M. Criado et al., 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, P NAS US, 96(13), 1999, pp. 7256-7261
Citations number
31
Categorie Soggetti
Multidisciplinary
Journal title
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
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.