural elements playing a role in individual steps of the exocytotic cascade.